Lectured by: Aldrin Jeff B.

Autencio, MSMT

Objectives:
 Define clinical chemistry  Enumerate the different biologic specimens used for
 Enumerate the areas of interest of clinical chemistry clinical chemistry analysis
 Identify the common analytes measured in the clinical  Differentiate the two chemistry systems
chemistry department
Clinical Chemistry
 All of the visible or invisible objects and materials around are constituted by the chemical composition, that includes the
human body. Analyzing the chemical composition of human body is the scope of clinical chemistry research.
 It is used to analyze the chemical composition of human body in order to understand the health status or to identify the disease.
 Clinical chemistry is defined as a quantitative science that is concerned with measurement of amounts of biologically
important substances (called analytes) in body fluids.
o The methods to measure these substances are carefully designed to provide accurate assessments of their concentration.
o The results of clinical chemistry tests are compared to reference intervals or a medical decision level to provide diagnostic
and clinical meaning for their values.
 The primary purpose of a clinical chemistry laboratory is to facilitate the correct performance of analytic procedures that
yield precise information, aiding patient diagnosis and treatment.
o The achievement of reliable results requires that the clinical laboratory scientists must be able to correctly use basic supplies
and equipment and possess an understanding of fundamental concepts critical to any analytic procedure.
 Clinical chemistry continues to be one of the most rapidly advancing areas of laboratory medicine.
o New technologies and analytical techniques have been introduced with a dramatic impact on the practice of clinical
chemistry and laboratory medicine.
 In addition, the health care system is constantly changing, therefore there is an increased emphasis on improving quality of
patient care, increased individual patient outcomes, increased financial responsibility, and increased total quality management.
o We must always remember that as medical technologists, we must always strive for good quality laboratory results.
o To ensure accurate and precise laboratory protocols, we must always follow the standard operating procedures to ensure that
we provide the best quality care to customers.
 Detailed procedures have been omitted because of the variety of equipment and commercial kits used in today’s clinical
laboratories. The commercial kits used commonly in today’s clinical laboratories are the package inserts or instrument
manuals and kit package inserts.
o They are the most reliable reference for detailed instructions on current analytic procedures.
o Through modernization, there has been constant change and improvement in quality service. This includes from outdated
chemicals, to modernized packaged kit inserts.
 In the clinical chemistry department, one must be able to identify what are the reagents used for different clinical chemistry
analysis.
 Reagents are known to be substances that are employed to produce a chemical reaction when coupled with other substances.
o Depending on the instrumentation, reagents come in several forms: liquid, dry, cartridge, or strip.
o Any agent that is prepared must be labeled with date and time of preparation, and the concentration being prepared.
o Preparation of any reagent, standard or chemical, requires precision and accuracy.

Testing by Manual Methods

 Manual means making the reagents and performing the test by hand.
 Disadvantages:
o Definitely slow o Definitely labor-intensive o Often not precise
Testing by Automated Methods
 Due to modernization there has been constant change in the clinical laboratory. Students in medical technology programs must
always learn manual methods to gain deeper understanding of the automated instruments to be used in the laboratory.
 Automated analyzers merely incorporate the methods previously used in manual methods for chemical analysis.
 Automation
o Even as late as 1960, most tests performed in the lab were done manually. Reagents were made by technologists, tests were
performed usually singly when needed.
o Slowly at first, automation became the driving force within the lab, yielding better, faster, and improved testing.
o Most current laboratories are now highly automated to accommodate the high workload typical of a hospital laboratory, and
tests performed are closely monitored and quality controlled.
 Common Benefits of Automation
o Increased number of tests performed by one technologist in a given time period.
 o Labor cost is decreased.
o Minimized variation in results between technologists due to slight variations in technique. This is all made possible by
performing quality control.
o Increased variety of techniques/tests being offered; improved patient care as a result
 Increased number of tests performed by one technologist pertains to increased walk-away capabilities (more multitasking for
the staff and allows the staff to perform other laboratory procedures for different specimens)
 Smaller amounts of samples and reagents are also required in automated analyzers.

Areas of Interest of Clinical Chemistry

1. Analytical Chemistry- is the science of obtaining, processing, and communicating information about the composition and
structure of matter.
2. Biochemistry- is the branch of science that explores the chemical processes within and related to living organisms.
3. Endocrinology- is a branch of biology and medicine dealing with endocrine system, its diseases, and its specific secretions
known as hormones.
4. Instrumentation- is a process by which the analytical instruments perform many tests with the least involvement of an analyst.
5. Toxicology- pertains to the measurement and analysis of potential toxins (intoxicating or banned substances) and prescription
medications present in a person's body.

Common Analytes Measured in the Clinical Chemistry Laboratory

 Clinical chemistry is the branch of laboratory medicine that focuses primarily on molecules.
 Analyte- is the chemical in the blood that you want to test for.
 Panel of Tests
o When an individual test alone is not sufficient to assess a medical condition, a combination of several tests may be used.
o The pattern of results from the combination of tests may provide better insight into the status of the patient, than any single
test result. Such tests done on the same sample are often ordered as a group called a panel or profile.
Common Biologic Specimens
 Blood
o Is the most common biologic fluid collected for clinical laboratory testing.
o It is usually drawn from a vein in the arm, directly into an evacuated tube or
syringe system.
o Typically a tube will hold about 5 mL of blood enough to perform many clinical chemistry tests,
since automated analyzers require only small amounts (usually from 2-100 μL) for a single test.
o Blood consists of two main parts- a fluid portion (called plasma, which contains the dissolved
ions and molecules) and a cellular portion (the RBCs, WBCs, and platelets).
o Most clinical chemistry analytes are found in plasma.
 Part of the preparation of blood testing for these analytes involves removing the cells. This is
done by centrifugation of the sample to pack the blood cells in the bottom of the collection tube and allow removal of the
liquid portion for testing.
 Depending on the laboratory test, the best yield for the analyte would either be plasma or serum (case-to-case basis).
o If a blood sample is collected in a tube containing an additive (which prevents blood from clotting), the fluid portion of the
blood is known to be as plasma.
o If blood is collected in a tube with no anticoagulant (which the blood is allowed to clot), it is known to be the serum.
o Upon centrifugation, the clot descends to the bottom of the tube along with the cells. The resultant liquid above the cells
and clot is called serum.
 o Some clinical chemistry tests are best performed using plasma, others are best performed using serum, and still others can be
performed using either plasma or serum.
 Urine
o It is especially suitable for tests that evaluate kidney functions, tests that look at waste products that are excreted by the
kidneys, and for metabolites that are cleared quickly from the bloodstream and accumulate in the urine, such as drugs of
abuse.
o Sometimes both serum and urine concentrations of a substance are useful to know in order to evaluate how well the analyte
is being excreted. Either to ensure that expected excretion is taking place, or to determine if unexpected leakage is occurring.
o Different Types of Urine and how these types of urine is being used:

o Urine is relatively easy to collect from most people, although special techniques may be needed for infants and small
children.
o Different types of urine samples, representing collection at different times of day and for different durations of time, are used
for laboratory analyses.
 Fluids other than blood and urine are used in limited clinical settings and are tested for only a special analyte.
 Amniotic Fluid- is typically used for tests of fetal health.
 Peritoneal/Pericardial/Pleural Fluids
o Chemical testing of these fluids is typically done to assess the origin of the fluid.
o This is to determine whether it has leaked from the blood vessels because of high pressure differences or because of
inflammation or injury.

Chemistry Systems
 Dry Reagent Strips
o Requires the comparison of a color change on the reagent strip color chart.
o These are most commonly used as a quick screening test, while their accuracy is only low to moderate.
 Wet and Dry Chemistry Systems
o Utilizes a spectrophotometer to mechanically measure color change.
o This is considered to be more accurate than dry reagent strips.

Types of Measurement
 Qualitative Measurement- Gives results in descriptive, non-numeric form
 Quantitative Measurement- Gives results in definitive form, usually in numbers with units
 Example: Capillary Blood Sugar taken from a patient
o Qualitative measurement: the blood glucose of the patient is high (relies on words to tell us quality)
o Quantitative measurement: the blood glucose of the patient is 208 mg/dL (relies primarily on numbers as
the main unit of analysis)
 Lectured by: Aldrin Jeff B. Autencio, MSMT
Objectives:
 List the commonly used laboratory supplies, and describe their specific functions.
 Differentiate between the terms “to contain” and “to deliver” (pertains to pipettes)
 Explain the difference between volumetric and graduated pipettes
 List a procedure for the preparation of reagent
 Describe the proper procedures for using the different types of pipettes
 Describe the proper operation and maintenance of centrifuge
 List examples of glass and plastic wares and describe their useful qualities
 List and describe the four grades of chemicals and explain how they differ in their degree of purity

Pipettes
 Are used to transfer or measure aliquots of a liquid (Aliquots- refers to a portion of a larger hole)
 Are glass or plastic utensils
 Can be disposable or reusable (Example of a disposable pipet routinely used in the laboratory: Pasteur pipettes)
 Pipettes vary in design, performance, linearity, ergonomics, and durability. The most well-made pipettes are often the highest priced
 2 organization groups responsible for the acceptance of the pipettes:
o National Institute of Standards and Technology (NIST) (also known as National Bureau of Standards/NBS)
 Established tolerances for different volumetric glassware used in a clinical chemistry laboratory
 Establishing tolerances pertains to the accuracy of marking. Example: if a pipette is said to
be tolerated for 25 mL, then that pipette will deliver 25 mL of an aliquot of a solution.
 Provides a calibration service for manufacturers of volumetric labware that directly links the
glassware standards to national and international standards
o American Society for Testing and Materials (ASTM)
 Is a membership organization that writes voluntary consensus standards.
 Each pipet is calibrated in accordance with ASTM E542 and meets accuracy requirements of ASTM E969.
 The pipettes shall consist (in general) of a suction tube and a delivery tube.
o When using the pipet, the tip must be immersed in the intended transfer liquid to a level that will
allow the tip to remain in the solution, even if after the volume of liquid has entered the pipette.
o This is to ensure no bubbles are acquired during the suction.
o Nominal volume- dictates the tolerance of the pipet
 Pipette aids/pipettors
o Are suction devices that are used to either suck liquids into or expel liquids out of pipettes.
o They are contoured shape and heavy-walled neck to increase the grip for easier use with pipettes.
o Caufield- most commonly used o Safety bulb
o Spectroline
 Pipette Classification
I. Design
 To contain (TC)
 Holds or contains a particular volume but does not dispense the volume indicated.
 Examples: micropipettes and Salhi’s pipette
 To deliver (TD)
 Will dispense the volume indicated
 Examples: Serological, Mohr pipettes, Volumetric pipettes, Ostwald-Folin pipettes
 The major difference is that TC devices do not deliver the same volume when the liquid is transferred into a container, whereas TD
designation means that the labware will deliver that amount.
 Whether the pipette is TC or TD, when using either pipet, the tip must be immersed in the liquid to be transferred to ensure that it
will allow the pipette to remain in solution after the volume of liquid has entered the pipette without touching the vessel wall (never
allow the pipette tip to touch the vessel wall)
II. Draining Characteristics
 Blow-out
 Has 2 continuous rings located near the top of the pipette (either marked as etched ring or etched band)
 The last drop of liquid should be expelled into the receiving vessel
 Examples: Serological and Ostwald-Folin pipettes
 In Ostwald-Folin pipettes in contrast with volumetric pipettes, has an etched ring indicating it is a blowout pipette
 Requires the aid of pipette aids/pipettors. The use of the pipettor/pipette aid will ensure that the last drop of liquid is expelled into
the receiving vessel. Using a pipette bulb/pipettor, a slight suction is applied to the opposite end until the liquid enters the pipette
and the meniscus is brought above the desired graduation line, suction is then stopped.
 Self-draining
 No ring markings located at the top of the pipettes (do not contain etched ring/etched band)
 Draining is solely by gravity
  Examples: Mohr pipette, Volumetric pipette, Van Slyke pipettes
 Mohr pipette is distinguishable in contrast to serological pipet because the increment does not reach the tip of the pipette
III. Type
 Measuring or Graduated
 Serologic
 TD and is generally a blow-out pipette
 Has graduation marks to the tip (needs a pipettor/pipette aid to dispense the last remaining droplets of
liquid found on the pipette tip to be expelled into the receiving vessel)
 In contrast to Mohr pipette, the increment is almost found at the tip of the pipet
 Mohr
 TD and self-draining pipette
 Does not have graduations to the tip
 The tip of the pipette should not be allowed to touch the vessel while the pipette is draining.
 Considered to be more accurate than serological pipettes.
 Bacteriologic  Ball, Kolmer, or Khan
 Micropipette
 Has a total holding volume of less than 1 mL
 Examples: Salhi-Hellige pipette, Lang-Levey pipette, RBC and WBC pipettes, and Kirk and Overflow pipette
 Transfer
 Volumetric
 TD and self-draining pipette
 Only measures one volume and is used for non-viscous fluids
 Have been used to add the diluent to a lyophilized control or to measure standards and controls
 Typically, volumetric pipette is more accurate than serologic pipettes.
 It has a bulb, no etched ring, and the reliability of volumetric pipet decreases as volume decreases, therefore
special micropipettes have been developed for microanalysis.
 Ostwald-Folin
 TD and blow-out pipette
 Bulb is closer to the delivery tip, reducing the surface area in contact with the liquid
 Used for viscous fluids (such as serum, plasma, and whole blood) and the final drop is blown out.
 In contrast with volumetric pipettes, an etched ring near the mouth indicates that this is a blow-out pipette.
 Van Slyke pipette
 Thick-walled capillary tubing with bulb in the center
 Salhi’s pipette
 Can deliver 20 μL of aliquot
 Pasteur pipettes
 Do not have calibration marks and are used to transfer solutions or biologic fluids without consideration of a specific volume.
 Automatic micro/macropipettes
 TC and measure small amounts of liquid
 Most routinely used pipette in the clinical laboratory, because they allow microanalysis since they
measure small amounts of a liquid and transfer that small amount of liquid from one container to the
next.
 Macropipette- piping capability of more than 1 mL
 Micropipette- pipetting capability of less than 1 mL
 Automatic as used here implies that the mechanism that draws up and dispenses the liquid is an integral part of the pipette
 Semi-automated pipettes
o Offer more convenience and efficiency to pipetting because they may be single channel or multichannel
o No pipetting bulb/pipettor is required nor do pipettes have to be washed.
o Require the use of plastic tips (propylene) because it is disposable and autoclavable.
o The automatic pipe is the most routinely used pipette in today’s clinical chemistry laboratory
o Automatic and semi-automatic pipettes have many advantages including safety, stability, ease of use, increased precision, the ability
to save time, and less cleaning required, as a result of the contaminated portions of the pipette often being disposed.
o Parts of the pipet
 Plunger/trigger/piston- used to aspirate and dispense the liquid into the pipette
 Ejector- used to remove the pipette tip
 Mechanical or automatic pipettes
o Positive displacement pipette
 Operates by mobbing the piston in the pipette or barrel, much like a hyperdermic needle
 Does not require a different tip for each use
 Recommended for saline, water, and phosphate buffers and is not suitable for very dense liquids
o Air displacement pipette (most common principle applied in the pipette)
 It relies on the piston for suction creation to draw the sample into a disposable tip
o Dispenser/dilutor pipette
 Obtain the liquid from a common reservoir and dispense it repeatedly
 Pipette Quality Control
Performance Verification- quality control ensures that the pipette that we are using is of good quality, giving accurate and precise
o
measurements of aliquots.
o Maintenance- includes disassembly, cleaning, replacement of worn, corroded or suspected parts, relubrication and reassembly
o Calibration- pipettes according to the College of American Pathologists (CAP) should be calibrated monthly
 For volumetric TD pipette, it should not be shaken or hit against the wall of the container during draining.
 Rinse the pipettes with distilled water before using and rinse it again using the desired solution to be aspirated (to ensure that the
aliquot is not diluted with the distilled water used to clean the pipettes first)
 Clean the pipettes with 5% sodium hypochlorite after each usage (unclean labwares may result to inaccurate laboratory results)
 Pipettes should be held vertically when dispensing the fluid into the receiving vessel.

Glasswares and Plasticwares

 Until recently, laboratory supplies consisted of some types of glass and could be correctly termed as glasswares.
 As plastic material was refined and made available to manufacturers, plastic has been increasingly used to make laboratory utensils.
 Glasswares are reusable and ideal for acidic solutions, high thermal, and corrosion resistance, and is often low cost.
 Plasticwares is ideal for alkaline solutions and are shock proof and shatter proof. They also are considered to be flexible.
 Tolerances of Accuracy by ASTM
o Class A- preferred for laboratory applications and do not need recalibration
o Class B- twice the tolerance limits of class A, and is often found in student laboratories
 Glasswares
o High thermal resistance glass
 Borosilicate glass is an example
 Strain point: 515°C; and low alkaline content
o Corex
 6x stronger than borosilicate glass
 Ideal for higher temperature thermometers, graduated cylinders, and centrifuge tubes
o Vycor
 Recommended for high temperature, drastic heat shock, and extreme chemical treatment with acids and dilute alkalis
 Ideal for ashing and ignition techniques
o High silica glass
 Used for radiation, because it is radiation resistant
 Ideal for high precision and analytical works and can also be used for optical reflectors and mirrors
o Glass with high resistance to alkalis (soft-glass)
 Boron-free for strong alkali solution and digestion
 Less thermal resistance than borosilicate glass
o Low actinic glass
 Amber or red-colored to reduce amount of light passing through the substance within the glassware
 Used for handling heat-labile substances (like bilirubin, carotene, and vitamin A)
o Standard flint glass
 Poorly resistant to high temperature and sudden changes in temperature
 Used as reagent bottles and disposable glasswares
 Plasticwares
o Plasticware is beginning to replace glassware in the laboratory setting. The unique high resistance to corrosion and breakage, as well
as varying flexibility has made plasticware most appealing.
o Polyolefins (polyethylenes, polypropylenes) o Tygon
o Polycarbonate resin o Teflon
o Polypropylene plastics are known to withstand higher temperature and can be sterilized, but this type of plasticware absorbs
pigment and tends to be discolored.
o Examples of plastic wares: water bottles, pipette tips, conical tubes
 In most laboratories, glass or plastic that is in direct contact associated with biohazardous material is usually disposable. If not, it must
be decontaminated according to appropriate protocols. Should the need arise however, cleaning of glass or plastic may require special
techniques.
 Immediately rinsing glass or plasticware supplies after use should be followed by washing it with powder or liquid detergent
designed for cleaning laboratory supplies. This must be paired with a couple and several of distilled water rinses to ensure that the
plasticware can be reusable.

Separation Techniques
 Centrifugation (most common separation technique)
o Is the process in which centrifugal force is used to separate solid matter from a liquid suspension. The centrifuge is used to carry out
this action.
o Different Parts of a Centrifuge:
 Rotor- which holds the container of mixture to be centrifuged  Motor- generates power
 Shield
 o Centrifugal force depends on three variables: Mass, Speed, and Radius
o The speed of the centrifuge is related to the relative centrifugal force (RCF) by the following equation:
��� = 1.118 � 10−5 � ������ � (���)2
 �. ��� � �� −�
is constantly an empirical factor determined from the angular velocity based solely on observation rather than
theory.
 Radius is in centimeters measured from the center of the centrifuge axis to the bottom of the test tube shield.
o Uses of centrifuge:
 Separate serum or plasma from blood cells  Separate two-immiscible liquids
 Separate supernatant from a precipitate  To expel air
o Tips to remember with the use of centrifuge
 Centrifuge care includes daily cleaning of any spills or debris such as blood or glass
 Ensuring that the centrifuge is properly balanced and free from any excessive vibrations. Balancing the
centrifuge load is critical.
o Types of Centrifuge
1. Horizontal-head or Swinging-bucket centrifuges
 Allows the tubes to attain horizontal position in the centrifuge when spinning, and in a vertical position
when head is not moving
 Commonly used for serum separator tubes
2. Fixed-angle or Angle-head centrifuges (most common)
 Holds the tubes at a specified angle, usually 25 to 50 degrees to the vertical axis of rotation.
3. Ultracentrifuge
 Are commonly used to separate lipoproteins (chylomicrons, VLDL, HDL, and LDL).
 Chamber is generally refrigerated to counter heat produced through friction.
 The centrifuge cover should remain closed until the centrifuge has come to a complete stop to avoid any aerosol
contamination.
 The speed of a centrifuge is easily checked using a tachometer or strobe light
 The hole located in the lid of many centrifuges is designed for speed verification using these devices but may
also represent an aerosol biohazard.
 Filtration

Chemicals Used for Regent Preparation

 Reagents are substances used to react with your sample
 Analytic Reagent Grade (ARG)
o Important for qualitative and quantitative analysis
o Specifications were established by the American Chemical Society (ACS)
o Uses: trace element analysis and preparation of standards; Standard solutions are important for quality control monitoring.
 Ultrapure reagents
o Have been put through additional purification steps
o Examples: Spectograde, Nanograde and for High Performance Liquid Chromatography (HPLC) pure analysis
o Uses: chromatography, atomic absorption, and immunoassays
 Chemically Pure (CP) or Pure grade
o Impurity limitations are not stated; preparation of these chemicals is not uniform
o Is not recommended for research and analytical chemistry unless purification or a reagent blank is included
 Technical or Commercial grade
o Is used primarily in manufacturing and it should never be used in clinical laboratory setting.
 United States Pharmacopoeia (USP) and National Formulary (NF)
o Is approved for human consumption but may not be applicable for laboratory analysis
o Purpose: drug monitoring
 Three Grades of Reagent Water
o Filtration is the first step before the processes are performed in reagent-grade water preparation.
o Processes involved in the preparation of reagent-grade water: Distillation, Ion exchange, Reverse osmosis, and UV oxidation
o Type 1 Reagent Water
 Is used for procedures that require maximum water purity for accuracy and precision
 Used for ultramicrochemical analysis, measurements of nanogram or subnanogram concentrations, tissue or cell methods
(microscopy), and preparation of standard solutions.
 Uses: Flame photometry, Atomic Absorption Spectrophotometry, Blood gases and pH, Enzyme studies, Electrolyte testing,
High Performance Liquid Chromatography (HPLC), Trace metal and Iron studies.
o Type 2 Reagent Water
 For hematology, microbiology, immunology and chemistry section
 Is acceptable for preparation of reagents and quality control materials
o Type 3 Reagent Water
 Routinely used for urinalysis, parasitology, and histology
 Commonly used for washing glasswares.
 Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Determine the rules in identifying significant figures  Solve for the given mathematical problems
 Memorize the units of measurement and their conversion factors

Significant Figures (significant digits)

 Very important part of scientific and mathematical calculations because they deal with accuracy and perception of numbers.
 If you are uncertain of your final answer, you have to take note of the number of the significant figures in order to determine what
significant figures you are going to place in your final answer.
 When making calculations, the final answer is rounded off to reflect the value with the least certainty (the fewest significant figures).
 For multiplication and division, the answer must have the same number of significant figures as the starting value with the least
number of significant figures.
 For addition and subtraction, the answer must have the same number of decimal places as the starting value with the fewest number
of decimal places.
 Rules in Identifying Significant Figures
o All non-zero digits are significant
o Leading zeroes (zeros before non-zero digit) are not significant
o Trailing zeroes (zeros after non-zero digit) are significant only if the digits have a decimal point
o Captive zeros (zeros that are in between of the non-zero digits) are significant
 Examples
o 10 →1 o 35 →2 o 0.00350 → 3
o 10. →2 o 0.35 →2 o 73.04 → 4
o 10.0 →3 o 0.0035 → 2
o Multiplication: 2.5 � 0.5250 = 1.3125 (2.5→ 2 significant figures, 0.5250→ 4 significant figures); Final answer: 1.3
o Addition: 15.5 �� + 25.92 �� = 41.42 (15.5 has the fewest decimal places); Final answer: 41.4

Exponential Notation
 Sometimes we are dealing with very high and very low numbers, in which if we have to write them as a whole figure it will be very
lengthy.
 Positive Exponential Value
o Example: 102 → multiply our value with 10 twice, whatever the exponent above a digit signifies the number of times you have
multiplied it to a certain value, if the exponent is a positive value
o 2.5 � 102 = 2.5 � 10 � 10 → Move the decimal points two digits to the right → 250
 Negative Exponential Value
o Example: 10-2 → divide the number depending on the value of the exponent
o 2.5 � 10−2 = 2.5 ÷ 10 ÷ 10 → Move the decimal point two digits to the left → .025
 Examples in Mathematical Operations o In division, the exponents are subtracted
o In multiplication, the exponents are added (4 � 103)
 (2 � 105) = 2 � 10−2
4 5 9
 3 � 10 5 � 10 = 15 � 10
Units of Measure
 SI Units
o Founded on 7 base units for 7 base quantities, and they are assumed to be mutually independent.
o Base quantity is an original unit of measure
o Selected derive is a derived quantity and is defined based on a combination
of base quantities and has a derived unit, that is the exponent, product, or
quotient of these base units.
o SI unit is the modern form of the metric system and it is the world’s most
widely used system of measurement. In 1966, the International Federation of
Clinical Chemistry and Laboratory Medicine (IFCC) recommended the use of
selected SI units for clinical laboratories.
 Prefixes
o Used to indicate a subunit or multiple of a basic unit.
o Positive exponents values are multiplied so they denote a multiple
of a base unit or a basic unit.
o Negative exponents values are divided and they represent a lower
value so they denote a subunit of a basic unit
 Conversion Factors
o In clinical trials, laboratory tests are performed as a tool for
diagnosing diseases and medical technologists are helping the
doctors to diagnose those diseases by having accurate and precise
 laboratory results.
o However, a common issue is the reporting units for laboratory tests, because some of the laboratories use SI units, while others use
conventional units.
 In order to have a clear distinction between the units and the interpretation of results, conversion factors are generated to identify
and then interpret the values in either conventional (customary unit/gravimetric unit) or SI
units (international system of units/system international).

Temperature Conversions
 Examples:
o 25°C→ °F o 98.6°F→ °C
9 9 5 5
°� = 5 � °C + 32 → °� = � 25 + 32 → 77°� °� = � °F − 32 → °� = � 98.6 − 32 → 37°�
5 9 9
o 20°C→ K � = 20 + 273 = 293�

Units of Concentration and Solution Preparations

 Molarity (M)
o Number of moles (mol) of solute per liter of solution. o �=
��� �� ������
o ��� =
����� �� ������
� �� �������� �� �� ������
 Molality (m)
o Number of moles (mol) of solute per kilogram of solvent. o �=
��� �� ������
�� �� �������
 Normality (N)
oTerm of concentration based on the ability of some o ���� ��. ��. =
�� �� ������

substances to release hydrogen or hydroxide ion in a �������

����� �� ������
solution. o ����������� �� =
���� ��. ��.
o Expressed as equivalents per liter o �= �
��
o � = � � ������� (if M is known)
o Valence- number of replaceable hydrogen ions (acid), number of replaceable hydroxide ions (base), number of replaceable anion
(salt, mixture of cation and anion)
 Ca(OH)2 → 2  H2SO4 → 2  NaCl →1
 HCl →1  NaOH → 1
 Sample Problems
1. What is the molarity of a solution containing 23.4 grams of potassium chloride dissolved in 500 mL of water? (MW of KCl=
74.5g/mol)
1�
 Given: grams of solute= 23.4 g KCl; vol. of solution= 500 mL → 500 �� � 1000 �� = 0.5�; MW of KCl= 74.5 g/mol
����� �� ������ 23.4 �
 ���� = → 74.5 �/��� = 0.31 ���
�� �� ������
����� �� ������ 0.31 ��� �����
 �= → = 0.63 �� 0.63 �
������ �� �������� 0.5 � �
2. What is the molality of a solution containing 5.0 moles of glucose and 4.0 kg of water?
 Given: moles= 5.0 glucose; wt. of solvent= 4.0 kg of water
����� �� ������ 5.0 ����� �����
 � = �� �� ������� → 4.0 �� = 1.2 �� �� 1.2 �
3. What is the normality of a 1-L solution containing 46.3 grams of Ca(OH)2? (MW of Ca(OH)2= 74.1 g/mol)
 Given: grams of solute= 46.3 g of Ca(OH)2; vol. of solution= 1L; MW of Ca(OH)2= 74.1 g/mol; valence of Ca(OH)2= 2
�� �� ������ 74.1 �/��� � �� ������ 46.3 �
 ���� ��. ��. = ������� → 2
= 37.05  ����������� = ���� ��. ��. → 37.05 = 1.25
�� 1.25 �� � 1.25
 �= → = 1.25 �� 1.25 �  � = � � ������� → � = → = 0.625 �
� �� �������� 1� � ������� 2
 Proportionally Equivalent Solutions
o Calculations of two solutions whose concentrations do not change.
o Weight per volume (W/V) is common, but weight per weight (W/W), volume per volume (V/V), and other combinations are also
used.
�1 �2
o The general formula for this is: �2 = �2
 Dilution Equations
oWhen making a dilution, the volume of the original solution is less than the volume of the diluted solution.
oAlthough the amount of solute remains the same, there is a change in concentration. The amount of solute is automatically the
concentration of solution.
o �1 � �1 = �2 � �2
 Sample Problems
1. If a 5.0 dL solution contains 4.5 mg of bilirubin, how much bilirubin would there be in a 2.0-dL aliquot of the solution?
 Given: W1= 4.5 mg bilirubin; V1= 5.0 dL; W2= ?; V2= 2.0 dL
�1 �2 �1 � �2 4.5 �� � 2.0 ��
 �1 = �2 → �2 = �1 → 5.0 ��
= 1.8 �� ���������
 If in 5 dL of serum, there is 4.5 mg of bilirubin, then in every 2 dL of serum, there is 1.8 mg of bilirubin
 2. The total carbon dioxide concentration of a serum sample is 25 mEq/L. How many milliequivalents (mEq) of CO2 are contained in
a 10-mL collection tube?
 Given: N1= 25 mEq/L; V1= 1000 mL; N2= ?; V2= 10 mL
�1 �2 �1 � �2 25 ��� � 10 ��
 = → �2 = → = 0.25 ���
�1 �2 �1 1000 ��
 In every 1000-mL of serum/plasma, there is 25 milliequivalents of carbon dioxide, but in every 10 mL of serum/plasma, there is
25 mEq of CO2
3. What volume of a 0.5M glucose solution can be made from 100 mL of a 3M stock solution?
 Given: V1= 100 mL; C1= 3M; V2= ?; C2= 0.5 M
�1 � �1 100 �� � 3�
 �1 � �1 = �2 � �2 → �2 = → = 600 ��
�2 0.5�
4. A 3.5L solution was diluted with 1.5L of water to create a 0.5M solution with a total volume of 5L. What was the concentration of
the original solution.
 Given: V1= 3.5 L; C1= ?; V2= 5.0 L; C2= 0.5 M
�2 � �2 5.0 � � 0.5�
 �1 � �1 = �2 � �2 → �1 = → 3.5 � = 0.7 �
�1

Ratio and Dilution

 Ratios can be used to express part-per-part or part-per-whole and are written with a colon between figures.
o Example: A blood sample was taken using a 3.2% sodium citrate as the anticoagulant, the ratio of the blood to anticoagulant is 9:1,
but the ratio of anticoagulant to blood is 1:9.
 A dilution can be expressed as a fraction of specimen volume (solute) divided by the sum of specimen volume and diluent (solvent).
o Dilution factor is the reciprocal of the dilution
o Example: 5g of salt is dissolved into 10 mL water
������ 5� 5 1
 �������� = �������� → 5 �+10�� = 15 = ; 3 �� �ℎ� �������� ������
3
 Sample Problems
1. In a solution containing 1 mL of serum and 4 mL of saline, identify the following: ratio of serum to total volume, ratio of serum to
saline, ratio of saline to total volume, ratio of total volume to serum, ratio of saline to serum and dilution.
 1 �� ����� + 4 �� ������ ������� = 5 �� (����� ������)
 Ratio of:
 Serum to total volume = 1:5  Total volume to serum = 5:1
 Serum to saline (diluent) = 1:4  Diluent to serum = 4:1
 Diluent to total volume = 4:5
 Dilution:
������ 1 1
 �������� = �������� → 1+4 = 5 ; 5 �� �ℎ� �������� ������
2. A serum sample for LDH determination was diluted by adding 0.1 mL of serum to 1.9 mL of diluent. Upon measurement, the
instrument showed a flag for upper limit. Further dilution was made by adding 2 mL of diluted sample and 3 mL of diluent. The
diluted sample was measured and showed a value of 120 IU? What should be the reported value?
 1st Dilution: 0.1 �� ����� + 1.9 �� �� ������� = 2.0 → there was a flagging (the result is either too low or too high, so the
machine cannot read the analyte) for upper limit (the LDH value is too high that the machine cannot read it)
 2nd Dilution: 2.0 �� ������� ����� + 3 �� �� ������� = 5.0 → �������� ����� = 120 ��
0.1 2 0.2 1
 �������� ������ = 2.0 � 5 = 10 = 50 ; �������� ������ �� 50
 �������� ����� = �������� ����� � �������� ������ → 120 � 50 = 6000 ��
3. 6 tubes were prepared and labeled A to F. Tube A contains 10 mL of pure/undiluted penicillin. 1 mL of penicillin from tube A is
transferred to Tube B with 9 mL of diluent awaiting and mixed. 1 mL of the mixture from Tube B was transferred to Tube C with 9
mL of diluent. Same procedure was done until Tube F. What is the final dilution of Tube F?
 Example of a dilution series in which the volumes used are constant
������ 1 1
 �������� �� ���� � = ��������
→ =
1+9 10
1 1 1 1 1 1
 �������� �� ���� � = � = → ���� � = → ���� � = → ���� � =
10 10 100 1,000 10,000 100,000
1
 ����� �������� �� ���� � =
100,000
 Lectured by: Aldrin Jeff B. Autencio, MSMT
Objectives:
• Identify the source and composition of blood specimens (plasma, serum, packed RBCs, buffy coat)
• Enumerate the different public relations and client interactions (pertains to being a phlebotomist)
• Enumerate the different types of venipuncture techniques
• List the different specimen considerations for venipuncture
• Enumerate the different troubleshooting for failed venipuncture
• Enumerate the pre-analytical considerations for venipuncture (pertains to activities done prior specimen processing like specimen
collection for venipuncture, specimen transport from the patient room to the laboratory)
• Enumerate the different procedural error risks
• Identify the different specimen handling and processing

Specimen Collection
• Blood is the most common specimen used in the clinical chemistry department
• Collection, handling, and processing of specimen represents a critical step in specimen analysis. Physicians rely on results obtained
from quality laboratory specimens to confirm health or diagnose and treat patients.
• Good quality- would mean that the pre-analytical phase should be done correctly (patient identification, non-hemolyzed specimens,
non-lipemic specimens, non-etheric specimens, etc.).
• The most sophisticated laboratory equipment can never deliver valid results if specimen integrity is compromised.

Collecting Blood Specimens (Phlebotomy)

• The process of collecting blood • Phlebotomy means “to cut veins”
• Two main procedures involved in blood collection: Venipuncture and Capillary puncture/Microcapillary puncture
• Done by phlebotomists (although laboratory technicians and technologists sometimes perform phlebotomy, some individuals called
phlebotomists have been specifically trained in blood collection techniques and they are employed primarily to collect blood specimens)
• Venipuncture
o Is the process where blood is collected through a needle inserted into a vein
o The process of collecting or drawing blood from a vein
o It is the most frequently used procedure performed by a phlebotomist
o Most important step: Patient identification
• Capillary puncture
o Is the process where blood is collected from a skin puncture with the use of a lancet
o Capillary punctures are ideal for small children when only a small volume of blood is needed
o Most common when monitoring blood glucose level. With the use of the kit, the patient’s blood sugar level can be identified
o Capillary blood sampling, which refers to sampling blood from a puncture on the finger, heel, or ear lobe is increasingly common in
medicine
o It enjoys several advantages over venous sampling: less invasive, requires smaller amounts of blood, and can be performed quickly
and easily
• Public Relations and Client Interaction
o As phlebotomists, we are to identify the public relations and client interaction (professionalism in the workplace)
o Upon entry, make sure to wear complete PPE. For girls who have long hair, it must be tied back and having good grooming.
o Blood collection personnel play an important role in public relations, for often they are the only contact to patient and the health care
facility. It is very important that when performing venipuncture or phlebotomy, make sure to assess the patient’s status, and give the
best quality of care that you can give or render to the patient because at the end of the day your skill and treatment to the patient or
your interaction to the patient is validated to the quality of service that the laboratory is giving.
• Professionalism
o Blood collection personnel must always project a professional image. This image involves appearance, attitude, communication skills,
and bedside manner
1. Appearance
▪ Lab coats should protect clothing underneath ▪ Shoes should be conservative and clean
▪ Close attention should be paid to personal hygiene
▪ Hair if long, must be pulled back and fingernails should be short for safety’s sake
2. Attitude
▪ Integrity/Honesty: doing what is right regardless of the circumstances
▪ Compassion: a deep awareness of the distress of others
▪ Motivation: having the drive to meet a need or achieve a goal
▪ Dependability and Work ethic: able to be relied upon, and being self-directed
▪ Diplomacy: skill in handling situations without creating hostility
▪ Ethical behavior: conforming to a standard of right and wrong conduct
▪ Attitude is very important because attitude dictates sometimes our actions. Attitude is the feeling or emotion of an individual has
with regard to something. A professional attitude involves these characteristics.
 3. Communication Skills
▪ As a medical professional, you always have to project professionalism, especially engage yourself in good conduct and good
communication skills with patients.
▪ The phlebotomist is one of the basis for quality of service done by the laboratory
❖ It is very important that when collecting the specimen, you must always have these qualities to ensure that the patient is reassured
that the phlebotomy or venipuncture procedure will be done correctly
▪ Listening forms the foundation for good interpersonal communication and builds rapport with the patients
❖ It is important to build rapport with your patients for reassurance. If reassurance is given to the patient, then the patient will give
his or her full confidence
▪ Using easily understood vocabulary encourages good verbal communication
4. Bedside Manner
▪ Discretion is important when dealing with significant others (SOs) of the patient
▪ Respect should be generated on both sides
▪ You may ask the SOs of the patient to leave the room or ask them to stay especially for pediatric patients
▪ Should the SOs of the patient yell at you, it is very important for you to keep yourself calm so that you will not be affected during
the specimen collection process. The patient is irritable, so that is why in bedside manner it is very important as a medical
technologist or being a healthcare professional to always have patience.
• Patient Confidentiality
o It is very important that being a professional, you have to make sure that any results generated from the machine should only be kept
between the patient and the doctor. Never spread rumors or never speak to it with anyone else .
o Gaining patient confidentiality protects patients and your practitioners. Healthcare workers are required to treat all patient information
as private and confidential.
• Infection Control
o Standard precautions must be taken with every patient to prevent the spread of infection
o This includes wearing personal protective equipment (PPE) when drawing blood or handling specimens and using proper hand
hygiene procedures. It is very important to never take granted of personal protective equipment because it protects you and your
patient and your colleagues.
• Personal Protective Equipment (PPE)
o Laboratory coats or gown, and gloves are required for phlebotomy procedures and during specimen handling.
o New gloves must be worn for each patient. Masks or respirators may be required when drawing blood from patients with certain
transmissible diseases.
o Proper hand hygiene is the most important means of preventing the spread of infection. Hands must be decontaminated frequently,
including before and after specimen processing, before and after glove removal, as gloves can contain defects.
o CDC guidelines allows the use of alcohol-based antiseptic hand cleaners instead of hand washing if hands are not visibly soiled. To
achieve antisepsis, cover all hand surface with ample cleaner and let it evaporate. Do not apply alcohol then dry it with tissue, because
that will not allow the alcohol to exert its activity towards the bacteria in the hands. If no hand washing facilities is available, clean
visibly soiled hands with detergent wipes followed by alcohol-based cleaners.
• Isolation procedures separates certain patients from others and limit their contact with hospital personnel and visitors.
o A description of required precautions is normally posted on the patient’s door and must be followed by all who enter the room without
exemptions.

The Vascular System

• Arteries
o Have thick walls to withstand the pressure of ventricular contraction, which creates a pulse that can be felt, distinguishing them from
the veins.
▪ When collecting a blood specimen from a patient, never look for the pulse because looking for a pulse would mean
aiming for an artery, and medical technologists are not allowed to perform arterial puncture.
o When arterial blood is collected by syringe, the pressure normally causes blood to “pump” or pulse into the syringe
under its own power. Normally, systemic arterial blood is bright red because it is oxygen-rich.
• Veins
o Have thinner walls than the same-size arteries because blood in them is under less pressure.
o Normal systemic venous blood is dark bluish red because it is oxygen-poor.
o Consequently, veins tend to collapse more than artery
• Capillaries
o Are only one-cell thick to allow the exchange of gases and other substances between the tissues and the blood.
o The capillary bed in the skin can easily be punctured with a lancet to provide blood specimens for testing.
• Phlebotomy Vascular Anatomy
o For patient extraction, the needle is positioned to the median antecubital fossa and the median
antecubital fossa has three different types of common veins.
o Median Cubital Vein (most preferred)
▪ Located near the center of the antecubital fossa
▪ Preferred vein for venipuncture because of the following reasons:
❖ Typically large and is closer to the surface
 ❖ It is most stationary, making it the easiest and least painful to puncture and the least likely to bruise.
o Cephalic Vein
▪ Located in the lateral aspect of the antecubital fossa
▪ Second-choice vein, although often harder to palpate or feel than the median cubital vein, it is fairly well-anchored and often the
only vein that can be felt in obese patients (vein of choice for obese patients)
o Basilic Vein
▪ Located on the medial side of the antecubital fossa
▪ Last choice of vein for venipuncture, although normally large and easy to feel, it is not well-anchored and rolls easily.
• Source and Composition of Blood Specimens
Arterial Blood Venous Blood Capillary Blood
Normally uniform throughout the body Is affected by metabolic activity of the Contains arterial and venous blood plus
tissue it drains and varies by collection site tissue fluid
It is primarily reserved for blood gas Differs most from arterial blood in its Capillary glucose is normally higher;
evaluation lower oxygen content calcium, potassium, and total protein
are normally lower
Needs special training (Respiratory Impaired blood flow that can affect other Squeezing the site can falsely elevate
therapists and physicians) analytes potassium levels, however
o Blood specimens can be obtained from arteries and veins and by puncturing the capillary bed in the skin. Composition varies by the
source in the following manner.
o When collecting specimens from capillary blood, the first drop of blood from the puncture have to be wiped off because it is
contaminated with tissue fluid.
o Registered respiratory therapists and physicians are allowed to perform arterial blood collection.
o Most common analyte in impaired blood flow affection is potassium. Prolonged tourniquet application or excessive fist clenching of
the patient could result to potassium alteration levels.
• Types of Blood Specimens
o Regardless of the source, blood is approximately 55% fluid and 45% blood cells. Tasks can be performed on either serum or plasma
derived from the fluid portion or on whole blood.
o Serum
▪ Is normally a clear, pale yellow fluid (non-fasting serum can be cloudy due to lipids) separated from clotted blood by
centrifugation.
▪ Many chemistry tests are performed on serum samples.
o Plasma
▪ Is normally clear to slightly hazy, pale yellow fluid that separates from the cells when blood in an anticoagulant tube is
centrifuged. It contains fibrinogen (while serum does not because it was used in clot formation).
▪ STAT and other tests requiring a fast turnaround time (TAT) are often collected in tubes containing heparin anticoagulant
(green-topped tube) because they can be centrifuged immediately to obtain plasma
o Whole Blood
▪ Contains both cells and plasma, like blood in the body
▪ Is used for most hematology tests and many point-of-care tests (POCTs), especially in acute care and STAT situations
▪ As with plasma, it must be collected in an anticoagulant tube to keep it from clotting

Venipuncture Equipment
• ETS is the preferred method because blood is collected directly from the vein into a tube, minimizing the risk of specimen contamination
and exposure to the blood.
• A needle and syringe/hypodermic needle are sometimes used on small, fragile, or damaged veins and
• A butterfly/winged infusion set can be used with the ETS or a syringe and is often used to draw blood from infants and children, from
hand veins, and in other difficult-draw situations.
• Tourniquet is applied to the patient’s arm during the acne puncture
o Tourniquets should be fastened tight enough to restrict blood flow/venous flow but not arterial flow. This distends the veins making
them larger and easier to find, and stretches the walls so that they are thinner and easier to pierce.
o As phlebotomists, a tourniquet must not be left on longer than 1 minute because the specimen quality will be affected.
• Needles are sterile, disposable and the size by length and gauge varies
o Gauge is a number that relates to needle diameter or bore. Gauge and bore are inversely related (i.e., the larger the gauge,
the smaller is the bore).
o Venipuncture needles include gauges 21 to 23, with a 21 gauge considered standard for routine venipuncture.
• Evacuated Tube System
o An ETS has three basic components—a multi-sample needle, a tube holder, and various types of evacuated tubes.
o Multi-sample needle- is specific for evacuated tube system. This allows collection of multiple tubes during
venipuncture.
▪ The multi-sample needle has two sharp edges- one is used to pierce the vein and the other end or the shorter
end is covered with a rubber sleeve which is used to puncture the rubber stopper of the evacuated tubes
▪ The multi-sample needle is threaded because it is used to screw the needle into a tube holder
▪ The purpose of the rubber sleeve in the shorter needle is used to prevent blood leakage when the tube is removed.
 o Tube Holder- is a plastic cylinder with a small opening for a needle at one end and a larger opening for tubes at the other end
▪ The tube end has flanges and they aid the medical technologist to place and remove tubes
▪ Holders are available with or without safety features and a holder without a safety device must be used with a needle
that has one.
o Evacuated tubes have a pre-measured vacuum that automatically draws the volume of blood indicated on the label
▪ A tube that has lost all or part of its vacuum will fail to fill with blood or will fill incompletely.
▪ When using evacuated tubes, it is very important to always check the expiration date.
▪ Tube stoppers are color-coded to identify a type of additive, absence of additive, or special tube property.
o Common Stopper Colors, Additives, and Departments Involved
STOPPER COLOR ADDITIVE DEPARTMENT(S)
Light blue (most common) Sodium citrate Coagulation
Red (glass) None Chemistry, blood bank, serology/immunology
Red (plastic) Clot activator Chemistry
Red/light gray (plastic) Nonadditive N/A (Discard tube only)
Red/black (tiger), Gold, Red/gold Clot activator and gel separator Chemistry
Green/gray, Light green Lithium heparin and gel separator Chemistry
Green Lithium heparin/Sodium heparin Chemistry
Lavender EDTA Hematology
Pink Blood bank
Gray Sodium fluoride and potassium oxalate/ Chemistry
Sodium fluoride and EDTA/Sodium fluoride
Orange, Gray/yellow Thrombin Chemistry
Royal blue None (red label)/EDTA (lavender label)/ Chemistry
Sodium heparin (green label)
Tan (glass tube) Sodium heparin Chemistry
Tan (plastic) EDTA
Yellow Sodium polyanethol sulfonate (SPS) Microbiology
Yellow Acid citrate dextrose (ACD) Blood bank/Immunohematology
• Syringe System
o The needle used for syringe system is the hypodermic needle.
o Although the preferred venipuncture method is the evacuated tube system, a syringe system which includes
the use of plastic syringe, a needle, and a transfer device is often used in certain situations.
o Syringe needles are available in a wide range of gauges and lengths for many different uses.
▪ Those appropriate for venipuncture are 21 to 23-gauge and hypodermic needle from 1 to 1 2/3 inches in length.
▪ Hypodermic needles must have a resheathing feature/safety feature so that they can be safely removed from
the syringe and a transfer device attached.
o Syringes are available in various sizes selected according to the size and condition of the vein and the amount
of blood needed.
o They have a barrel with graduated markings in either milliliters (mL) or cubic centimeters (cc) and a plunger
that fits snugly to it.
• Winged Infusion Set/Butterfly System
o This is used for pediatric patients.
o Butterfly/winged infusion set is a short needle with plastic part resembling the butterfly wings, and the length
of the tubing with a Luer fitting for syringe or a Luer adapter for the ETS.
▪ During use, the plastic wings are typically held together with the thumb and index finger, allowing the user to achieve the shallow
needle angle needed to access smaller veins.
▪ Same with the syringe and ETS, they come in various gauges with 23-gauge as the most commonly used for phlebotomy.
❖ Drawback: Smaller needles (e.g. 25-gauge, which has a smaller bore) increase the risk of specimen hemolysis
• Tube Additives
o An additive functions optimally when the tube is filled to its stated volume and gently inverted immediately after collection to mix
the additive with the blood.
o If additive is an anticoagulant→ blood will not clot and the specimen will be whole blood→ can be centrifuged to obtain plasma
o All other additives and additive-free tubes (e.g. glass red-topped tube)→ produces serum specimens
o Specimen quality will be compromised if a tube is partially filled. Shaking or vigorously mixing can hemolyze the blood, making it
unsuitable for testing. Always check the expiration date.
o Anti-glycolytic Agents
▪ Prevent glycolysis, which can decrease glucose concentration by up to 10 mg/dL per hour
▪ It preserves glucose for up to 3 days and inhibits bacterial growth.
▪ The most common antiglycolytic agent, sodium fluoride which is often combined with potassium oxalate.
o Clot Activators
▪ Clot activators in gel separator tubes and plastic red-topped tubes are typically silica.
▪ Are coagulation factors such as thrombin and substances such as glass (silica) particles and inert clays like diatomite (Celite) that
enhance clotting by providing more surface for platelet activation.
 o Thixotropic Gels
▪Are inert substances contained in or near the bottom of certain tubes
▪During centrifugation, the gel lodges between the cells and the fluid, forming a physical barrier that
prevents the cells from metabolizing substances in serum or plasma
• Order of Draw
1. Sterile tube 5. Plasma separator tube (PST)
2. Light blue 6. Green
3. Red 7. Lavender
4. Serum separator tube (SST) 8. Gray
o PST Tubes- contains spray coated lithium heparin and a polymer gel for plasma separations;
▪ Samples processed in these tubes are used for plasma determination in chemistry department
o Order of draw- is a special sequence of tube collection that reduces the risk of specimen contamination.
There are tests that are affected if the order of draw is not followed properly.
o EDTA carry over (lavender-topped tube)- causes more problems than that of any other additive
o Heparin- causes the least interference in testing, because it also occurs in blood naturally

Multiple Venipuncture Attempts

• Phlebotomy can only be attempted twice. If the second attempt is unsuccessful, ask
someone else to take over (etc. senior phlebotomist).
• Unsuccessful venipuncture attempts frustrate both the patient and the phlebotomist.
• If a second person is unsuccessful in two attempts, give the patient a rest and try
later (unless the test is STAT or timed). If a specimen can never be obtained by
both the phlebotomists, notify the nurse or physician accordingly.
• If you are unable to obtain a specimen on the first attempt, try again below the first
site, on the other arm, or on a hand or wrist vein.

Pediatric Venipuncture
• Pediatric venipuncture poses a challenge even for the most professional phlebotomist
• Requires the expertise and skill of an experienced phlebotomist.
• If a child is under age 2, venipuncture should be limited to superficial veins of the antecubital fossa and forearm. They should never
be deep and should never be hard to find veins.
• An infant or young child has a small blood volume and every effort must be made to collect the minimum amount of blood required
for testing. Manufacturers develop microcontainers, which are specifically designed for micro-collection to collect the minimum
amount of blood required for testing.
• Interacting with a Child
o Approach the child slowly and determine his or her degree of anxiety or fear before handling equipment or touching arms to look for
a vein.
o Explain procedure to the child (and also to the parents) in terms the child can understand and answer questions honestly.
o Calm a crying child quickly, however, because crying can erroneously alter blood composition.
▪ Never tell a child that it will hurt and that it will not hurt. Just tell to the patient that it will be over quickly. Never be afraid of
sticking the needle to the pediatric patient.
• Immobilizing a Child
o Immobilization of pediatric patients is critical to successful venipuncture and helps ensure their safety.
o An infant can be wrapped in a blanket. A toddler can be restrained while sitting on a parent’s lap

Geriatric Venipuncture
• The veins of geriatric patients are very fragile and the patient is also irritable. Physical effects of aging can represent challenges to a
phlebotomist’s interpersonal skills and technical expertise.
• Changes During Geriatric Condition:
o Alzheimer’s disease
o Arthritis- make sure to carefully position the patient; arthritis can lead to a difficulty in getting in and out of blood drawing chairs
o Coagulation problem- increases the risk of prolonged bleeding and hematoma formation
▪ Always tell the patient to apply pressure until bleeding stops in the venipuncture side
o Dim vision (cataracts)- there may be a need to guide the elderly patient to the drawing chair/station
o Hearing loss- patient will have difficulty in answering questions, extra time to answer questions may be needed
o Less elastic skin and veins- skin and veins are a less elastic in geriatric patients so there is increased risk of injury
▪ Veins for these patients are usually narrowed, more fragile, and apt to collapse, therefore as a skill, tout the skin or anchor the vein
securely but gently
o Slower nerve conduction- affects the learning and reaction time and diminishes pain perception
o Parkinson’s disease- a communication ineffectiveness; when dealing with geriatric patients, you have to be very compassionate,
patient, and you have to reassure the patient that what you’re doing is for the benefit of his or her good health
Pre-analytical Considerations
• In addition to technical skills necessary to collect a blood specimen, anyone who collects blood specimen must be able to recognize
problem sites, hematoma sites, edematous area, mastectomy patients, identify what site is preferred, and what site is to be avoided, as
well as know the procedural error risk associated, and handle patient complications during blood collection.
• Problem Sites
o Burns, Scars, and Tattoos
▪ Never collect from these sites for they are difficult to palpate and draw from, and these areas have impaired circulation that can
affect test results.
▪ Recently burned or tattooed areas are susceptible to infection. Tattoos contain dyes that can interfere in testing. Areas with dye
should be avoided unless no other site is available.
▪ If you are not certain in sticking the patient on his/her non-dominant hand, then proceed to the dominant hand. It is better to perform
venipuncture once, but you are sure of than perform it twice then you are uncertain.
o Damaged veins: Sclerosed (hardened) or Thrombosed (clotted)
▪ Are occluded (obstructed) so they feel hard and cordlike, lacking resiliency. They are difficult to palpate, have impaired blood flow.
▪ True for cancer patients.
o Edema
▪ Makes vein hard to locate and may yield erroneous test results because the swelling alters blood composition
▪ Edema is the swelling caused by the abnormal accumulation of fluid in tissue
o Hematoma
▪ Never draw blood through a hematoma site because it is painful and leads to inaccurate test result.
▪ Hematoma is a swelling or mass of blood that escapes the vein during venipuncture.
▪ If no other side is suitable, draw the specimen distal to the hematoma so that free-flowing blood is collected
o Mastectomy (removal of breast)
▪ Lymph node removal, typically part of the procedure, can cause lymphostasis (stoppage of lymph flow), which makes the arm
susceptible to swelling and infection.
▪ Always make sure to ask the patient if she had mastectomy and which side was the mastectomy performed. As protocol, never ever
perform venipuncture on the mastectomy side because this will increase the risk of the patient’s health to deteriorate due to infection.
• Vascular Access Devices (VAD)
o Only specially trained personnel should draw blood from a VAD, although a phlebotomist may assist by transferring the blood to the
appropriate tubes (true in patients who undergo hemodialysis). Only a trained nurse is allowed to extract blood from these vascular
access devices, so what the phlebotomist do is to wait on the side of the patient while the nurse is collecting blood, and once collection
has been done, transfer that blood to the designated appropriate tubes.
o Arterial line (A line)
▪ Commonly located in the radial artery; used to collect blood gases and other blood specimens
o Arteriovenous (AV) shunt or fistula
▪ Created by a surgical procedure that permanently fuses a vein and artery together to provide access for dialysis (common in
hemodialysis patients)
o Heparin or saline lock:
▪ A catheter connected to a stopcock, or a cap with a diaphragm, through which medication is given or blood drawn.
▪ Using heparin lock, always discard 5-mL of blood before collection
o Intravenous line:
▪ Tubing connected to a catheter inserted in a vein and used to administer fluids.
▪ IV fluid can contaminate the specimen and affect test results.
▪ If the patient has an IV line, it is best preferred to extract the blood in the non-IV site. If it is difficult, ask the nurse if the IV line
can be stopped. If the IV line can be stopped, then stop the IV line for 5-10 minutes and use discard tubes before inserting the
appropriate tubes for the test.
▪ A previous IV access site also should not be used for venipuncture within 24 to 48 hours because this is prone to error.
o Central vascular access device (CVAD) or indwelling line
▪ Line inserted into a main vein or artery that is used primarily to administer fluids and
medications, monitor blood pressure, and draw blood.
▪ PICC line- common in pediatric patients so that to avoid sticking the patient, they collect
through the PICC line, and then transfer that blood to the appropriate evacuated tube.
• Procedural Error Risks
o The following can result from the procedural error and have adverse effects on the patient
o Hematoma
▪ Rapid swelling at or near the venipuncture site due to blood leaking into the tissue (very common)
o Iatrogenic anemia: Anemia as a result of treatment (e.g., frequent blood draws or removing large quantities at a time)
o Inadvertent arterial puncture
▪ Accidentally sticking an artery; often the result of deep or blind probing or attempting to draw from the basilic vein
▪ Never collect from an arterial site. Venipuncture must be discontinued and pressure applied for 5 minutes. Identify specimen as
arterial blood if submitted for testing again.
 o Infection of the site
▪
Infection risk can be minimize by using aseptic technique, including cleaning the site properly and not touching it again before
needle insertion (always apply alcohol prior to venipuncture).
o Nerve Injury
▪ Results from poor site collection, inserting the needle too deeply or quickly, patient movement on needle insertion, excessive needle
redirection or blind probing (patient might feel a burning sensation or an electrical shock).
o Reflux
▪ Backflow of blood from the tube into the patient’s vein that can occur if blood in the tube is in contact with a needle during a blood
draw. To prevent reflux, the patient’s arm must be in downward position.
o Vein damage
▪ Scar build-up that can result from many venipunctures in the same area for an extended period, improper redirection of the needle,
or probing.
• Patient Complications and Conditions
o Allergies to supplement or equipment
▪ Use an alternate antiseptic if required (some patients are allergic to alcohol, so use iodine, and vice versa).
▪ Paper tape placed over folded gauze or self-adhesive bandage material can be used in place of adhesive bandages.
▪ Never use latex items on latex-sensitive patients or even bring them into the room.
o Excessive bleeding
▪ Apply pressure to the site until bleeding stops. If it continues beyond 5 minutes, notify the appropriate personnel.
▪ Some patients tend to bleed for more than 5 minutes because they are in anticoagulant therapy.
o Fainting (Syncope)
▪ Warning signs that a person may faint include perspiration beads on the forehead, hyperventilation, and loss of color
o Nausea or vomiting
▪ Reassure a nauseous patient and provide a container of some sort to hold and as a precaution.
▪ Ask the patient to breathe slowly, and apply a cold compress to his or her forehead.
▪ If the patient vomits, terminate the procedure and notify first aid personnel.
o Obese patients
▪ Focus on the cephalic vein, which is more easily located by rotating the patient’s arm
▪ If there is no easily palpable vein, ask the patient what sites have been successful for past blood draws
o Pain
▪ A slight amount of pain is expected during a routine venipuncture or capillary puncture.
▪ A stinging sensation can be avoided by allowing the alcohol to dry completely after cleaning the site.
▪ If pain persists apply an ice pack and notify appropriate personnel.
o Petechiae
▪ The spots are actually minute amounts of blood that escape from the capillaries and come to the surface of the skin as a result of
platelet abnormalities or a defect in the capillary walls. They do not indicate that the phlebotomist has done anything wrong.
▪ When a tourniquet is applied to certain individuals, tiny red spots (petechiae) appear on the arm below it.
o Seizures/Convulsions
▪ Discontinue blood collection immediately if a patient has a seizure or goes into convulsion.

Capillary Specimen Collection

• Capillary specimen collection (also called dermal or skin puncture) is especially useful in pediatrics where removal of large quantities
of blood can have serious consequences.
• Drops of blood for testing can be obtained by puncturing the capillary bed of the skin with a lancet.
• Collection sites include: fingers (for adults and children over the age of 2) and heels (for infants).
• CLSI recommends using 70% isopropyl alcohol to clean capillary puncture sites. Gauze or gauze-type pads are used to wipe away
the first blood drop to eliminate alcohol residue and excess tissue fluid.
• Lancets are sterile, disposable, sharp instruments used for capillary puncture. Their blades or points must permanently retract to prevent
sharps injuries.
• Microcollection tubes (microtubes) are special small plastic tubes often referred to as “bullets.” Instead of collecting 2 mL of blood
for CBC analysis, with the microcollection tubes, 0.25mL or 0.5mL of blood can be collected.
• Capillary Order of Draw
o Specimens must be collected quickly to minimize the effects of platelet clumping and microclot formation and to ensure that an
adequate amount of specimen is collected before the site stops bleeding.
o The CLSI order of draw:
1. EDTA specimens 3. Serum specimens
2. Other additive specimen
o The order of draw for collecting multiple capillary specimens differs from venipuncture. Hematology specimens are collected first
because they are most affected by clotting. Serum specimens are collected last since they are supposed to clot.
• Indications of Capillary Puncture
o Not appropriate for patients who are dehydrated or have poor circulation to the extremities from other causes such as shock.
o Performed when:
▪ There are no accessible veins
 ▪ Available veins are fragile or must be saved for other procedures such as chemotherapy.
▪ The patient has thrombotic or clot-forming tendencies.
▪ Blood is to be obtained for POCT procedures such as glucose monitoring.
o Capillary puncture is the preferred way to obtain blood from infants and very young children for the following reasons:
▪ Infants have a small blood volume; removing quantities of blood typical of venipuncture or arterial puncture can lead to anemia or
threaten life if over 10% of blood volume is removed.
▪ Infant or child venipuncture is difficult and can damage veins and surrounding tissues.
▪ An infant or child can be injured by the restraining method used during venipuncture.
▪ Capillary blood is the preferred specimen for some tests, such as newborn screening tests.

Specimen Handling and Processing

• Improper handling or processing can negatively affect test results and cause delays in patient care.
• Mixing Tubes
o Additive tubes must be mixed immediately after collection by inverting them from 3-8 times
depending on the additive type
o If tubes are not quickly and thoroughly mixed, microclots can form in anticoagulant tubes, and
clotting may be incomplete in clot activator tubes.
o Gentle inversion is essential, as vigorous mixing can cause hemolysis. Some tests, including
potassium, magnesium, and most enzyme tests, cannot be performed. If a hemolyzed sample
was obtained, the medical technologist in charge in the clinical chemistry department will not
run the sample and instead will ask to recollect the sample again.
• Transporting Specimens
o Blood specimen tubes must be transported to the laboratory in a plastic bag with a biohazard logo, a liquid-tight closure, and a slip
pocket for paperwork.
o Regardless of delivery method, blood specimen tubes must always be transported carefully to prevent breakage and protect specimen
integrity.
o Agitation or rough handling of the specimen can cause hemolysis or lead to platelet activation that can affect coagulation tests.
• Delivery Time Limits
o STAT specimens must be transported, processed, and tested immediately
o Routine blood specimens, however, should ideally be delivered to the laboratory within 45 minutes of collection and centrifuged
within 1 hour of arrival if serum or plasma is needed. Prompt separation is essential to minimize the effects of metabolic processes,
such as glycolysis.
• Special Handling
o Analyte protection:
1. Cooling below body temperature (37°C) can cause precipitation or agglutination in some specimens
❖ These specimens are typically collected in pre-warmed tubes and transported in 37°C heat blocks or wrapped in special warming
material
2. Other specimens require chilling to slow down metabolic processes that can negatively affect and analyze
❖ These specimens must be transported in crushed ice and water slurry.
3. A number of analytes can be broken down by light, resulting in false low values (e.g. Bilirubin)
• Centrifugation
o Specimens for tests that require serum must be completely clotted before centrifugation
o At room temperature, complete clotting normally takes 30-60 minutes. Incomplete clotting results in latent fibrin formation that
can interfere with testing.
o Unless the task is performed on whole blood, a blood specimen must be centrifuged to separate the serum or plasma from the cells.

• Normal serum
• These specimens are unacceptable for clinical chemistry analysis
o Hemolytic serum- presence of hemolysis (red serum part/supernatant)
o Icteric serum- there is bilirubin pigment, so instead of being pale yellow, the serum is in dark yellow
indicating high bilirubin concentration
o Lipemic serum- instead of clear, it is cloudy or turbid in appearance, denoting that the patient has
increased lipids
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MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

ACCURATE RESULTS
POSITIVE True Positive False Positive
Ø The number of patients that Ø The number of patients that
were tested positive and were were tested positive but were
PREDIC actually positive for the actually negative for the
TIVE disease. disease.
RESULTS Ø Diagnosed with particular Ø You do not have the disease.
disease and lab results reflect Negative for a disease but result
that you are indeed actually shows positive results for the
positive. disease.
Ø Diagnosis: Ø Diagnosis:
Ø Laboratory Results: Ø Laboratory Results:
NEGATIVE False Negative True Negative
Ø The number of patients that Ø The number of patients that
were tested negative but were were tested negative and were
actually positive for the actually negative for the
disease. disease.
Ø Doctor diagnosed and you Ø You don’t have the disease, with
really have the disease but laboratory results showing you
laboratory results show you really don’t have the disease.
don’t have the disease. Ø Diagnosis:
Ø Diagnosis: Ø Lab results:
Ø Lab results:

Actual Results – the diagnosis of the patient.

Predicted Results – released in the laboratory.

Examples:
1. Glucose result is above reference range and the patient has DM. = TRUE POSITIVE
2. Glucose results above reference range and patients are diagnosed to have no DM. = FALSE POSITIVE
3. Glucose results are within reference range and patients are diagnosed to have no DM. = TRUE NEGATIVE
4. Glucose results are within reference range and patients are diagnosed to have DM. = FALSE NEGATIVE

ANALYTICAL SENSITIVITY VS. ANALYTICAL SPECIFICITY

Analytical Sensitivity
Ø The ability of an analytical method to measure the smallest concentration of the analyte of interest; the
machine can detect the lowest level of the analyte.
Ø If the sensitivity of the machine depends on the limit it was set to; usually concerning the low limits.
Example: some machines can detect glucose as low as 10 mg/dL or 25 mg/dL.
Ø Analytes are substances that we detect or measure in the laboratory. Examples: glucose, uric acid, protein,
vitamins, hormones, etc.

Analytical Specificity
Ø The ability of an analytical method or machine to measure only the analyte of interest.
Ø The machine will only detect what you want to measure.
Ø If one wants to detect the glucose in the sample of a patient; if the machine is not specific, it will detect
other sugars like fructose, etc.
Ø If a machine has high specificity, it will only measure glucose and nothing else.
QA & QC: PRINCIPLES OF MEASUREMENTS 05
MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

Formula Definition
Sensitivity Ø Percentage of patients with the disease that receive a positive
result.
Ø Percentage chance that the test will correctly identify a person
who actually has the disease.
Ø Patient is truly diagnosed with the disease.
Ø Results of the laboratory showed that the patient is indeed positive.
Ø TRUE POSITIVE RATE
Ø Patient is diagnosed with a particular disease.
Ø The machine or method is sensitive enough to detect the analyte in
the sample and tell that the patient is indeed positive for that
disease.
Ø Ability of the test to correctly identify those with the disease.
Specificity Ø Percentage of patients without the disease that receive a negative
result
Ø Percentage chance that the test will correctly identify a person
who is disease-free.
Ø Ability of the test to identify those without the disease.
Ø TRUE NEGATIVE RATE

ACCURACY AND PRECISION

Accuracy
Ø The degree of agreement between a measured value and a “true/consensus” value.
Ø “ TRUE /CONSENSUS VALUE “ = TARGET VALUE
o Value we want to achieve in a measurement.
Ø MEASURED VALUE = OBTAINED VALUE
o The value we get from a measurement or the value obtained when measuring something.
Ø The closeness or nearnest of the obtained value to the target value.
Ø Pertains to the veracity or truthfulness of the result.

Precision
Ø It refers to the agreement between replicate measurements.
Ø Measuring something; we have obtained values during repeated measurements.
Ø The nearness of the obtained values to each other; regardless how many times we repeat.
Ø Degree of repeatability or reproducibility.

Goal of Quality Control: achieve a high level of both precision and accuracy over an extended period of time = high
reliability. Meaning that all data and results released are reliable and truthful. We are maintaining quality control.

Examples for Accuracy and Precision:

QA & QC: PRINCIPLES OF MEASUREMENTS 05
MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

DESCRIPTIVE STATISTICS: MEASURES OF CENTER, SPREAD AND SHAPE

Measures of Center of a Data Set Examples:

Mean or average Mean = sum of the terms/number of terms

Ø Most commonly used measurement of the center.
Median Find the median:
Ø Middle point of the data; used for skewed data
after the data have been rank ordered; least to 1. 5, 4, 6, 5, 3, 7, 5 = 3, 4, 5, 5, 5, 6, 7 = 5
greatest. 2. 5, 4, 6, 8, 9, 7 = 4, 5, 6, 7, 8, 9 = 6 +7 = 13/2 = 6.5
Ø It divides the data in half.
Mode Find the mode.
Ø Measure of the data center but most often used
to describe data with 2 centers. 1. 3, 4, 5, 5, 5, 6, 7 = 5
Ø Bimodal 2. 3, 4, 5, 5, 5, 6, 7, 8, 9, 9, 9 = 5 and 9
Ø Frequently occuring value in a data set.

MEASURES OF SHAPE AND SPREAD: APPLICATION OF REFERENCE

RANGE
Ø How data are distributed.
Ø Spread refers to the relationship of all the data points to the
mean.
Ø 3 most commonly used measure of spread: range, standard
deviation and coefficient of variation.

GAUSSIAN DISTRIBUTION CURVE

Ø Johann Karl F. Gauss
Ø “ bell shaped curve “
Ø Normal distribution curve
Ø Help us determine the reference values of the data we are measuring.
Ø Normal value or reference range
o Range of values one would expect in a apparent population with no clinical problems.
Ø Distribution of data:
o Total area under the curve: 1 or 100%
o Much of the area under 68.3 % under the normal curve is between the + 1 SD and – 1 SD.
o Most of the area under 95.4 % under the normal curve is between +/- 2 SD.
o Almost all of the area under 99.7 % under the normal curve is between +/- 3 SD.
QA & QC: PRINCIPLES OF MEASUREMENTS 05
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Formula
Mean

Standard Deviation
Ø Reflects precision since it
can provide the lab an
estimate of test consistency
at specific concentration.
Ø We will know a test
repeatability is consistent
because we will get a low
standard deviation which
could mean low imprecision.
Ø Repeatability of a test is
inconsistent because we will
get a high SD, high
imprecision.

Coefficient of Variation
Ø Monitored in order to assess
the consistency of the
precision of an assay.
Ø The lower the CV, the better
precision of assay, lower
variability of the values.
QA & QC: PRINCIPLES OF MEASUREMENTS 05
MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

REFERENCE RANGES AND SD VALUES

Under the Gaussian Curve, much of the area under 68.3% is 17.9-18.1
95.4% = 17.7 – 18.3
99.7% = 17.6 – 18.4

After determining different ranges and SD value, we plot it in a Gaussian curve.

To visualize how values are spread over the curve.
QA & QC: PRINCIPLES OF MEASUREMENTS 05
MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

SKEWED DATA
Ø Data that is having a long tail on one side or another.

1. Negative skew
o The long tail is on the negative side of the peak.
o The mean is on the negative side or left hand side of the peak.

2. Positive skew
o The long tail is on the positive side of the peak.
o The mean is on the positive side or right hand side of the peak.
o Referred to as skewed to the right.

3. No skew
o The center is the normal distribution curve.
o Not skewed and perfectly symmetrical and mean is located
at the peak.

DESCRIPTIVE STATISTICS OF GROUPS OF PAIRED OBSERVATIONS

Ø To examine a certain method in a laboratory.
Ø We compare two methods frequently, encountered in COM experiment.

COM experiment
Ø involves measuring patient specimens by using two methods, both an existing (reference) method and a new
(test) method.
Ø The difference is obtained and is what we call, the error.

Error
Ø the difference between test and reference method.
Ø Visualized using linear regression equation.

Ø Y axis = test method

Ø X axis = reference method
Ø Correlation coefficient = R value, measure of strength of the relationship between the two methods; can
have values ranging from -1 to +1.
Ø The signs only indicate the direction of the relationship between the two variables or methods.
o A positive r value indicates that both method increase and decrease together.
o A negative r value indicates that one variable increases, the other decreases.
o An r value of 0, indicates no relationship.
o An r value of 1 is indicated as a perfect relationship. A perfect agreement between test and
comaparative method.
§ 0.75-1.0 = strong association
§ 0.5-0.75 = moderate
§ 0.25-0.5 – weak
§ O.95 = excellent
§ Most clinical chemistry labs require a 0.98 to say that the reference method is comparable to
the test method.
o Positive and negative signs only indicate the direction of the relationship.
QA & QC: PRINCIPLES OF MEASUREMENTS 05
MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

Two types:
Ø Random error
o Calculated as the standard deviation of the points about the regression line.
o If the points are perfectly aligned with the regression line, we get an sy/x = 0, meaning there is no
random error.
o A sy/x is high, we have a scatter of points from the regression line and also there is a high amount of
random error.
Ø Systematic error
o Constant error – exists when there is a continual difference between the test and reference method
values regardless of the concentration.
o Proportional error – exists when the differences of the test and comparative method are
proportional to the analyte concentration.
o Clue if it exists because slope is equal to 1.0.

METHOD EVALUATION
Ø is used to verify the acceptability of new methods prior to reporting patient results.
Ø To maximize the usefulness of the test; a method is also selected and evaluated to its usefulness.
Ø To produce results within medically accepted errors to help doctors help their patients.

Regulatory Aspects of Method Evaluation

Ø CMS (Centers for Medicare and Medicaid Services) – regulates the CLIA (Clinical Laboratory Improvement
Amendments)
Ø FDA (Food and Drug Administration) – regulates laboratory instruments and reagents
o “Office of In Vitro Diagnostic Device Evaluation and Safety” (OIVD) regulates diagnostic tests.
§ Waived – cleared by the FDA to be simple, they are most accurate and post risk to patient if
not performed correctly. Examples: dipstick test, glucose monitoring.
§ Moderate complexity – includes automated methods.
§ High complexity – manual and methods that require more monitoring.
Ø Final rule of CLIA:
o Waived test should follow manufacturer’s instruction. Perform accurately.
o Moderate – validated whether FDA approve or not.
QA & QC: PRINCIPLES OF MEASUREMENTS 05
MT 116: CLINICAL CHEMISTRY 1 LECTURE / CUISON

Flow of Procedures for method validation:

1. Select testing methods and compare new method and reference method.
a. If new method is at par with than reference, validate it but process is costly, labor intensive. So why
select a new method? Several reasons:
i. Reduce the cost of reagents and materials or test itself.
ii. Increase client satisfaction.
iii. Increase efficiency as medtechs.
iv. Improve quality
2. Validate method.
a. Should be precise, accurate and reportable range.
b. Important we get data from colleagues, scientific presentations, researches, etc.
c. Consider the volume of specimen, disposal needs, PPE, safety considerations, personnel
requirements.
d. Should meet criteria for quality.
3. Implement method.
4. Perform routine testing.
5. Monitor performance daily with quality control prior.
a. If results are not in quality, go back to adjusting and maintaining procedure.
 MT 116 LECTURE 6: QA AND QC: INTRO AND CALCULATIONS
Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Differentiate sensitivity and specificity  Differentiate accuracy and precision
 Determine the reference ranges of samples based on the given data
 Familiarize the process of method selection, evaluation and monitoring.

 This is particularly important because it will help ensure that the generated laboratory data are
accurate and conform to the quality standards
 True positive- the number of patients that were tested positive and were actually positive for the
disease (you are diagnosed with a particular disease and the laboratory results also suggest that you have the disease).
 True negative- the number of patients that were tested negative and were actually negative for the disease (you do not have the
disease and your laboratory result showed that you are negative for that disease).
 False positive- the number of patients that were tested positive but were actually negative for the disease (you do not have the
disease or your diagnosis is you are negative for a particular disease but the result shows that you are positive for the disease)
 False negative- the number of patients that were tested negative but were actually positive for the disease (you have the disease,
your doctor diagnosed you with that disease but your lab results showed that you do not have a disease)
 Examples:
1. Glucose result is above the reference range and the patient has DM (Diabetes Mellitus)- True positive
2. Glucose results above reference range and patients are diagnosed to have no DM- False positive
3. Glucose results are within reference range and the patients are diagnosed to have no DM- True negative
4. Glucose results are within reference range and the patients are diagnosed to have DM- False negative

Analytical Sensitivity vs Analytical Specificity

 Analytical Sensitivity- the ability of an analytical method to measure the smallest concentration of the analyte of interest
o Analytes- substances detected or measured in the laboratory (e.g. glucose, proteins, uric acid, creatinine, vitamins/drugs)
o It means the machine could measure even the smallest concentration of the substances in the patient’s sample. No matter
how little or how low the concentration of the analyte in the patient’s sample, the machine can still detect it.
o Example: Blood glucose level of 18 mg/dL; this blood glucose level is very low that sometimes the machine cannot measure
or detect it, that is why flagging is seen in the machine
 If the sensitivity of the machine can only measure up to 25 mg/dL, the machine will not detect the blood glucose level of 18
mg/dL because it is not that sensitive enough.
 But if the sensitivity of the machine can detect up to 10 mg/dL glucose, then the 18 mg/dL of glucose can be measured
because the machine is sensitive enough that it could even measure as little as 10 mg/dL of glucose.
 Analytical Specificity- the ability of an analytical method to measure only the analyte of interest
o The machine will only detect what you want to measure or it is the ability of the machine or the method to detect only the
substance or the analyte that you want to measure.
o Example: You only want to measure the glucose level of the patient
 If the machine is not specific, it could measure other sugars such as fructose, sucrose, or maltose for example
 If the machine is having a high specificity for glucose, then it will only measure glucose and nothing else
 Sensitivity
o Percentage of patients with the disease that receive a positive result (true positive rate)
o Percentage chance that the test will correctly identify a person who actually has the disease (ability of a test to
correctly identify those with the disease)
 Specificity
o Percentage of patients without the disease that receive a negative result (true negative rate)
o Percentage chance that the test will correctly identify a person who is disease-free (ability of the test to correctly
identify those without the disease)

Accuracy and Precision

 Accuracy- the degree of agreement between a measured value and a “true/consensus” value (target value)
o Target value- is the value that we want to achieve in a measurement
o Measured value- is the value that we get out of a measurement (value that we obtain when we are measuring something)
o Closeness or the nearness of the obtained value to the target value. It also refers to the veracity/truthfulness of the result.
 Precision- refers to the agreement between replicate measurements
o Nearness of the obtained values to each other, regardless how many times the measurement is repeated using the same sample.
o Degree of repeatability or degree of reproducibility
 Goal of QC: Achieve a high level of both precision and accuracy (Reliability)
o Reliability- maintain accuracy and procession in the laboratory over extended period of time
 Example (Accuracy): A 20 item quiz and the passing is 18; target value= 20; a perfect/passing score is needed to
say that there is accuracy in the answers
o A score of 18 means that there is accuracy in the answers because 18 is near to the target value which is 20. A
score of 19 is closer to 20 (the target value), then the answers are more accurate.
o A score of 15 or 12 or 16 are not near to the target value, so the answers in the quiz are not that accurate.
 Example (Precision): A 20 item quiz and still the passing is 18, and the teacher said she will give 2 chances for
those who did not pass the quiz; your scores=15, 14, and 16 (did not pass)
o Your scores are near to each other, that means the obtained values are near to each other so there is precision
but these are not accurate since 15, 14, and 16 are not close to the target value.
 In the clinical chemistry laboratory, we need to have both accuracy and precession that is to ensure that the data
that we are releasing are reliable and that we are maintaining quality in our laboratory.

Descriptive Statistics
 Measures of Center
o Mean- average, most commonly used
o Median- middle point after the data have been rank ordered (the value that divides the data in half),
used for skewed data
o Mode- most frequently occurring value in a data set; rarely used as a measure of the data center but
is more often used to describe data that seem to have two centers (bimodal)
o Examples:
 Find the Median:
1. 5, 4, 6, 5, 3, 7, 5→ 3, 4, 5, 5, 5, 6, 7→ 5 2. 5, 4, 6, 8, 9, 7→ 4, 5, 6, 7, 8, 9→ 6+7 = 13/5= 6.5
 Find the Mode
1. 3, 4, 5, 5, 5, 6, 7→ 5 2. 3, 4, 5, 5, 5, 6, 7, 8, 9, 9, 9→ 5 and 9
 Measures of Shape and Spread
o After describing the center of the data set, it is very useful to indicate how the data are
distributed.
o Spread represents the relationship of all the data points to the mean
o Three most commonly used measure of spread: Range, Standard Deviation (SD), and
Coefficient of Variation (CV)
o Gaussian Distribution Curve (Normal distribution curve)
 Introduced by Johann Karl F. Gauss  Bell-shaped curve
 Could help determine or know the reference values of the data that are measured
 Normal value or Reference range- refers to the values or range of values one would expect in
a defined population with no apparent clinical problems
 Distribution of data in a normal distribution curve
 For a normal distribution curve, the total area under this curve is 1 or 100%
 Much of the area (around 68.3%) is under the normal curve between the ± 1SD
 Most of the area (around 95.4%) under the normal curve is between the ± 2SD
 Almost all of the area (around 99.7%) under the normal curve is between the ± 3SD
o Skewed Data
 Refers to data that are having a long tail on one side or another
 Negative skew- the long tail is on the negative side of the peak, and also the mean is
on the negative side (left hand side) of the peak (skewed to the left)
 Positive skew- the long tail is on the positive side of the peak, and also the mean is on
the positive side (right hand side) of the peak (skewed to the right)
 Not skewed- it is perfectly symmetrical and the mean is located exactly at the peak
 Example: Income Distribution
 Positively skewed data because the long tail is located at the positive side (right side) of the
peak and also the mean is on the right side of the peak
o Formulas used in order to determine the reference ranges of the values
Mean (𝑿 ̅ ) ∑= sum Standard Deviation (S) S= standard deviation
X= each value in the 𝑋̅ = mean (average) of the QC values
∑𝑥 data set ∑(𝑋 − 𝑋)̅ 2 ∑ (𝑋 − 𝑋̅)2 = the sum of the squares of differences
n= number of values in 𝑆= √ between individual QC values and the mean
𝑛 𝑛−1
each data set n= number of values in the data set
 Standard deviation also reflects precession because it could provide the laboratory an estimate of tests consistency at
specific concentration (Consistent: ↓SD, ↓Imprecision; Inconsistent: ↑SD, ↑Imprecision)
 Coefficient of Variance (CV)
𝑆𝐷 𝑥 100 CV is monitored in order to assess also the consistency of the precision of an assay.
𝑋̅ The lower the CV, the better the precision of an assay
 Example: CV of Test A= 17.03; Test B= 28.35→ Test A has better precision, has better variability of values than Test B
 Example: This table shows the different control values that were obtained for 10 consecutive days and to get the reference
ranges of these control values, the mean and the SD has to be computed first and determine the precision of the assay by
getting the CV value.
Day X (mg/dL) |𝑿 − 𝑿̅ | (𝑿 − 𝑿 ̅ )𝟐
1 18.0 0 0
2 18.1 0.1 0.01
3 18.2 0.2 0.04
4 17.9 0.1 0.01
5 18.0 0 0
6 17.8 0.2 0.04
7 18.1 0.1 0.01
8 18.0 0 0
9 18.2 0.2 0.04
10 17.9 0.1 0.01
Total: ∑= 180.2 ∑(𝑿 − 𝑿 ̅ )𝟐 = 0.16
 Descriptive Statistics of Groups of Paired Observations
o Even though in the laboratory we could use the basic descriptive statistics for examining a single
method in the laboratory, we frequently need to compare two different methods.
o Comparison of Methods (COM) experiment- involves measuring patient specimens by both
an existing (reference) method and a new (test) method.
o Data obtained from the COM are visualized graphically
 By convention: values obtained by the reference method are plotted on the x-axis and the values obtained by the test method
are plotted on the y-axis
 Correlation coefficient (r)
 A measure of strength of the relationship between the two methods (reference and test methods)
 Can have values from -1 to +1 (signs only indicates the direction of the relationship between the 2 variables/methods)
 Positive r value- both methods increase and decrease together (directly proportional)
 Negative r value- as one variable/method increases, the other decreases (inversely proportional)
 r value of 0- no relationship
 r value of 1- perfect relationship
 Dispersion of values in a linear regression and could also indicate the association/strength of relationship between the two
variables (reference and test methods are used)
 r= 1- perfect association (tperfect agreement between the test and the comparative method)
 r= 0.75 to 0.1- strong association  r= 0.25 to 0.5- weak association
 r= 0.5 to 0.75- moderate association  r= 0.95- considered excellent (some laboratories)
 r= >0.98- required in most clinical chemistry comparisons to say that the reference method is comparable to the test method
o Error- the difference between test and reference method
 Random Error- calculated as the standard deviation of the points about the regression line
 If the points were perfectly in line with the regression line; Standard Error of the Estimate Sy/x= 0 (no random error)
 ↑Sy/x= wider scatter of points from the regression line and also a high amount of random error
 Systematic Error
 Constant Systematic Error- exists when there is a continual difference between the test and the reference method values
regardless of the concentration
 Proportional Systematic Error- exists when the differences between the test method and comparative method are
proportional to the analyte concentration; Proportional error is present when the slope is 1.

Method Evaluation
 After determining the differences and agreement between the test and reference methods, we can now check if the new method
can be used to generate reliable laboratory results.
 Is used to verify the acceptability of new methods prior to reporting patient results.
o Before using the new method tested, it needs to be verified first if it can really provide reliable and accurate laboratory data.
o To maximize the usefulness of a test, this time this new method is selected and it will be evaluated for its usefulness to those
who will be using the test, especially medical technologists.
o This process is carefully undertaken to produce results within medically acceptable error to help the doctors maximally benefit
their patients
 Regulatory Aspects of Method Evaluation
o Centers for Medicare and Medicaid Services (CMS)- regulates the Clinical Laboratory Improvement Amendments (CLIA)
o U.S. Food and Drug Administration (FDA)- regulates laboratory instruments and reagents
 Office of In Vitro Diagnostic Device Evaluation and Safety (OVID)- regulates diagnostic tests
 Three types of diagnostic tests:
 Waived Test- these are cleared by the FDA to be simple (most likely accurate and would pose negligible risk or harm to
the patient if not performed correctly) e.g.: dipstick test, glucose monitoring
 Moderate Complexity- automated methods
 High Complexity- manual methods and the methods which require more interpretation
 The final rule of the CLIA requires that:
 Waived tests should just simply follow the manufacturer’s instructions (as long as you know how to perform it and
performed accurately, and following manufacturer’s instruction)
 For moderate and high complexity tests, they should be validated whether they are FDA approved or not.
 For the FDA-approved non-waived tests, they must undergo a shorter validation process. A more extensive process is
required for tests developed by laboratories.
General CLIA Regulations of Method Validation
Nonwaived FDA-Approved Tests 1. Demonstrate test performance comparable to that established by the manufacturer
a. Accuracy
b. Precision
c. Reportable range
2. Verify reference (normal) values appropriate for patient population
Nonwaived FDA-Approved Tests 1. Determine
Modified or Developed by a. Accuracy
Laboratory b. Precision
c. Analytic sensitivity
d. Analytic specificity (including interfering substances)
e. Reportable range of test results
f. Reference/normal ranges
g. Other performance characteristics
h. Calibration and control procedures
o Flow of Procedures for Method Validation
1. Compare the new method and the reference method
 If found out that the new method is at par or better than the reference method,
the new method can be validated, but this process is labor intensive and costly.
 Why select a new method?
 To reduce the cost of the reagents, materials, equipment or even the cost of
the test itself
 To increase the satisfaction of our clients/patients
 To increase efficiency as medical technologists working in the clinical
laboratory
 To improve the quality
2. Validate Method
 It is not only enough to get the precession, accuracy, or the range of values. It is also important that we get data from our
colleagues, from scientific presentations, researchers, or scientific literature.
 Consider the volume of the specimen, disposal needs, PPE requirements, safety considerations, personnel
requirements.
 The test performed in a new method should meet the criteria for quality.
3. If everything have already been tested, that is the time the method can be implemented and perform the routine clinical
testing
 Performance needs to be monitored daily (need to perform quality control before releasing results
 If results are not in quality, go back to adjusting and maintaining the procedure to make sure that everything is accurate
and reliable.
 MT 116 LECTURE 7: QA AND QC: LEVY-JENNINGS/CONTROL CHARTS
Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Identify the different variables involved in the total testing process  Differentiate QA and QC
 Determine how to use the Westgard rules in identifying errors in quality control run
 Familiarize the steps in proficiency testing and determine its importance in maintaining quality in the laboratory

The Total Testing Process

 A simplified, cyclic framework and has three phases
 Pre-analytical phase
o Includes all the processes prior to the actual testing of a specimen
o Things done in the laboratory before testing the specimen include:
 Sample Transport  Personnel Competency Test Evaluations
 Sample Receipt and Accessioning  Patient-Client Preparation and Sample Collection
 Analytical phase- Consists of all the processes which are involved in the testing of the specimen
 Post-analytical phase- includes all the processes involved after the analytic phase or after the test analysis
 Monitoring the processes in all these three phases of testing is the key to ensuring patient safety and reducing the possibility of medical
errors related to laboratory testing.
o These processes must be monitored from the time you receive the request from the doctor or from the patient up to the time you release
the result, because we need to maintain quality in the laboratory.
 Pre-analytical Variables
o Variables associated with entire steps, procedures and considerations in handling a test request/laboratory requisition form.
o The test request comes from the doctor and includes demographic data of the patient and also the test being requested for by the doctor.
Once the patient goes to the laboratory, the patient will give this request to the med tech and the med tech will inform the patient on
the necessary things that he/she must do before the conduct of the tests.
o Test Ordering, Patient Information
 Make sure that all the data seen in the test requisition are correct and if ambiguous information are noticed, ask the patient to write
those information in order to correct the information present in the requisition.
 When you want to know the name of the patient, do not ask the patient questions answerable by yes or no. Always let the patient state
his/her name, just to make sure that you are asking the correct questions from your patients.
o Patient preparation (very critical)
 Examples: abstinence or fasting when the patient is tested for FBS or lipid profile
 Explain to the patient the hours of fasting, the preparation, and the food intake of the patient
o Sample collection, handling, transport, and storage
 Includes proper techniques in phlebotomy, the tubes used for the test, proper mixing of the tube containing the specimen, the correct
anticoagulant used, and the temperature of handling and when or where to store the specimen.
o Processing of specimen, centrifuging, and separation of specimen
 Examples: when separating cells from the plasma or serum, make sure to avoid hemolysis, pipetting techniques
 These variables are difficult to monitor and control because most occur outside of the laboratory
 Analytical Variables
o Concerns the laboratory personnel, equipment, reagents, quality controls, verification and sometimes interpretation and diagnosis
(specimen processing and running quality control materials)
 Interpretation and diagnosis are sometimes included in the analytical variables because sometimes by just looking at the specimen
during analysis, we already have an idea on the possible result of the test of the patient
 Chylous/milky specimen would tell that the specimen of the patient contains a high cholesterol or triglyceride level
 Ecteric specimen indicates a high bilirubin concentration
o Primarily depend on instrumentation and reagents
o The way daily and monthly preventive maintenance are conducted (includes Specimen analysis, Commercial Controls)
 Post Analytical Variables
o The final phase of the total testing process and involves evaluation of lab test results.
o Reporting out specimen results
 When a med tech signed the result, counter signed by the pathologist, and give it to the patient to the nurse/attending physician
o Physician contact- when relaying test results to the doctors or even to the nurse in charge
o Reference ranges- when the doctor interprets the result of the patient
o This is very important because releasing of test results in a timely manner to appropriate individuals, especially if it is a critical result,
then that is very necessary to support clinical decision making because when a report is lost or delayed in reporting of the result, then
that could jeopardize the patient condition or the diagnosis of the patient.
o Main advantage of technology: Errors in the post-analytical phase have greatly decreased because of automation and computer-
generated patient reports.

Quality Control
 A statistical process that is used to monitor and evaluate the analytical process that produces patients result.
 Focuses mainly on the analytical phase (systematic monitoring of the analytical process to detect analytical errors that occur).
 Main purpose: Prevent the reporting of inaccurate or incorrect patient test results
 Requirements for the Statistical Process
o Regular testing of quality control products along with patient sample
o Comparison of quality control results to specific statistical limits (ranges).
 QC results are used to validate whether the instrument is operating within predefined specifications, so that could infer that the patient
test results are reliable. Once the test system is validated, the patient’s results can then be used for diagnosis, prognosis and planning for
the treatment of the patient.

QC vs QA
 Goal of QC and QA
o To ensure the reliability of results that are generated by the facility (results have precision and accuracy)
o This is also a requirement for the renewal of license to operate a clinical laboratory or blood bank
Quality Assurance Quality Control
 Total testing process (Pre-analytical to post analytical)  Analytical phase
 Process-oriented  Product-oriented
o From the time you get the requisition from the o You are ensuring that you are conducting the test correctly to
doctor/patient, up to the time you release the result create a good product/test result
o Makes sure you are doing the right things the right way o Makes sure that the results are what you expected
 You plan to avoid the defect in the first place  You try to find defects and correct them while making the product
 QA is all about prevention  QC is all about detection of error
 QA involves processes managing quality  QC is used to verify the quality of the product

Quality Control Products

 Control- is a substance or a solution that contains an established amount of the analyte being tested
 Patient-like material because it is ideally made from human serum, urine, spinal fluid and other body fluids
 Can be liquid or lyophilized (freeze dried or dehydrated into powder)
o That is why it requires reconstitution (add distilled water to it and mix it carefully to avoid incorrect control values)
 Composed of one or more analytes of known concentration
 Should be tested in the same manner as patient samples
 Purpose: Validate the reliability of the test system and to evaluate the performance in the analytical phase that might impact the result.
 2 Types of Controls
o Normal Control- contains normal levels of the analyte being tested
o Abnormal control- contains analyte at a concentration above (abnormally high control) or below (abnormally low control) the normal
range for the analyte
 A good laboratory practice requires testing normal and abnormal controls for each test at least daily to monitor the analytical process
(some of the laboratory would test control per shift)
 Different types of control solutions
o Pool serum- comes from the idea that the serum specimens were obtained from different individuals and then they are mixed together,
creating one control solution; can come from human or animal sources
 Human sources- more expensive but preferred; closely resembles patient samples
 Animal sources- bovine serum (serum of the cow or other cattle) is the most common animal source
o Commercial control (more stable)
 Already prepared by the manufacturer and usually are lyophilized for stability
 Requires reconstitution (usually distilled water); incorrect mixing results to incorrect control values
 Two types of commercial control
 Assayed- more expensive; company-analyzed control and establish reference range at ± 2SD
(company which created the control already established the reference ranges)
 Unassayed- cheaper, without values; control is lyophilized and has to be reconstituted and it is
the laboratory that will get the reference range for that particular control solution
o Example of a QC Log with Patient results with patient results for potassium
 When the daily QC result obtained for the normal control is compared to the range calculated for
that normal control, it becomes apparent that each result lies somewhere within the expected
range. This indicates that the analytical process is in control at the normal level on that day of testing.
 When the daily QC result for the abnormal control for the high potassium is compared to the defined range for that abnormal control,
the analytic process is shown to be in control for each day of testing (except for the last day on November 7).
 That means the patient results in November 1-6 could be reliably reported.
 However, the laboratory was out of control for abnormal high potassium on November 7, because the value obtained for the QC
material is 8 mmol/L (outside the expected range).
 This means that some error occurred, which may have made the patient’s results unreliable and should not be reported, until the
error is resolved and the abnormally high samples are retested.
 Scenario is very common because a test system can malfunction/begin to malfunction at any time since the last successful QC.
  It would be a good laboratory practice to retest all patient samples that were reported with abnormally high potassium levels or
near the upper limit of normal since the last QC was performed. A retest using a random sample of patients vs all the samples
that we are testing (it is acceptable, although it is a risky practice just like when dealing with potassium and the amount of time
the plasma or serum has been in contact with cellular elements must be taken into considerations).
 A sample can randomly be selected, but the requirements for the specimen and analyte tested should also be put into consideration.

Tools Needed to Achieve Accuracy

 Standard Solution
o 100% pure substance of a that contains a specified concentration of a parameter
o Accurately weighed and prepared
o Certified by professional organizations
o Used as a reference for unknown (patient’s sample)
 Before running/testing a patient’s sample, you have first to check your standard value, especially if you are doing it manually or you
are conducting the test manually using for example a spectrophotometer.
 When a standard is used in place of a sample and then tested, the result should match the concentration of the standard (matching
result) because that will give you a confidence that the test is working correctly, but if there is any discrepancy (like a discrepancy
greater than 10%), then that could indicate a problem that must be investigated.
 Blank Solution
o Used in order to achieve accuracy in the testing
o Is used in order to cancel out or zero (0) the absorbance of all the other components in the sample except the component whose
absorbance is to be measured.
 Contains everything that is in the sample, except that one material that is measured.
 Related to the use of spectrophotometer because before using the spectrophotometer, it is very important to set the absorbance reading
at 0 to eliminate any contamination, any artificial sources of error to be introduced to the spectrophotometer.
o Contains very little amount, or sometimes it does not even contain any detectable amount of the analyte of interest.
o Main purpose: It is used to calibrate the instrument (e.g. spectrophotometer)
o Water blank- if all reagents are colorless
o Sample blank- can correct for potential error from existing color or turbidity in the sample before
reagents are added, used if the sample is having color or turbidity (e.g. Icteric/Chylous sample)
o Reagent blank- corrects for the absorbance caused by the color of the reagents. The absorbance of
reagents is automatically subtracted from each unknown reading.

Levy-Jennings Chart and Westgard Rules

 Levy-Jennings Plot (Shewhart Plot)- considered as the most common intra-laboratory quality control chart
used in clinical chemistry laboratories considered as the Gaussian curve on its side.
o Used to graph successive quality control values day-to-day or depending on the protocol of the laboratory
o In order to prepare this, we need to have at least 20 control values, and then calculate the mean and the standard deviation and set
the confidence limit (usually set at ±2SD). If you have already all those data, plot the control values on the y-axis against the day or
time on the x-axis.
o Plotting the control values in a Levy-Jennings chart
***Values used for the graph are from the ones in Lecture 6***
 Plot the values and connect them, then analyze these control values using the rejection
criteria (Westgard Rules)
 Drawbacks of Levy-Jennings Chart
o Cumbersome
o After the data are obtained, the individual points are plotted on graph and analyzed visually
o Time consuming and subjective (because the interpretation will be based on the medical technologist analyzing the Levy-Jennings
chart.
 Westgard Rules are developed to improve quality monitoring and decrease subjectivity in data analysis
o Will help test/determine if the analysis is in control or out of control based on the values plotted.
o For convenience, a shorthand notation is adapted to abbreviate different decision criteria/control rules.
o 12S Rule- two (2) control value exceeds ±2SD from the mean. This rule is used as a warning rule to trigger careful
inspection of the control data by the following rejection rules (random or systematic error may be present in the test system)
 The relationship between this value and other control results within the current and previous analytical runs must also be examined.
 If no relationship can be found and no source of error can be identified, we can assume that a single control value outside the ±2s
limits is an acceptable random error (and the patient results can be reported)
o 13S Rule- two (2) control value exceeds ±3SD from the mean
 Identifies an acceptable random error, or possibly the beginning of a large systematic error.
 Any QC result outside the ±3s violates this rule.
o 22S Rule- two (2) consecutive control values exceed the same limit, either the +2SD or -2SD.
o R4S Rule- one (1) control measurement in a group exceeds the mean +2SD and another exceeds the mean -2SD, and
they are consecutive values.
o 41S Rule- four (4) consecutive control values exceed either +1SD or -1SD. The value must all be in the same direction.
 o 10X Rule- ten (10) consecutive control values all lie on the same side of the mean.
o 8X Rule- eight (8) consecutive control measurements fall on one side of the mean.
 Modification of the 10X rule to make it fit more easily with Ns of 4.
 N- represents the total number of control measurements that are available at the time
a decision on control status is to be made
 Example: N=2 (2 measurements on one control material or 1 measurement on each of two different control materials), N=3 (it
involved one measurement on each of the three different control materials), N=4 (2 measurements on each of two different control
materials, or 4 measurements on one material, or 1 measurement on each of four materials)
o 12X Rule- twelve (12) consecutive control measurements fall on one side of the mean.
 The 8X and 12X are usually used with Ns of 2 or 4, which means they are appropriate when two
different control materials are measured 1 or 2 times per material.
o 2of32S Rule- 2 out of 3 control measurements exceed the same mean plus 2s or mean minus 2s control limit
o 31S Rule- three (3) consecutive control measurements exceed the same mean plus 1s or mean minus 1s control limit
o 6X Rule- six (6) consecutive control measurements fall on one side of the mean.
o 9X Rule- nine (9) consecutive control measurements fall on one side of the mean.
 Modification of the 10X rule to include a larger number of control measurements that still fit with an N of 3
o 7T Rule- seven (7) control measurements trend in the same direction, i.e., get
progressively higher or progressively lower
o Oftentimes there are only 6 rules (12S, 13S, 22S, R4S, 41S, and 10X) and the rest are just
some modifications of the first 6 rules.
o Example: Levy-Jennings Chart of Test3
 Mean= 252.3
 Violations found:
 22S- because two consecutive control values lie outside of the +2SD
 R4S- because one of the control values is outside of the +2SD and another one is outside
of the -2SD
 10X- 10 consecutive control values that lie one side of the mean
o Levy-Jennings chart reflects our analysis that is out of control
 Multirule QC
o Uses a combination of decision criteria, or control rules, to decide whether an analytical run is in-control or out-of-control
o If any violation of the warning rule 12S is seen, that means look carefully before proceeding with analysis.
o The next time around, if another control rule violations are seen that means stop or reject the run because
it is are already out of control and the patient’s result should not be reported.
o If the only violation is 12S rule and nothing else, then proceed and can say that the run is in control.
o The N must be at least 2 to satisfy the US CLIA QC requirements.
 Two Types of Error
o Random Error
 No trend or means of predicting  Non-repeating error  Usually it is a human error
 Only occurs once  Easy to spot  Ex: 12S, 13S, and R4S
 Other causes: Mislabeling of sample, Improper mixing of sample and reagent, Voltage fluctuations, and Temperature fluctuations
 To assess the situation to know if there is really a random error occurring, the sample is re-assayed using the same reagents, and if
a random error occurred, the same mistake may not be made again and the result will be within appropriate control limits.
o Systematic Error
 Repeating error  Will be seen as a trend in data  Ex: 22S, 41S, and 10X
 Other causes: Improper calibration, Deterioration of reagents, Sample instability, Instrument drift, and Changes in standard materials
 When doing a re-assay, this type of error will not correct the problem instantly. The data should be
further analyzed on how to initially resolve (prepare new control materials/new reagents, re-
standardize the assay, check the wavelength and instrument settings (of the machine), and the
reagents that are close to expiration date should be discarded)
 Shift- 6 or more values on one side of the mean, but maintain a constant level (usually indicates
deterioration of standard)
 Trend- control values that increase or decrease over a period of 6 consecutive days (usually pass
through the mean and usually indicates deterioration of reagents)
 Proficiency Testing (Interlaboratory Comparison)
o Compares the measuring results obtained by different laboratories because some laboratories
participate in accreditation.
o Accrediting agency will send unknown samples in the participating laboratory facilities
 Information is provided on the reconstitution of the samples and the assays to be performed.
 Once the laboratory receive these samples, they will analyze it the way they do it routinely in their laboratory
o After the analyses are carried out, the results are mailed back to the accrediting agencies for comparison with those from other
laboratories involved in the program.
o Data are returned to the laboratory with comments and recommendations
o Determines the performance of individual laboratories for specific tests and usually it is used to monitor the continuing performance
of the laboratory.
 Lectured by: Aldrin Jeff B. Autencio, MSMT

Objectives:
 Discuss the importance of proteins  Know the different plasma proteins and its functions
 Identify the different classification of proteins  Discuss the different methods of analysis for protein
determination

Amino Acids
 Are building blocks of proteins (without amino acids, proteins cannot be formed)
 Responsible for growth, repair, and maintenance of all cells are dependent on the amino acids.
 The chemical properties of the amino acids of proteins determine the biologic activity of the protein.
 Protein can either be acute phase reactant, transport protein, or immunoglobulins
 Proteins catalyze almost all of the reactions in living cells. They control all the cellular processes that occurs in the human body.
 Amino Acid Basic Structure
o An amino acid contains at least one of both amino and carboxylic acid functional groups
o The n-terminal end of amino group and the c-terminal end of the carboxyl group bond to the
carbon with the amino group of one amino acid, linking with a carboxyl group of another, forming a peptide bond.
o A chain of amino acids is known as polypeptide. They are described to be large and are the one responsible to constitute a protein.
o In human serum, proteins average about 100 to 150 amino acids in the polypeptide chains.
 Amino Acid Metabolism
o We get amino acids via our dietary intakes.
o Essential amino acids must be supplied by the diet in the form of proteins. About half of the 20 amino acids needed by humans
cannot be synthesized at a rapid rate enough to support growth. Therefore, food is essential to supply the amino acids.
 Arginine
o Plays an important role in cell division, the healing of wounds, stimulation of protein synthesis, enhance immune function, and the
release of hormones. A complex of amino acid that is often found at the active site in proteins and are abundant in enzymes, due to
its amine-containing side chain.
o Is required for the generation of urea, which is necessary for the removal of toxic ammonia from the body and is also required for
the synthesis of creatine.
 Histidine
o Is the direct precursor of histamine, one of the proteins involved in the immune response.
o Is also an important source of carbon atoms in the synthesis of purines.
o Is needed to help grow and repair body tissues and to maintain the myelin sheaths that protect nerve cells.
o Helps manufacture red and white blood cells and help protect the body from heavy metal toxicity.
o One of the basic amino acids due to its immediate side chain.
o Stimulates the secretion of the digestive enzyme, gastrin and acts as a catalytic site in certain enzymes.
 Isoleucine
o In the group of branch chain amino acids that are needed to help maintain, heal, and repair muscle tissue, skin, and bones.
o Is needed for hemoglobin formation (hemoglobin is responsible for RBCs), and it helps to regulate blood glucose levels and
maintain energy levels.
 Leucine
o In conjunction with valine and isoleucine, leucine boosts the healing of muscle, skin, and bones; aids in recovery from surgery.
o Lowers blood glucose levels. Necessary for the optimal growth of infants and for nitrogen balance in adults.
o Also part of the branched chain amino acids along with Valine and Isoleucine.
o Considered to be the second most common amino acid found in protein, besides glycine
 Lysine
o Plays a role in the production of antibodies (vital for the immune system) and lowers triglyceride level
o Helps in the absorption and conservation of calcium and plays an important role in the formation of collagen
o A positive charge ion which makes it one of the three basic charged amino acids.
 Methionine
o Is a source of sulfur, required by the body for normal metabolism and growth.
o Considered an important amino acid which helps to initiate the translation of mRNA (messenger ribonucleic acid) by being the
first amino acid incorporated into the n-terminal position of all proteins.
o Assists in the breakdown of fats, helps to detoxify lead and other heavy metals, helps diminish muscle weakness, and prevents
brittle bones
 Phenylalanine
o Promotes alertness and vitality, elevates mood, decreases pain, aids memory and learning, and is used to treat arthritis and
depression
o Used by the brain to produce norepinephrine (a neurotransmitter that transmits signals between nerve cells)
o Uses an active transport channel to cross for the blood brain barrier and, in large quantities interferes with the production of
serotonin.
 Threonine
Has a vital role in the production of neurotransmitters. This also is important for the health of nervous system.
o
Helps maintain proper protein balance in the body and it aids in liver function, metabolism, and assimilation.
o
Is an alcohol-containing amino acid that is an important component in the formation of protein, collagen, elastin (a connective
o
tissue protein), and tooth enamel.
 Tryptophan
o Is also a precursor for serotonin and melatonin.
o Is a natural relaxant; it helps alleviate insomnia by inducing sleep, soothes anxiety, and reduces depression
o Formed from proteins during digestion by the action of proteolytic enzymes.
o Used in the treatment of migraine headaches, aids in weight control by reducing appetite, and helps control hyperactivity in children
 Valine
o Another branched chain amino acid that is a constituent of fibrous protein in the body.
o Is needed for muscle metabolism and coordination, tissue repair, and maintenance of nitrogen balance.
o Used by the muscle tissue as an energy source.
o Used in treatments for muscle, mental, and emotional problems, insomnia, anxiety, and liver and gallbladder disease.
 Amino Acid Analysis
o Specimens: Blood, Urine, Amniotic Fluid
 Blood samples for amino acids should be drawn after or at least 6-8 hours fasting to avoid the effect of the absorbed amino acids
originating from dietary proteins.
 Anticoagulant of choice: Heparin
o Screening Test: Thin Layer Chromatography (TLC) o Specific and Sensitive Test: Mass Spectrophotometry (MS/MS)

Proteins
 All biochemical reactions are catalyzed by enzymes, which contain protein. Protein is made up of amino acids.
 The structure of cells and extracellular matrix that surrounds all cells is largely made of protein group
collagens. Collagens are the most abundant protein in the human body.
 The transport of materials in body fluids depends on proteins such as transferrin, receptors for hormones are
transmembrane proteins, and transcription factors needed to initiate the transcription of a gene, are proteins.
 Proteins make up antibodies (which plays a major role in the combat of foreign substances in the body), which are a
major component of the immune system.
 Every function in the living cell depends on proteins. They are vital and essential to the human body.
 A typical protein contains 200-300 amino acids.
 Proteins are macromolecules (molecule with molecular mass of several thousand or more)
o Polymers built from more than one or more unbranched chains of amino acids.
 Synthesis
o Most plasma proteins are synthesized in the liver and secreted by the hepatocyte into the circulation.
o Immunoglobulins are exemptions because they are synthesized by the plasma cells.
o Protein synthesis occurs at the rate of approximately 2-6 peptides per second.
 Structure
o In order to function properly, proteins must have the correct sequence of amino acids.
o 4 distinct levels of protein structure:
 Primary structure- represents the number and types of amino acids in the specific amino acid sequence.
 Secondary structure- is regularly repeating structures stabilized by hydrogen bonds between the amino acids within the protein.
 Common secondary structures are the α-helix, β-pleated sheets, and turns with the most serum proteins forming a helix.
 Secondary structures add new properties to a protein such as strength and flexibility.
 Tertiary structure- refers to the overall shape, or conformation, of the protein molecule.
 The conformation of the tertiary structure is known as the fold, or the spatial relationship of the secondary structures to one
another.
 Tertiary structures are three-dimensional.
 Quaternary structure- is defined as the shape or structure that results from the interaction of more than one protein molecule, or
protein subunits, held together by noncovalent forces, such as hydrogen bonds and electrostatic interactions.
 Denaturation
o Can be caused by heat, hydrolysis by strong acid or alkali, enzymatic action, exposure to urea or other
substances, or exposure to ultraviolet light.
o When the secondary, tertiary, or quaternary structure of a protein is disturbed, the protein may lose its
functional and chemical characteristics. This loss of its native, or naturally occurring, folded structure is called denaturation.
o Folded protein becomes unfolded
 Nitrogen content
o Proteins consist of the elements: carbon, oxygen, hydrogen, nitrogen, and sulfur.
o The nitrogen content of serum protein is, on average, approximately 16%. This measurement of nitrogen content is used in one
method for total protein.
o Protein contains nitrogen that sets them apart from pure carbohydrates and lipids which do not contain any nitrogen atoms.
 Charge and Isoelectric point
Proteins can be positively and negatively charged
o
Proteins contain many ionizable groups on the side chains of their amino acids as well as on their N-terminal and C-terminal ends.
o
The pH of the solution, the pK of the side chain, and the side chain’s environment influence the charge on each side chain.
o
In general terms, as the pH of a solution increases, deprotonation of the acidic and basic groups on proteins occurs, so that
o
carboxyl groups are converted to carboxylate anions (R-COOH to R-COO-) and ammonium groups are
converted to amino groups (R-NH3+to R-NH2). Cations- positively charged cells; Anions- negatively
charged cells
o The relationship between pH, pKa, and charge for individual amino acids can be described by the Henderson-Hasselbalch
equation.
o The pH at which an amino acid or protein has no net charge is known as its isoelectric point (pI).
 Isoelectric point (pI) is the point at which the number of positively charged groups equals the number of negatively charged
groups in a protein. When there is an equal amount of positively charged group and negatively charged group, the pH of the
amino acid will have no net charge.
 If a protein is placed in a solution that has a pH greater than the pI, the protein will be negatively charged; at a pH less than the pI,
the protein will be positively charged.
 Proteins differ in their pI values, but for most proteins, it occurs in the pH range of 5.5 to 8.
 Classification by Protein Functions
o Enzymes- proteins that catalyze chemical reaction
o Hormones- proteins that are chemical messengers that control the actions of specific cells or organs
o Transport proteins- proteins that transport movement of ions, small molecules, or macromolecules, such as hormones, vitamins,
minerals, and lipids, across a biological membrane
o Immunoglobulins (antibodies)- proteins produced by B-cells (lymphocytes) in the bone marrow that mediate the humoral immune
response to identify and neutralize foreign objects
o Structural proteins- fibrous proteins that are the structures of cells and tissues such as muscle, tendons, and bone matrix
o Storage proteins- proteins that serve as reserves of metal ions and amino acids that can be released and used later without harm
occurring to cells during the time of storage
 Ferritin- most widely tested and studied storage protein; stores iron to be later used in the manufacture of hemoglobin.
o Energy source- plasma proteins serve as a reserve source of energy for tissues and muscle
o Osmotic force- plasma proteins function in the distribution of water throughout the compartments of the
body. Their colloid osmotic force, due to their size, does not allow protein to cross the capillary membranes.
 Protein plays a vital role especially in its osmotic force.
 When there is low osmotic force, tissue fluids will leak into other surrounding tissues, and cause a
problem such as edema.
 Classification by Protein Structure
o Simple proteins- contain peptide chains composed of only amino acids
 Simple proteins may be globular or fibrous in shape.
 Globular proteins are globe-like, symmetrical proteins that are soluble in water and they are transporters, enzymes and
messengers.
o Conjugated proteins- consist of a protein and a nonprotein (prosthetic) group
 The prosthetic group is the non-amino part of a conjugated protein. It may be lipids, carbohydrates, porphyrins, metals, and
others.
 It is the prosthetic groups that define the characteristics of these proteins.
 Plasma Proteins
o The most frequently analyzed of all proteins.
o The major measured plasma proteins are divided into albumin and globulins.
o There are four types of globulins, each with specific properties and actions. A typical blood panel
will provide four measurements such as: Total Protein, Albumin, Globulin, Albumin-globulin
Ratio
o Prealbumin (Transthyretin)
 Prealbumin is the transport protein for thyroxine and triiodothyronine (thyroid hormones); it also binds with retinol-binding
protein to form a complex that transports retinol (vitamin A) and is rich in tryptophan.
 A low prealbumin level is a sensitive marker of poor nutritional status.
 “Pre”- Prealbumin is so named because it migrates ahead of albumin in the classic electrophoresis of serum or plasma proteins.
o Albumin
 It is the protein present in highest concentration in the plasma. Albumin also exists in the extravascular (interstitial) fluid
compartment.
 Albumin also buffers pH and is a negative acute-phase reactant protein
 Albumin transports thyroid hormones; other hormones, particularly fat-soluble ones; iron; and fatty acid
 Amino acids or proteins in general are synthesized in the liver, so basically albumin is synthesized in the liver.
 Another prime function of albumin is its capacity to bind various substances in the blood.
 Albumin is a very good protein, it functions to have four binding sites on itself and is actually responsible for transporting
different substances throughout the body.
 Albumin is decreased in the adequate source of amino acids, liver disease, protein losing enteropathy, kidney loss, and others
o Globulins
 The globulin group of proteins consists of alpha-1, alpha-2, Beta, and Gamma fractions.
 Alpha-1-Antitrypsin
 Is an acute phase reactant, therefore it increases in inflammatory reactions, pregnancy, and contraceptive use.
 A glycoprotein mainly synthesized in the liver, has as its most important function in the inhibition of the protease neutrophil
elastase.
 Abnormal form of 1-antitrypsin can also accumulate in the liver and can cause cirrhosis.
 Is the major component (approximately 90%) of the fraction of serum proteins that migrates immediately following albumin.
 Alpha-1-Fetoprotein (AFP)
 Is synthesized in the developing embryo and fetus, and then by the parenchymal cells of the liver
 The physiologic function of AFP has not been completely identified, but it has been proposed that the protein protects the fetus
from immunologic attack by the mother.
 Elevated AFP levels includes spina bifida, neural tube defects, abdominal wall defects, absence of the major portion of the brain,
and general fetal distress.
 Low AFP levels, on the other hand, is prone to the risk of down syndrome and trisomy 18.
 Alpha-1-Acid Glycoprotein (Orosmucoid)
 Major plasma glycoprotein, is negatively charged even in acid solutions
 Is produced by the liver and is an acute-phase reactant
 Provide a useful diagnostic tool in neonates with bacterial infections
 Alpha-1-Antichymotrypsin
 Inhibits the activity of the enzymes cathepsin G, pancreatic elastase, mast cell chymase, and chymotrypsin.
 Is produced in the liver and is an acute-phase protein that is increased during inflammation
 Haptoglobin
 Is synthesized in the hepatocytes (in the liver). One of the proteins used to evaluate the rheumatic diseases.
 Is also considered as an acute-phase reactant that is elevated in many inflammatory diseases such as rheumatic disease,
ulcerative colitis, heart attack, and severe infection.
 Haptoglobin testing is used primarily to help detect and evaluate hemolytic anemia and to distinguish it from anemia due to
other causes.
 Ceruloplasmin
 Is an acute-phase reactant
 Is primarily ordered along with blood and/or urine copper tests to help diagnose Wilson’s disease.
 A copper-containing analyte that is synthesized in the liver.
 Lipoproteins
 Are complexes of proteins and lipids whose function is to transport cholesterol, triglycerides, and phospholipids in the blood.
 They are subclassified according to the apoproteins such as Chylomicrons, VLDL (Very Low Density Lipoprotein), IDL
(Intermediate Density Lipoprotein), LDL (Low Density Lipoprotein), Lipoprotein(a), and HDL (High Density
Lipoprotein/Good cholesterol)
 Fibrinogen
 One of the largest proteins in blood plasma. It is synthesized in the liver, and it is classified as glycoprotein.
 Fibrinogen is one of the acute-phase reactants and is determined as a clottable protein.
 C-reactive protein (CRP)
 Is synthesized in the liver and is one of the first acute-phase proteins to rise in response to inflammatory disease.
 Named because it precipitates with the C substance of a polysaccharide of pneumococci.
 High-Sensitivity C-Reactive Protein (hsCRP)
 High-sensitivity CRP (hsCRP) is the same protein but is named for the newer, monoclonal antibody-based test methodologies
that can detect CRP at levels below 1 mg/L. Determines risk of cardiovascular diseases.
 Immunoglobulins
 Are glycoproteins composed of 82%-96% protein and 4%-18% carbohydrate produced by white blood cells, known as B
cells.
 Are not synthesized to any extent by the neonate, and are for immune response.
 Myoglobin
 Is a single-chain globular protein of 153 amino acids, containing a heme (iron-containing) prosthetic group.
 Is the primary oxygen-carrying protein (approximately 2% of total muscle protein) found in striated skeletal and cardiac
muscle.
 Since this is part of the cardiac muscle, this is used in conjunction with troponin to rule out or help diagnose a heart attack.
 Troponin (cTn)
 Has established itself firmly as the “gold standard” in the diagnosis of cardiac disorders.
 Cardiac troponins can be measured on serum or heparinized plasma by ELISA of myocardial injury.
 Total Protein Abnormalities
o Hypoproteinemia- a total protein level less than the reference interval. One cause of a low level of plasma proteins is excessive
loss (“Hypo”- Low levels; is where there is less concentration of protein caused by excessive loss)
 o Hyperproteinemia- an increase in total plasma proteins, is not an actual disease state but is the result of the underlying cause,
dehydration. (“Hyper”- High levels)
o Total Protein Test is a rough measure of all proteins in plasma. Total protein measurements can reflect nutritional status, kidney
disease, liver disease (because almost all proteins are derived from the liver except for immunoglobulins), and many other
conditions.
 Methods of Analysis
o Total Nitrogen
 Proteins are unique because they contain nitrogen. Measures all chemically bound nitrogen in the sample.
 The method can be applied to various biologic samples, including plasma and urine
 In plasma, both the total protein and nonprotein nitrogenous substances are being measured
 Method: Chemiluminescence
o Total Proteins
 Specimen: Serum
 The specimen most often used to determine the total protein is serum (rather than plasma), because there are a lot of
interferences accompanied in plasma because this contains the nonprotein nitrogenous compounds
 The reference interval for serum total protein is 6.5-8.3 g/dL (65-83 g/L) for ambulatory adults. In the recumbent position, the
serum total protein concentration is 6.0-7.8 g/dL (60-78 g/L)
 A fasting specimen is not needed but one must prevent the analysis of lipemic or hemolyzed samples for this will cause falsely
elevated results because of the release of red blood cell proteins into the serum.
o Kjeldahl
 Determines nitrogen content of the analyte (the classical method for quantitation total protein)
 Obsolete method (not really performed nowadays because there are more specific assays, e.g. Dye-binding technique)
 Not used in clinical laboratory because it is time-consuming and too tedious for routine use.
 The method also requires the assumption that no proteins of significant concentration in the unknown specimen are lost in the
precipitation step.
 The serum proteins are precipitated with an organic acid such as TCA or tungstic acid.
o Refractometry
 Is useful when a rapid, easy method that requires a small volume of serum is needed (refractometer is used)
 The total protein is commonly measured with a handheld refractometer.
 How to perform:
 Add a drop of serum in the refractometer between the covered glass and prism. Refractometer is held so that the light is refracted
to the serum layer. Read by the number of g/L at the line in the refractometer on the internal scale and then record.
o Biuret
 Is the most widely used method and the one recommended by the International Federation of Clinical Chemistry expert panel
 Wavelength of the spectrophotometer: 540 nm
 The color varies from a pink to a reddish violet. The color that is formed is proportional to the number of peptide bonds.
o Dye-binding
 Are based on the ability of most protein and serum to bind to the dye’s color change.
 Bromphenol Blue  Amido Black 10B  Coomassie brilliant blue
 Ponceau S  Lissamine green
 Fractionation, Identification, and Quantitation of Specific Proteins
o A reversal or significant change in the ratio of albumin and total globulin, the albumin/globulin (A/G) ratio, is found in diseases of
the kidney and liver. To determine the A/G ratio, total protein and albumin are measured and globulins are calculated by
subtracting the albumin from the total protein (����� ������� − ������� = ���������).
o Salt Fractionation
 Is done using precipitation.
 Globulins are separated from albumin by salting out, using sodium salt to cause precipitation of the globulins.
 The albumin that remains in the solution (in the supernatant) is measured in routine total protein methods.
 Obsolete method, not widely used today because of the direct methods that are available that reacts specifically with albumin.
o Albumin
 Dye-binding methods
 The pH of the solution is adjusted so that albumin is positively charged.
 Albumin is attracted to and binds to anionic dye by electrostatic force, so when albumin binds to the dye, it binds to the free dye
and it emits color change.
 A variety of dyes have been used, including methyl orange, 2,4-hydroxy-azobenzenebenzoic acid (HABA), bromocresol
green (BCG), and bromocresol purple (BCP)
o Total Globulins
 Total globulin level in serum is determined by a direct colorimetric method using glyoxylic acid.
 Glyoxylic acid, in the presence of Cu2+ and in an acid medium (acetic acid and
H2SO4), condenses with tryptophan found in globulins to produce a purple color.
o Electrophoresis
 Separates proteins on the basis of their electric charge densities (buffer is needed)
 Protein, when placed in an electric current, will move according to their charge density,
 which is determined by the pH of a surrounding buffer.
 The direction of movements depends on what the charge is, positive or negative.
 Cations migrate to the cathode (negative terminal).
 Anions migrate to the anode (positive terminal).
 Cellulose acetate or Agarose gel- is the support media use in today’s laboratory.
o Serum Protein Electrophoresis (SPE)
 All major serum proteins carry a net negative charge at pH 8.6 and migrate
toward the anode.
 Using standard SPE methods, serum proteins appear in five bands: albumin
travels farthest to the anode, followed by alpha-1-globulins, alpha2-globulins,
beta-globulins, and gamma-globulins, in that order.
 The width of the band of the proteins in a fraction depends on the number of
protein present in that fraction.
 Homogenous protein gives a narrow band.
 To make the color visible, a variety of dye is used (e.g. Ponceau S, Amido Black 10B and Coomassie
brilliant blue). The protein appears as bands, on the support medium.
 No color means negative
 Measured using densitometers.
 Many scanning densitometers compute the area under the absorbance curve for each band and the
percentage of total dye that appears in each fraction.
 The computation also may be made by cutting out the small bands from the membrane and eluting the dye
from each band in 0.1 mol/L NaOH. The absorbances are added to obtain the total absorbance, and the percentage of the total
absorbance found in each fraction is then calculated.
 Reference values for each fraction are as follows:
1. Albumin: 53-65% of the total protein (3.5-5.0 g/dL) 4. Beta-Globulin: 8-14% (0.7-1.1 g/dL)
2. Alpha-1-Globulin: 2.5-5% (0.1-0.3 g/dL) 5. Gamma-Globulin: 12-22% (0.8-1.6 g/dL)
3. Alpha-2-Globulin: 7-13% (0.6-1.0 g/dL)
o Capillary Electrophoresis
 Is a collection of techniques in which the separation of molecules takes place in silica capillaries.
 In capillary zone electrophoresis, the capillaries are filled with a conducting solution, usually an
aqueous buffer (important to this test).
 The separated molecules are detected by their absorbance, as they pass through a small window near
the detection end of the capillary. Absorbance is being read.
o Immunochemical Methods
 Specific proteins may be identified using the different immunochemical methods.
 Radial Immunodiffusion (RID)  Electroimmunodiffusion
 Immunoelectrophoresis (IEP)  Immunoturbidimetry
 Immunofixation (IFE)  Immunonephelometry
 Proteins in Other Body Fluids
o Urinary Protein
 Since the kidneys are used to dispel any toxic substances/waste of the human body, proteins can normally be found in urine.
 Plasma proteins appear in the urine because they have passed through the renal glomerulus and have not been reabsorbed by the
renal tubules.
 Measured by test strips
 Methods: Biuret method, Folin-Ciocalteu reagent, Coomassie blue, ELISA, RIA, Zone Immunoelectrophoresis
o CSF Protein
 Reference range: 14-45 mg/dL
 Routinely measured when a CSF is sent to the laboratory, because increased and decreased levels will come into mind when it
does not meet the reference range
 Increased levels: bacterial, viral, and fungal meningitis; traumatic tap; multiple sclerosis; obstruction; neoplasm; disk herniation;
and cerebral infarction
 Decreased levels: hyperthyroidism and when fluid is leaking from the CNS
 Protein measurement is one test that is usually requested on CSF in addition to glucose level.
 Most frequently used methods: Turbidimetric methods using TCA, Sulfosalicylic acid with sodium sulfate or benzethonium
chloride, and Dye-binding such as Coomassie brilliant blue method.
 Lectured by: Alessandra Kamille P. Mallari, MD, DPSP

Objectives:
 To briefly discuss the different types of plasma proteins.
 To discuss the common causes of abnormal levels of each protein described.

Plasma Proteins
 The most frequently analyzed of all the proteins.
 The major serum proteins are those components that are readily resolved and detected on electrophoretic
gels stained by conventional clinical laboratory techniques.
 These are divided into 2 major categories: Albumins and Globulins
 Normal Capillary Serum Electrophoresis
o The electrophoretic zone is divided into several zones: Albumin, α-globulins (α-1-globulins and α-2-
globulins), β-globulins, and γ-globulins
 Stained Agarose Gel Electrophoresis- The highest (predominant) plasma protein is albumin.

Albumin (39 to 51 g/dL)

 Albumin is the single most abundant protein in the plasma
 Amounts to about 2/3 of the total plasma protein.
 Synthesized the liver and it is present at highest concentration in the plasma.
 Functions of albumin:
1. Maintain the intravascular oncotic pressure- it prevents the water or the fluid in the vascular spaces from going out into the tissues
2. General transporter protein
3. Repository of amino acids
 Variants of Albumin
o Differ from the most common allotype- by a single amino acid substitution
o 77 variants identified and may cause either bisalbuminemia (two different types of population of albumin present in the blood that
will be seen in electrophoresis), analbuminemia (absence of albumin in the blood), or strong binding
capacity of thyroid hormones (causes abnormalities in thyroid function).
o Most of these variants does not produce any clinical manifestation.
 Analbuminemia
o Congenital absence of albumin
o Does not lead to problems in fluid retention because of the compensatory mechanism present from birth.
o Increased levels (infrequent): Due to dehydration or prolonged application of tourniquet during blood extraction.
 Hypoalbuminemia (Decreased Levels, most common abnormality)
o Usually seen in sick individuals (especially in hospitalized patients)
o Decreased synthesis in the liver (due to liver pathology/liver diseases such as cirrhosis, hepatitis or liver failure)
o Malnutrition or malabsorption
o Dilution by excess (due to excess IV fluid from administration)
o Nephrotic syndrome (an abnormality in kidney function wherein it is marked by massive proteinuria)
 Marked by massive proteinuria
 Caused by a derangement in glomerular capillary walls, wherein there is increased permeability to plasma proteins (all of the
albumins will go out)
 Increased excretion of proteins in the urine, and one of these proteins is albumin.
 When albumin is excreted in the urine, there is a subsequent decrease in albumin levels in the blood.
 Since the main function of albumin in the blood is for fluid retention (keeping the fluid inside the blood vessels), so decreased or
absence of albumin in the blood causes fluid (water) to go out of the blood vessels and into the surrounding tissue, causing edema
(periorbital edema, which is common in nephrotic syndrome patients)
 Other areas that will commonly have edema is dependent portions of the body such as feet (in kidney failure patients). In patients
who have kidney failure, they will excrete the albumin in blood and presence of albumin in the urine is almost always abnormal.
 Pre-Albumin (0.15 to 0.36 g/dL)
o Also called thyroxine-binding prealbumin (TBPA) or transerythrin (TTR)
o Very low in plasma in that there may be trouble seeing it in electrophoresis.
o Quantified by immunologic nephelometry
o Early indicator of change in nutritional status
 Usually has a very fast lifespan (short half-life, only 2 days)- usually decreases very rapidly
o Negative acute phase reactant protein o Binds retinol to form a complex with Vitamin A.
o Usually a transport protein for thyroxine for thyroid hormones. o Rich in tryptophan
o Increased levels (seen in Type I Familial Amyloidotic Polyneuropathy)
 Caused by prealbumin gene mutation
 Prealbumin has considerable β-pleated sheet conformation- becomes source of β-fibrillar amyloid
 Amyloid accumulates in the nerves (nervous tissue)
  Pathogenic variants cannot be distinguished from normal by serum protein electrophoresis (usually identified by other methods).
 Other causes: Patients who are receiving steroids, alcoholism, and in chronic renal failure

Globulins
 Group of proteins consisting of α-1, α-2, β, and γ fractions. Each consists of a number of different proteins with different functions.
 α-1-Antitrypsin (2 to 4 g/dL)
o A glycoprotein mainly synthesized by the liver o Acute phase reactant
o Most important function: Inhibition of the protease, neutrophil elastase
 Neutrophil elastase is released from leukocytes during infection, but when it is left unchecked, it can destroy the alveoli.
o Decreased levels (α-1-Antitrypsin deficiency)
 Mutation in SERPINA1 gene, which causes either complete (homozygous) absence or decreased (heterozygous) levels (AATD)
 Causes Emphysema
 Destruction of the elastic support of the alveoli due to uncontrolled activity of neutrophil elastase.
 Since the main function of α-1-Antitrypsin is the inhibition of neutrophil elastase, without α-1-Antitrypsin, the neutrophil elastase
becomes unchecked/uncontrolled and will start attacking (has a preponderance to) the alveoli in the lung.
 It destroys the walls of the alveoli resulting in individuals primary manifestation of difficulty in
breathing.
 Other causes of emphysema: Smoking and Chronic bronchitis (eventually leads to emphysema)
 Diagnosis: Serum Protein Electrophoresis (SPE)
 In SPE: almost a complete absence of α-1-globulin fraction
o Increased levels (Acute phase reactant; seen in: Inflammation, Pregnancy, and Oral Contraceptive Pill use)
o Mutations
 Formation of abnormal forms that may accumulate in the liver and cause cirrhosis.
 Characteristic features in the liver: Presents as round to oval, intracytoplasmic eosinophilic inclusions in the hepatocytes.
 These mutated forms usually becomes impacted in the cytoplasm of hepatocytes.
 Seen as the very pink lobules inside the hepatocytes.
 α-2-Macroglobulin (1.5 to 3.5 g/dL)
o Largest non-immunoglobulin protein in the plasma, seen in the α-2 region of the electrophoresis
o Increased levels (seen in: Nephrotic Syndrome)
 Inversely proportional to albumin (hypoalbuminemia/decreased levels of albumin in blood is associated
with Nephrotic syndrome).
 Selective proteinuria- loss of low molecular weight protein with retention of the larger proteins
 Abnormal glomerular processes usually permeates only the low molecular weight proteins.
 It just selectively strains/excretes the smaller protein proteins that are found in the blood.
 α-2-Macroglobulin cannot be excreted in the urine because it is very large and is usually reabsorbs back in the
body, causing increased levels.
 Decrease in albumin because it is excreted in urine and increase in α-2-Macroglobulin because it is very large
and it cannot be excreted in urine.
 Haptoglobin (0.4 to 2.9 g/dL)
o Main function is to combine with hemoglobin after RBC lysis to preserve iron and protein stores.
o An α-2-glycoprotein synthesized by the liver o Acute phase reactant
o Increased levels (seen in: Stress, Infection, Acute inflammation, and Tissue necrosis)
o Decreased levels (seen in: Massive hemolytic episode, Hemolytic transfusion reaction, Thermal burns, Autoimmune hemolytic
anemia)
 Since its main function is to combine with hemoglobin after lysis, all of them are involved in lysis of RBCs. Because of the massive
lysis, all of the haptoglobins are being used (saturated), that is why they are decreased when testing.
o Testing is used primarily to help detect and evaluate hemolytic anemia and to distinguish it from anemia due to other causes.
 Transferrin (200 to 300 mg/dL)
o A glycoprotein and a negative acute phase protein. Also called siderophilin.
o Main function is to transport ferric ions from iron stores of intracellular or mucosal ferritin to the bone marrow.
o Measured as total iron binding capacity (TIBC)
o Increased levels
 Iron Deficiency Anemia- increased up to 2x its normal levels
 Hemochromatosis
 Can be caused by a hereditary mutation or a secondary event.
 In hereditary mutations causing hemochromatosis, there is a gene mutation involved.
 In primary hemochromatosis, more iron is absorbed than excreted.
 In secondary hemochromatosis, there is iron overload due to frequent blood transfusions.
 There is excess free iron in the blood in both of these disorders, resulting in oversaturation of the iron binding capacity. Due to this,
iron cannot be mobilized and cannot be excreted, therefore they are deposited into the different tissues of the body.
 Usually associated with bronze skin abnormalities in the liver. Presents with liver cirrhosis, diabetes, cardiomyopathy, arthritis,
and endocrine disorders (such as diabetes mellitus).
 In hemochromatosis, the body absorbs excess iron from food. It is also called bronze diabetes because in patients with increased
levels of iron in the blood, it can lead to darkening of the skin (bronze-like) and hyperglycemia (increased levels of glucose).
  Diagnosis: Screening of this disorder is recommended to halt the progression of the disease.
o Decreased levels
 Atransferrinemia
 There is congenital deficiency of transferrin in the blood.
 Rare disorder and is characterized by microcytic anemia and iron overload.
 Protein-losing Nephropathy
 Transferrin is lost from the circulation into the urine along with iron (similar to nephrotic syndrome).
 Fibrinogen (1.0 to 4.0 g/dL)
o One of the largest proteins in the plasma. o It is synthesized in the liver.
o It is the end target of the coagulation pathway to transform fibrinogen to fibrin in forming a stable clot.
o One of the acute phase reactants usually increased in plasma during acute phase of inflammatory process.
o Variants
 Dysfibrinogenemia
 Forms an abnormal fibrinogen molecule with an altered amino acid sequence.
 Associated with abnormality in clot formation
 Clinical Manifestations: hemorrhagic diathesis with increased tendency from thrombosis
 Congenital Afibrinogenemia (congenital absence of fibrinogen)
 No fibrinogen is synthesized
 Results in a hemorrhagic disorder (not as severe as in hemophilia disorders)
o Increased levels (Acute phase reactant; seen in: Inflammation, Pregnancy, or Oral Contraceptive Pill use)
o Decreased levels (seen in: Disseminated Intravascular Coagulation/DIC)
 A serious medical condition where in it is usually secondary to overwhelming sepsis, malignancy,
trauma, obstructive complications, or intravascular hemolysis.
 Extensive activation of coagulation factors.
 Eventually, there is consumption of fibrinogen.
 Always forming clots but one of the main clinical manifestation is bleeding, because eventually, when
all clotting factors are consumed, there is nothing to stop from bleeding.
 Diagnosis: Measurement of fibrin spit products (measured for assessment).
 Ceruloplasmin (20 to 40 mg/dL)
o Copper-binding protein, that is then synthesized in the liver. o Important function in iron metabolism.
o Contains most of the copper in plasma. o Migrates to the α-2 region of the SPE.
o Increased levels (Acute phase reactant; seen in: Inflammation, Pregnancy, or Oral Contraceptive Pill use)
 Wilson’s Disease
 One of the prominent conditions wherein ceruloplasmin is ordered along with urine copper test is to diagnose Wilson’s disease.
 An autosomal recessive disorder  Mutation in ATP7B gene
 Disordered copper metabolism- Hepatic excretion of copper in bile is impaired, leading to
accumulation of toxic levels of copper in the blood, and eventually will deposit into tissue.
 Organs involved: Liver, brain, cornea, kidneys, bones, and parathyroid
 Clinical Manifestations: Presence of Kayser-Fleischer ring in the cornea (most prominent),
Neurologic disorder, Osteopenia, and Liver Cirrhosis
 Diagnosis: Low ceruloplasmin and increased copper concentration in urine and liver.
 Treatment: Long term chelation with penicillamine or liver transplantation (in hepatic failure or cirrhosis)
 Fatty liver in Wilsons Disease- if left untreated, cirrhosis may ensue.
 C-Reactive Protein (100 mg/dL at birth; 170 ng/mL in children; 470 to 1340 ng/mL in adults)
o Acute phase reactant- first to rise in response to inflammation o Although not specific, valuable as a general indicator.
o High or increasing amounts suggests acute infection or inflammation.
o In inflammatory diseases (rheumatoid arthritis and SLE) used to assess effectiveness of treatment.
o Also increased in: Rheumatic fever, Bacterial infections, Malignancy, Gout, and Viral infections
 Complement
o Part of the complement system, which is one of the natural defense mechanisms that protects the body from infection.
o Synthesized in the liver
o Complement C3 is the most abundant protein in the human serum with, Complement C4 being the second.
o Increased: Inflammatory states o Decreased: Malnutrition, Hemolytic anemia
o Decreased C3 Levels: Neonatal respiratory distress syndrome, Bacteremia, Tissue injury, and Chronic hepatitis
o Decreased C4 Levels: DIC, Hepatitis, Nephritis, and SLE
 Immunoglobulins (Antibodies)
o Produced by B-cells o Involved in humoral activity
o These are glycoproteins and are categorized into 5 different types: IgM, IgD, IgA, IgG, and
IgE
o Increased levels (Gamma Gammopathy)
 Known as paraproteinemia, is the presence of excessive amounts
of myeloma protein or monoclonal gamma globulin in the blood.
 Seen as a spike in the γ region of the SPE, called as an M spike
 Usually caused by overwhelming infection or most commonly it is caused by abnormalities in
the plasma cells, such as multiple myeloma (a neoplastic disorder of plasma cells)
 Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Identify the parts and functions of kidneys
 Determine the different non-protein nitrogenous substances and their clinical implications
 Differentiate pre-renal, renal, and post-renal azotemia.

 Non-Protein Nitrogenous Compounds (NPNs) can also reflect the functions of the kidneys, that is why they are also called as the
kidney function tests.

NPNs vs Proteins
 Proteins- High in molecular weight, colloidal in nature (opaque and large, causes turbidity or haziness)
 NPNs- Low molecular weight, crystals in nature (do not cause turbidity or haziness to the specimen)

The Urinary System

 Kidneys- their main function is to filter the blood and attached to them are the ureters
 Urinary bladder- area where the urine is being stored
 Urethra- from where the urine is being excreted
 The blood from the heart will pass through the large artery known as the aorta, and it will be delivered towards the kidneys, via the
renal artery. Once the blood reaches the kidneys, it will be filtered so all toxic materials and unnecessary substances will be excreted
through the ureter, down to the bladder, and to the urethra. The filtered blood, which contains substances that are needed by the body
will be returned to the bloodstream via the renal vein.
 Kidneys only weigh less than 1% of the total human body, but it receives about 25% of cardiac output.
 The kidneys are full of blood vessels (artery, arterioles, and capillaries that surround the entirety of the kidneys)
o Example: Having approximately 1.1L/min of blood flowing through the kidneys. For a normal person who has about 5L of blood, it
would mean that within 5 minutes, all of the blood has already passed through the kidneys and it
has already been filtered before returning to the bloodstream.
 Parts of the Kidneys
o The blood coming from the heart will be delivered to the kidneys via the renal artery; the blood
is oxygenated because it comes from the heart.
o The renal artery will branch off into smaller vessels called the renal arterioles.
o Once the blood reaches the kidneys, it will be filtered and the reabsorption of ions and
necessary nutrients will also happen.
o All the filtered blood will be collected into a vessel called as the renal vein. From the renal vein, it will be delivered to the
bloodstream, and to the rest of the body. The blood coming from the renal vein has lesser oxygen (deoxygenated) because the
oxygen has already been used up in the filtration and the absorption process occurring in the kidneys.
o Outer part is the renal cortex, and the middle part is the renal medulla.
o Nephron- the functional unit of the kidney; situated between the renal cortex and the renal medulla
 First, it will be in the renal cortex, then it falls down to the renal medulla, back to the renal cortex, down to the renal medulla.
o The first part of the kidney where urine is present (comes into contact with the urine) is the tips called renal calyx (calyces).
o All the urine that was gathered from the renal calyx will be collected to the central part of the kidney called renal pelvis.
o The urine will be collected from the pelvis, down to the ureter, down to the urinary bladder, and then to the to the urethra.
o If there are tubes/vessels coming out in an organ (in this case the kidneys), it is called the hilum (hila). The renal artery, renal vein,
and the ureter together come out in a renal hilum (hila).
o Blood Flow in the Kidneys
 First, the renal artery will branch off into smaller arteries, called arterioles. The arterioles will meet the tuft of capillaries, called as
glomerulus. The glomerulus filters the blood.
 The arteriole that first meet the glomerulus (going towards the glomerulus and kidney) is specifically known as the afferent
arterioles, and the one that is coming out or has left the glomerulus is called as the efferent arterioles.
 The blood coming from the heart will be delivered to the renal artery, towards the afferent arterioles, towards the glomerulus, and
towards the efferent arteriole. Eventually, the efferent arteriole will turn into a capillary, then as
a venule, and eventually it will become as a renal vein. The renal vein will return the filtered
blood to the bloodstream, to the rest of the body.
o Fluids like sodium, glucose, amino acids, and other smaller molecules can leak out from the arteriole in the area towards the
bowman’s space (and it does not happen anywhere else in the body).
 Inside of the glomerular capillaries are smaller molecules (such as sodium, glucose) and large protein molecules (since they are
large, normally they cannot pass through the blood vessels). The lining of the capillary are filled with endothelial cells that are
fenestrated (contains fenestrations/holes/pores). The holes will allow smaller molecules to pass through the glomerular capillaries
toward the Bowman’s space, and other proteins are also allowed to leak through it, but larger proteins cannot leak towards the
Bowman’s space, because of another layer sits in between these endothelial cells (membrane, not a complete barrier) that is called
basement membrane.
  Basement membrane is semi-permeable and makes sure that small things can pass through the glomerular capillaries (e.g. sodium,
amino acids, and glucose) but the bigger proteins bounce back either because they cannot make it through the fenestration, or the
basement membrane prevents them from leaking into the Bowman’s space.
 Another layer called the tubular cells that make up the interaction point on the end of the Bowman’s capsule.
 Sometimes their structure looks like they are anchored to the endothelial cells so they have leg like
projections called as podocytes (podo- means food), which will help that the connection between
the endothelial cells and the basement membrane will stay close.
o The nephron (functional unit of the kidney) lies between the renal cortex and renal medulla, and each
kidney contains 1.5 millions of nephrons.
 Parts of the nephron: Main site of filtration is the tuft of capillaries known as Glomerulus,
Bowman’s capsule, Proximal Convoluted Tubules (PCT, proximal because it is near to the
glomerulus and is convoluted, because the structure has a lot of twists and turns), Loop of Henle,
Descending Limb, Ascending Limb, Distal Convoluted Tubules (DCT, distal because it is away
from the glomerulus), and Collecting Duct (contains many DCTs).
 The PCT absorbs a lot of electrolytes, such as the sodium and also other nutrients such as the glucose, and because sodium is
reabsorbed in this area, water is also reabsorbed in the PCT.
 In the Loop of Henle, the descending limb and ascending limb have an opposite direction and they also reabsorb different kinds of
things. The renal medulla is very salty because there are a lot of ions reabsorbed in this area.
 Descending limb- reabsorbs mainly water; it is permeable to water, but impermeable to ions
 Ascending limb- is permeable to ions, such as sodium chloride and potassium, but is impermeable to water
 Because of the opposing direction of the ascending and descending limb, there is a system called counter current multiplication
(counter current because the limbs of the Loop of Henle have opposing direction, multiplication because that means when ions are
reabsorbed in the ascending limb, it will make the medulla salty).
 By not reabsorbing water in the ascending limb, and only ions are allowed to be reabsorbed here, that drives water to be
reabsorbed passively in this descending limb (passive means the water is reabsorbed without the expenditure of energy), and in
the ascending limb, active transport is used to reabsorb ions, and by actively pumping ions into the medulla and no water is
reabsorbed in the ascending limb, the medulla will become very salty so the amount of water that are passive passively reabsorbed
in the descending limb can then be multiplied. All the substances that are reabsorbed in the kidney go to the space, called
interstitium/interstitial space.
 The DCT also reabsorbs other ions such as sodium and chloride (picks up more of the important nutrients needed in the
bloodstream). The DCT passes through or comes closely to the glomerulus, and when the DCT comes closer to the glomerulus, it
creates the structure known as the Juxtaglomerular apparatus, which is mainly responsible for controlling blood pressure.
 The collecting duct gathers all materials that has not returned to the blood, and dispose it as urine.
 From the nephron, the urine will go out and will touch first the renal calyces, down to the renal pelvis, down to the ureter.
 Water and Urea are also reabsorbed in the collecting duct.
 Urea is one of the main waste products of the kidney. It is reabsorbed in the collecting duct just to help maintain the osmolarity
of the medulla. It will drive again the passive reabsorption of water in the Descending Loop of Henle.
o Summary
 The blood coming from the Renal Artery will pass through the Renal Arteriole, which will
eventually pass through the Glomerulus and will be filtered in here, and the toxic substances
and unnecessary materials will be caught in the Bowman’s space down to the Proximal
Convoluted Tubules, down to the Descending Loop of Henle, towards the Ascending Loop
of Henle, towards the Distal Convoluted Tubule, and eventually to the Collecting Duct, and
that filtrate will now be discarded as urine.
 The filtered blood (those that contains necessary materials and substances needed by the body)
will pass through the Efferent Arteriole, and then that efferent arteriole will branch off into smaller capillaries, which will collect all
the remaining materials coming from the interstitium, because it surrounds the nephron and is called as peritubular capillaries.
Eventually, the peritubular capillaries will branch off as the renal vein. The renal vein will return the filtered blood coming from the
kidney, towards the bloodstream, to the rest of the body.
 Nephron
o Functional unit of the kidney
o Consists of glomerulus (mainly functions for filtration) and the tubules (mainly functions for tubular reabsorption
and tubular secretion)
o The glomerulus is wrapped in the Bowman’s capsule and they are collectively called as the renal corpuscle.
o The main difference between reabsorption and secretion:
 Tubular Reabsorption- all the materials in the tubules and the materials reabsorbed from the interstitium will be returned to the
blood (from the tubule to the blood)
 Tubular Secretion- from the blood towards the tubule
 Functions of Kidneys
o Excretion- elimination of metabolic waste products through the formation of urine; the job of the nephrons
 3 main functions of the kidneys: Glomerular Filtration, Tubular Reabsorption, and Tubular Secretion
o Synthetic- the kidney is capable of producing a lot of substances
 Substances produced by the Kidneys
  Erythropoietin- produced by the kidneys, but the action of erythropoietin is towards the bone marrow to increase the production
of RBCs
 Renin- regulates the water and sodium balance in the body, especially in low blood volume or low blood sodium; renin will be
activated in order to maintain homeostasis in these substances
 Prostaglandins- also have a function in maintaining the balance of the fluid and electrolyte in the body; prostaglandin also
functions for uterine contractions and also in lowering the BP
o Metabolic
 Inactivation of aldosterone, glucagon, and insulin.
 Aldosterone- its main function is it reabsorbs sodium and excretes potassium, so any defect/imbalances in aldosterone levels
will lead to imbalances in sodium and potassium.
 Glucagon- increases blood sugar  Insulin- lowers blood sugar
 Main site for the activation of vitamin D to its active form vitamin D3
 To make this happen, the vitamin D obtained from sunlight or the supplements that are taken, the kidney will transform it into its
active form, because without the kidney, the vitamin D cannot be used up by the body, since the vitamin D that is acquired from
the external environment is still inactive (low levels of vitamin D are expected in chronic kidney diseases)
 Formation of creatine (important for muscle contractions)
o The kidney is mainly responsible for:
 Acid-base balance  Toxin removal  Vitamin D metabolism
 Water balance  Blood pressure control
 Electrolyte balance  Erythropoietin production

Non-Protein Nitrogenous Compounds

 Constituents (in decreasing order, Mnemonic: All Underarms Create Cheesy Aroma):
o Urea o Uric Acids o Creatine
o Amino acids o Creatinine o Ammonia
 Urea (major NPN compound found in the plasma)
o One of the major products of excretion of protein catabolism.
 The body is unable to store proteins/amino acids so that means when excessive amount of proteins are ingested, the excess amino
acids produced from the digesting proteins are transported from the small intestine towards the liver.
o Urea is formed in the liver from CO2 and the ammonia (a toxic product) generated from the deamination of amino acids.
 The basic structure of an amino acid has a central alpha carbon, a hydrogen atom, an amine group, a carboxyl group and
the R-group. When the amino acids are absorbed by the liver cells, a series of chemical reactions begin:
 The amino acid is oxidized in the presence of an enzyme catalyst in the liver.
 Deamination- there is a removal of the amine (NH2) group, and together with the hydrogen atom (H)
 This reaction will produce a toxic product called as ammonia (product of deamination of the amino acid)
 Since ammonia is highly toxic, it is not allowed to accumulate. With the help of specific catalysts in the liver cells, the carbon
dioxide will react chemically with the ammonia that was produced. Once the carbon dioxide reacts to the ammonia, a less toxic
nitrogenous compound, urea is produced together with water.
o Over 90% is excreted, and partially around 10% are reabsorbed along with water.
o Readily filtered from the plasma by the glomerulus (an NPN, smaller in size as compared to proteins)
o One of the most popular tests for assessing renal function (but not that specific and sensitive)
o 70-80% of glomerular destruction must occur first before there is an increase in the level of plasma urea.
o Concentration of urea in the plasma is an indicator of renal function and perfusion (perfusion refers to the blood flow), dietary intake
of protein, and the level of protein metabolism (major product of protein catabolism)
o Measurement of plasma urea is further enhanced when the results are considered together with serum creatinine.
 Since urea is not that sensitive because 70-80% of glomerulus has to be destroyed before urea increases, unlike creatinine.
 Urea is greatly affected by the intake of proteins in diet, whereas creatinine is not affected by protein diet.
o Clinical Significance
 Azotemia- an elevated concentration of urea in the blood; divided into 3 types:
 Pre-renal azotemia
 Usually related to the renal circulation (the flow of blood to the kidneys)
 Normal renal function (the kidneys do not have any problem, the problem is on the flow of the blood towards the kidneys)
 In normal conditions, urea goes through the kidneys for excretion and the absorption. If there is a prerenal problem, the urea
doesn't make it to the kidneys to be filtered, so there is an increase or build-up of urea in the blood
 Diseases: Congested Heart Failure, Shock, Hemorrhage, and Dehydration
 These conditions slow down the flow of the blood towards the kidney
 Other related conditions:
 High protein diet (urea is greatly affected by protein consumption)
 Muscle wasting (in cases of starvation)
 Glucocorticoid treatment
 Increased protein breakdown (in cases of stress and fever)
 Renal azotemia
 Involves the kidneys- there is a lack of ability to function correctly
 Kidneys have a decreased ability to excrete substances (like urea)
  Diseases: Acute/chronic renal failure, Glomerulonephritis, Tubular necrosis, Chronic nephritis, and Polycystic kidney
 In these conditions, kidneys are affected so it has no capability to filter the urea, and the urea will stay in the blood
 Post-renal azotemia
 Usually, the function of the kidney is also normal, and there are no problems in the blood flow towards the kidneys.
 Obstruction in the flow of the urine from the kidneys
 Increased diffusion of urea from the renal tubules into the circulation (because there is obstruction/blockage and post renal
azotemia usually comes from the issues in the ureters and bladder)
 Diseases: Kidney stones, UTI, Tumors (In these conditions, urea cannot be excreted out to the urine, so it stays in the blood)
 Uremia- a very high plasma urea concentration in the blood, accompanied by renal failure (Uremic syndrome)
 Creatinine
o Main storage component of high energy phosphate needed for muscle metabolism.
o Anhydride of creatine- creatinine is formed once creatine loses water and is not reused in the body’s metabolism, that is why
creatinine is only a waste product
o Synthesized mainly in the liver from three amino acids: arginine, glycine, and methionine
o Filtered and secreted, not reabsorbed (only very little amount of creatinine in blood)
o Less affected by intake and excretion (such as the intake of proteins)
o Most commonly used in the assessment of glomerular filtration rate (GFR)
o Clinicopathologic Correlation (Increased Creatinine in blood in these cases)
 Skeletal muscle necrosis or atrophy.
 Creatinine is the anhydride of creatine, and the body content of creatine in normal men is proportional to the muscle mass.
 Decrease in glomerular filtration rate
o Creatinine Clearance
 Two specimens are needed here: Serum/plasma and a 24-hour urine specimen
 Example: A patient with 24 hour urine volume is 2000 mL and a creatinine level of 50mg/dL. The serum creatinine is 1.0mg/dL.
��
�� 50 � 200��
Determine the Creatinine Clearance. �� = ��1440 → 1.0����� 1440 ���� = 69 ��/���
��
 Uric Acid (Urate)
o The breakdown product of nucleic acid and purine catabolism in humans.
 Purines are normally found in foods such as the liver, dried beans, peas, and beer
o At pH 7.4, more than 95% of uric acid in the body fluids exist as monosodium urate.
o Uric acid formation occurs only in tissues that contain the enzyme, xanthine oxidase
 Xanthine oxidase is mainly responsible in purine metabolism and it will catalyze the oxidation of xanthine to uric acid
 The highest levels of xanthine oxidase are found in the liver primarily but it can also be found in the heart, pulmonary, and adipose
tissues. Uric acid formation could also occur in all tissues that contain xanthine oxidase
o 90% of the uric acid are reabsorbed through active transport. Uric acid is synthesized in the liver and intestine/intestinal mucosa.
o Clinicopathological Correlation
 GOUT- comprises of heterogeneous group of disorders such as:
 Hyperuricemia (increase uric acid)
 Attacks of acute inflammatory arthritis (inflammations in the hands, knees, foot (usually in the joints)
 Deposition of monosodium urate crystals throughout the body
 Nephrolithiasis or Kidney stones or calculi
 Ammonia (toxic to the body and is disposed as urea)
o Derived from bacterial action on the contents of the colon
o Metabolized by the liver normally (with the process of deamination and with the help of carbon dioxide and water molecule)
o Increase in plasma ammonia is very toxic to central nervous system
o Most ammonia is ultimately disposed as urea
o Clinicopathologic Correlation
 Altered ammonia metabolism occurs in severe liver diseases because ammonia cannot be removed by the liver, so it stays in the
blood and it is increased in the blood and could affect the central nervous system.
 Elevated in Reye’s Syndrome (in children)
 Acute encephalopathy associated with hepatic dysfunction but without hyperbilirubinemia
 Survival reaches to 100% if plasma ammonia concentration remains below 5 times the normal
 Normally, cause is unknown is usually associated with aspirin consumption, especially in children who were affected with viral
illnesses. In this condition, both the liver and the kidneys are affected.
 Amino Acids
o Readily absorbed in the renal tubules by active transport
o Only < 5% percent are excreted in the urine (most abundant after urea)
o Clinicopathological Correlation
 Increased urinary excretion of amino acids fall under 2 major types:
 Overflow aminoaciduria
 Usually caused by increased urinary excretion (of amino acids) because there is an increased plasma concentration.
 Acquired secondary or inborn error of metabolism.
 Renal aminoaciduria
 Diminished tubular absorption of amino acids
  Acquired secondary or inborn specific or nonspecific disorder of renal tubular absorptive mechanism
 The problem lies in the fact that the renal tubules cannot easily reabsorb amino acids, that is why there is increased urinary excretion
of amino acids.
 Lectured by: Alessandra Kamille P. Mallari, MD, DPSP
Non-Protein Nitrogen
 The term originated in the early days of clinical chemistry when analytic methodology required the removal
of protein from the sample before analysis.
 Although measurement of total urinary nitrogen is of value in the assessment of nitrogen balance for
nutritional management, more useful clinical information is obtained by analyzing the patient’s specimen for
the individual components of the NPN fraction.
 NPN fraction comprises about 15 compounds.
o Majority of these compounds arise from the catabolism of proteins and nucleic acids of the body (waste products).
o Most of the NPNs are often used to monitor renal function.
o Based on the common clinically measured NPN compounds, the majority is composed of urea (45 to 50% of plasma concentration in
blood), amino acids (25%), uric acid (10%), creatinine (5%), creatine (1-2%), and ammonia (0.2%).
 Creatinine
o Creatinine is formed from creatine and creatine phosphate in muscle, and it is excreted into the plasma from catabolism at a constant
rate related to the muscle mass (the higher muscle mass of the individual, the increased creatinine levels in plasma).
o Primarily used as a diagnostic indicator of kidney function, and the most widely used marker for glomerular filtration rate (GFR).
o PROS (Good measure for GFR)
 Endogenous substance with constant rate of production.
 Not bound to plasma protein, freely filtered by the glomerulus (the amount filtered=amount excreted)
o CONS (Not a perfect analyte in determining GFR)
 Rate of production is constant, but dependent on muscle mass; unreliable with severe muscle wasting (e.g. patients who are
chronically ill, bedridden, amputees who cannot walk)
 Muscle mass on each individual varies considerably and is widely known that men have more muscle mass than females, and the
younger generation have more muscle mass than the older population.
 Affected by diet
 Partially secreted by PCT (overestimation, about 10% creatinine is secreted or reabsorbed by the PCT).
o Plasma Creatinine Concentration
 The measurement of plasma creatinine concentration is used to determine the sufficiency of the kidney function.
 Function of: Relative muscle mass, Rate of creatine turnover, and Renal function
 Although reasonably stable, protein content in the diet does influence plasma levels.
o What is “Clearance”?
 Rate of urinary excretion of material to the plasma concentration of that material.
 Defined as the volume of plasma that would have been “cleared” of the substance.
o What is “Glomerular Filtration Rate (GFR)”?
 Glomerular filtration rate (GFR) is generally considered the best overall indicator of the level of kidney function.
 Acts as a colander/filter which filters out products that is found in the blood, and it filters out those analytes/products that should be
reused or recirculated in the body from those that are to be secreted.
 When the product is filtered out or passes through the glomerulus, it goes to the urine.
o Since creatinine clearance is freely filtered and 90% of the creatinine is excreted in the urine. Since the function of filtration is a
marker for kidney function, that is why creatinine clearance is widely used to determine the status of the kidney.
 The less creatinine excreted into the urine, the higher the creatinine levels will be in the plasma. If there is kidney damage, the
creatinine cannot be excreted into the urine so it accumulates in the plasma.
o Creatinine Clearance
 Plasma creatinine level and glomerular filtration rate (creatinine clearance) is inversely proportional.
 A damaged kidney can no longer filter the solutes or products in the blood, so it accumulates in the blood and the
clearance is decreased (inversely proportional).
 Measure of amount of creatinine eliminated in the body.
 Provides a reasonable approximation of the GFR, and thus gauges the renal function of the individual.
 Creatinine Clearance is reported in units of mL/minute, and can be corrected by body surface area.
 Overestimates GFR- a small amount of creatinine is reabsorbed (up to 10%)
 Since there are a lot of variables/factors to be considered in the levels of creatinine in the blood and the levels that the rate that
creatinine is excreted, there are 2 commonly used formulas in the correction of those variables.
o Estimated GFR (eGFR)
 Based on the Modification of Diet in Renal Disease (MDRD), which is an estimate of the GFR.
 The results are normalized to a standard body surface area of 1.73m2. The formula assumes and gives a standard body surface area
to all patients. The formula is valid only for patients 18-70 years old. It estimates the GFR of how much does the glomerulus filters
per minute in a person would with a standard of 1.73 m2.
 It was developed from non-hospitalized patients known to have chronic kidney disease.
 4 variables in the equation: Plasma concentration, Age of the patient, Gender/Sex of the patient, and Ethnicity
 As one grows old, the creatinine secretion is decreased because muscle mass decreases as one ages (but will depend on lifestyle
modifications and the gender of the patient).
 Men generally have more muscle mass than female (but some females have higher muscle mass than males)
 It is believed that African-Americans (Caucasians) have more muscle mass than patients who are Asians.
 o Cockcroft-Gault formula for Estimating Creatinine Clearance
Instead of measuring the rate of glomerular filtration, it measures the creatinine clearance.

 It measures the amount of creatinine excreted in the urine.
 Estimates the creatinine clearance.
 Similar to eGFR because it factors in the Age, and the Sex of the patient, but
it also factors in the Lean body weight of an individual
 Unlike in MDRD wherein it assumes a standard of 1.73m2 but not everyone
has a body surface area of 1.73m2, (MDRD overestimates the GFR)
 Same errors resulting from the variations of creatine production rate caused
by diseased states will be found in both formulas.
 Not applicable to children less than 18 years old, patients who are obese, bedridden, or amputees.
o The reference intervals are method dependent and vary among age and gender.
o Creatinine concentration decreases with age beginning the 5th decade of life.
 Urea
o Main waste product of nitrogen-containing chemicals in the body.
o In the urea cycle, urea is produced from the conversion of arginine to ornithine by the enzyme
arginase, and its rate of production is affected by food.
o Widely used as a measure of renal dysfunction; however measure of GFR is not very good
 Urea concentration depends on the renal function and rate of production (dependent on protein intake).
 Vegetarian patients have lower urea values than patients who are carnivorous and eat animal products (has higher protein values).
 Rate of protein intake varies widely.
o Blood Urea Nitrogen (BUN)
 Concentration of urea is expressed by the nitrogen content of urea (serum or urine urea nitrogen) = Blood Urea Nitrogen
 The normal level of urea in blood is measured as Blood Urea Nitrogen (BUN) = 8 to 23 mg/dL
 BUN: Creatinine Ratio
o In the absence of renal dysfunction and severe dehydration, urea clearances have only about 50% of creatinine clearance, and is
representative of only 50% of GFR because 50% of filtered urea is reabsorbed into the body.
 The bulk of the reabsorption takes place in the PCT, which amounts to 40% of its filtered load.
o Normal ratio of BUN: Plasma creatinine is 10 to 1 (value of BUN is much higher in the plasma than creatinine)
o Urea clearance underestimates GFR. o Creatinine clearance slightly overestimates GFR.
o Urea clearance is a better predictor of advanced renal dysfunction (than creatinine clearance), but it is highly insensitive for the
detection and monitoring of renal diseases.
 Creatine
o Creatinine is a byproduct of the metabolism of creatine in the muscle.
o Abnormal levels of creatine will relate to any muscular problems, because they are produced and originated from the muscle.
o Plasma and urinary creatinine levels are increased in: Muscular dystrophy, Poliomyelitis, Hyperthyroidism, and Trauma
o Measurement of creatine kinase- a diagnosis of muscle disease
o Plasma creatine concentration is not increased in renal disease
 Disease Correlation: Creatinine, BUN, and BUN: Creatinine Ratio
o Increased Creatinine
 Creatinine is produced from the conversion of creatine and creatine phosphate in the muscle, and it is excreted in the urine.
 Any abnormalities of the two will cause abnormal levels of creatinine. If there is abnormality of kidneys, it cannot filter out or
excrete creatinine and will increase creatinine levels in the blood.
 Any problems in the kidney, associated with abnormal renal function, and usually indicates renal damage
 Plasma creatinine is inversely proportional to clearance of creatinine
 Insensitive marker because it is only increased when more than 50% of the kidney function is deteriorated.
 The kidney is highly adaptable because when there are any abnormalities in one kidney, the others will compensate.
 In renal damage: Plasma creatinine is increased; GFR is decreased; Creatinine clearance is decreased
 Chronic Kidney Disease
 CrCl is widely used in staging patients with chronic kidney disease, because chronic kidney disease is diagnosed by 2 criteria:
1. If creatinine clearance is more than 90 mL/min/1.73m2
2. If kidney damage is present for more than 3 months
 Decreasing GFR means worsening of the kidney problem.
 End-Stage Renal Disease or End-Stage Renal Failure
1. If GFR is less than 15 mL/min or
2. If the patient is dependent on dialysis
 Patients in chronic dialysis cannot excrete metabolic waste products from the body, so they accumulate in blood and affect organs
so they go through dialysis so those metabolic waste products will be cleared out from the blood.
o Increased Urea
 Azotemia- elevated urea concentration in the blood (divided into 3 according to the cause of the increased urea).
 Uremia or Uremic Syndrome- very high plasma urea concentration accompanied by renal failure
 A condition that is eventually fatal if not treated by dialysis or concentration.
 BUN/Creatinine Ratio
 Less than 10:1- intrinsic renal disease  More than 20:1- hypoperfusion of the kidney (pre-renal failure)
  Prerenal Azotemia (Increased BUN; Normal Plasma Creatinine; >20:1 BUN: Creatinine)
 BUN: Creatinine ratio increases because BUN is the only one increased.
 Caused by reduced renal blood flow (renal hypoperfusion)
 Critically ill patient with renal hypoperfusion with normal tubular function
 Causative Factors: Congestive heart failure, Shock, Hemorrhage, Dehydration, High protein diet
 Any factors resulting in decrease in blood volume
 There is no problem in the kidney, but there is problem in the perfusion of the kidney, so no blood is going out to the kidney, and
no urea is to be filtered out (Less Blood flow= Less Urea filtered= Increased Urea in the blood)
 Increased protein catabolism
 Since urea is a waste product of purine protein metabolism, any events that has increased protein catabolism may increase urea
concentration. They have an increased urea concentration, thereby disrupting the BUN: Creatinine ratio.
 Usual causes: Stress, Fever, Major illness, Corticosteroid therapy, GI hemorrhage, individuals who have high protein diets
(especially those who are going to the gym and take protein shakes)
 Increased protein catabolism/Increased protein intake= Increased urea blood concentration
 Renal Azotemia (Increased BUN; Increased Plasma Creatinine; >15:1 BUN: Creatinine)
 Since the problem is in the kidney, both BUN and plasma creatinine will be increased because both of them can no longer be
filtered out by the kidney, thereby the BUN and creatinine ratio will be within normal range (since both increase together).
 Acute and chronic renal failure
 Due to decreased renal function- increase in plasma urea concentration secretion to compromised urea excretion
 Renal diseases (e.g. glomerular nephritis, tubular necrosis)
 Decreased renal function= Compromised urinary excretion= Increased plasma urea
 Postrenal Azotemia (Increased BUN; Increased Plasma Creatinine; >15:1 BUN: Creatinine)
 Due to obstruction of urine flow anywhere in the urinary tract
 Although there are normal levels in blood, kidneys can filter it out, but they cannot be passed out in the urine
 Obstructions in urinary tract (Renal calculi, Tumors of the bladder or prostate, and Severe infection) causes increased BUN
o Decreased Urea (Decreased BUN; Decreased Plasma Creatinine; >10:1 BUN: Creatinine)
 Major pathologic causes: Low protein intake, Acute tubular necrosis, and Severe liver disease
 Physiologic causes (normal in these patients): Late pregnancy, infancy (due to increased protein synthesis)
 Uric Acid
o A product of the catabolism of purine nucleic acid.
o Immediately produced by xanthine, which is acted upon by xanthine oxidase, forming the uric acid.
o Although it is filtered by the glomerulus and secreted/reabsorbed by the DCT in the urine, most uric acid
is reabsorbed in the PCT, and is mostly reused.
 Not a good measurement of renal function because they are not filtered out in the kidney.
o Relatively insoluble in plasma, and at high concentrations it can be deposited in joints tissues causing
inflammation.
o Physiologic conditions that can increase uric acid levels:
 Eating purine-rich foods (liver, anchovies, mackerel, dried beans, peas, drinking alcohol)
 Diuretic drugs
o Abnormalities in Uric Acid Levels: Hyperuricemia (increased uric acid levels) and Hypouricemia (decreased uric acid levels)
o Hyperuricemia
 Most commonly defined as serum uric acid concentration levels of:
 Men: >7.0 mg/dL (0.42mmol/L); Women: >6.0 mg/dL (0.36 mmol/L)
 Can be either due to increased formation or decreased excretion, can be primary or secondary
 Disorders in Purine Metabolism
 Inherited disorders, rare
 Usual symptoms:
 Kidney failure or stones in a child or young adult
 Gravel or sandy material in an infant’s diaper
 Unexplained neurological problems in an infant, child, or adolescent
 Gout presenting in individuals younger than 30 years old
1. Lesch-Nyhan syndrome
 High serum uric acid
 X-linked genetic disorder (females are mostly carriers) but usually seen in males.
 Complete deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT)- major
enzyme of purine salvage pathways
 Clinical Manifestation: mental retardation, abnormal muscle movement, behavioral problems
 The clinical manifestations is related to the abnormal or incomplete catabolism of such products.
 Infants (first few weeks)- crystalluria, acute kidney failure, gout, hyperuricemia, decreased activity
 Diagnosis
 Decreased activity of HGPRT (RBCs and fibroblasts)
 DNA technology during prenatal diagnosis (1st trimester)
2. Partial HGPRT deficiency
 Similar to Lesch-Nyhan, except that there is only partial deficiency. Severe X-linked gout.
  Usually present with early gout, kidney failure, or nephrolithiasis
3. PRPP Synthetase Superactivity
 Inherited as an X-linked recessive trait
 Increased intracellular PRPP activity causing increased uric acid concertation
4. Glucose-6-Phosphatase deficiency
 Both hyperuricemia due to overproduction and underexcretion of uric acid
 Gout
 Polygenic basis- with 99% cases of unknown ethology
 Characterized by occasional attacks and long periods of remission
 Due to:
 Metabolic overproduction of purine (thereby increased uric acid levels)
 Decreased renal excretion of patient with decreased renal tubular secretion (or reabsorption) of uric acid
 Increased dietary intake (e.g. patients who always eat peanuts)
 Gouty Arthritis (most common manifestation)
 Associated with urate crystals in joint fluid and deposits of crystals (tophi) surrounding the joint or other soft tissue.
 Uric acid is insoluble in plasma, so they usually deposit in these areas causing inflammation, and they exhibit the 5 cardinal
signs of inflammation (calor/heat, dolor/pain, rubor/redness, tumor/swelling, and functio lassa/loss of function)
 Monosodium urate crystals in synovial fluid- appears as square to rectangular elongated crystals under polarizing microscope
 Big toe (first metatarsophalangeal) joint- classic site
 First sign of gouty arthritis is usually inflammation of the big toe (big toe that is red, warm to touch, tumor-like appearance
and very painful) because deposition of uric acid crystals in the joint elicits an intense inflammatory response.
 It may also accumulate in other joints, as seen in the hand.
 May elicit an intense inflammatory response consisting of polymorphonuclear leukocytes (PMNs) and macrophages.
 Microscopic appearance: When these areas are biopsied, they appear as pink amorphous material in soft tissue which are
surrounded by polymorphonuclear leukocytes.
 Kidney disease with Hyperuricemia
 Since they are insoluble in water, so when they accumulate or they have high concentrations of
uric acid in the kidney, they may form renal calculi.
 Management
 Lifestyle management- Avoid food with high purine content; avoid drugs that affect urate excretion
 Nonsteroidal anti-inflammatory drugs (NSAID)- pain management and reduce inflammation
 Uricosuric drugs (probenecid, sulfinpyrazone)- enhance renal excretion of uric acid
 Allopurinol (choice of treatment for gout)- xanthine oxidase inhibitor (stops the formation of uric acid)
o Hypouricemia
 Less common than hyperuricemia
 Usual causes:
1. Defective hepatocellular disease with reduced purine synthesis or xanthine oxidase activity
2. Defective renal tubular reabsorption of uric acid
a. Fanconi syndrome
b. Secondary to injection of radiopaque contrast media
c. Chronic to exposure to toxic agent
d. Overtreatment with allopurinol, uricosuric drug or cancer chemotherapy (i.e. 6-mecaptopurine or azathioprine)
 In these cases, there is decreased reabsorption of uric acid in the blood so most of them are secreted into the urine.
 Ammonia
o Formed in the deamination of amino acids during protein metabolism.
 Amino acids and protein are deaminated in the tissues or gut, forming ammonia
 Ammonia will be processed in the liver and is converted to urea and excreted in the kidneys.
 If liver is damaged, ammonia level rises and because ammonia will passively diffuse into the CNS, it will cause mental objurgation
o Removed from circulation as urea, that is processed in the liver (only organ that can convert ammonia to urea for excretion)
o Free ammonia (NH4) is toxic (but found in low concentrations in the plasma)
o Severe Liver Disease (most common cause)
 If parenchyma liver cell function is severely impaired, ammonia is not removed from concentrations. High concentrations of
ammonia are neurotoxic, and often associated with encephalopathy.
 Ammonia is one of the analytes measured in patients with end-stage liver disease. Toxicity of ammonia maybe partially a result of
increased extracellular glutamate concentration and subsequent depletion of ATP in the brain.
 In severe liver diseases, monitoring blood ammonia levels used to determine the prognosis (high ammonia levels= poor prognosis)
 Arterial ammonia- better indicator of the severity of liver disease (than plasma ammonia)
o Reye’s Syndrome
 Common in children, can be fatal
 Preceded by viral infection and administration of aspirin
 Acute metabolic disorder of the liver with severe fatty infiltration (steatosis)
 White spaces are fatty infiltration in the liver.
 Since there is severe fatty infiltration in the liver, there is reduced or decreased liver function
 Ammonia levels are correlated with the severity and prognosis.
 Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Classify carbohydrates into their respective groups
 Determine the metabolism of carbohydrates in the body and the mode of action of hormones in carbohydrate metabolism
 Differentiate the types of diabetes mellitus by clinical signs and symptoms

Carbohydrates
 Carbohydrates are compounds of Carbon, Hydrogen, and Oxygen joined together to form molecules (shortcut: CHO)
o CHO stands for carbohydrates and CHON for proteins (proteins has nitrogen which is not found in carbohydrates)
o There are 2 molecules of Hydrogen and 1 molecule of Oxygen (just like in water and glucose/C6H12O6)
 The concentration of hydrogen is twice as much as those of the carbon molecules.
 Immediate source of energy, especially the brain, erythrocytes, and retinal cells of the eyes
o The nervous tissues cannot concentrate these carbohydrates nor store them, therefore it is very critical to maintain a steady supply of
glucose and other carbohydrates to the tissues.
 Serves as a major entry point to the metabolic pathway
o Any disturbances to the levels of carbohydrates may lead to some pathologic conditions, like diabetes mellitus.
 Fiber, which is also a form of carbohydrate, is essential for the elimination of waste materials and toxins from the body
o Fiber is very important because it decreases the absorption of cholesterol and bile.
o It also decreases intake of fatty foods by making us feel full (feeling of satiety)
 Carbohydrates come from the process of photosynthesis (from plant sources).
o The sun’s energy becomes part of the glucose molecule (its calories in a sense). The chlorophyll in the
plant captures the light energy coming from the sun, which is transformed into a chemical energy in
the form of ATP. This chemical energy is used to combine the CO2 and water in the environment to
form the glucose molecule. The byproduct of that process is oxygen, that is why plants could release
oxygen, and then the extra glucose is stored in plants as starch (storage form of carbohydrates in
plants).
 Classification of Carbohydrates
o Monosaccharides- building blocks of carbohydrates; sugars containing approximately 3-6 carbon atoms; reducing sugars
 Examples: Glucose, Fructose, Galactose, Ribose, Deoxyribose
 Glucose is considered as the central to carbohydrate metabolism because in a way the total carbohydrate used by the body is
measured when measuring for glucose because galactose and fructose are also converted to glucose before it can be used up by
the body. Example: In galactosemia, the patient will have a difficulty in converting galactose to glucose causing a buildup of
galactose in the blood of that patient.
 Ribose and deoxyribose are part of the structures of RNA and DNA, which are involved in genetic code
 Glucose, fructose, and galactose are hexoses because approximately they have 6 carbon atoms, and ribose and deoxyribose are
pentoses because approximately they have 5 carbon atoms in their structures.
o Disaccharides- are double structures of monosaccharides (2 monosaccharides per molecule of disaccharide)
 Examples: Sucrose (Glucose + Galactose), Maltose (Glucose + Glucose), and Lactose (Glucose + Galactose)
 They have two monosaccharides in their molecule, and each one of them has a glucose molecule
 Sucrose is used every day, common table sugar and it can also be found in honey. Maltose are usually found in grains. Lactose
are found in the dairy products and in milk (milk sugar).
o Polysaccharides- are long chains of monosaccharides (polymers of monosaccharides); most abundant type of carbohydrates
 Examples: Starch, Glycogen, and Cellulose
 Starch is considered as the storage form of carbohydrate in plants.
 Glycogen is considered as an animal starch, because it is a storage form of carbohydrates in animals.
 Cellulose is from the plants, not digested by humans, they just provide proper form or bulk in stool for proper intestinal
functioning.
 Dietary Sources of Carbohydrates
o The main dietary sources of carbohydrates are the starch and disaccharides.
o Cellulose (also a polysaccharide) remains undigested; it comes from the plants and it is not changed in the digestion process
 Its main function is it contributes to the bulk of stool, because unlike termites, we do not have necessary enzymes (such as
cellulase) to degrade this type of cellulose
o Glycogen is a type of polysaccharide. It is an animal starch and it is the excess carbohydrate stored in the muscles and liver.
 The glycogen in the muscles will be used up by the muscles only, whereas the glycogen in the liver will be used up by the cells
of the body because the liver has glucose-6-phosphatase (not in muscles), which is necessary for the metabolism of glucose.
 Glycogen is also known as endogenous carbohydrates, that means at those times when the body is not taking in carbohydrate
from eating, the glycogen will break down to form glucose to help maintain the proper blood levels of glucose.
 Meats are NOT good sources of carbohydrates because upon death, glycogen/animal starch easily disintegrates in the animal
body.
o Digestion of Starch (Polysaccharide)
 Starch- is a long branching chain of glucose molecules which are linked together.
  Amylase- necessary for the breakdown of this carbohydrate (only pancreatic amylase)
 Salivary amylase is not involved in the digestion of starch because it is inactivated by the acidity when it reaches the stomach.
 Digestion of starch occurs in the small intestine. When the food reaches the stomach and the intestinal tract, it will send
biochemical signals to the pancreas.
 Pancreatic secretions and bile from liver will neutralize the acidity in the stomach and will change the pH into a more basic one
to digest the starch.
o Digestion of Disaccharides
 Food is taken in as a polysaccharide, usually in the form of starch. Upon reaching the intestinal tract, these polysaccharides will
be broken down and will be converted into disaccharides, then into monosaccharides, and into simple sugar (e.g. glucose).
 For the digestion of disaccharides, there are enzymes which are produced in the walls of small intestine.
 Glucose + Fructose= Sucrose→ Sucrase  Glucose + Glucose= Maltose→ Maltase
 Glucose + Galactose= Lactose→ Lactase
 Role of Liver in Carbohydrate Metabolism
o Glycogenesis- Glucose is converted to glycogen for storage in the muscles and liver (buildup of glycogen)
o Glycogenolysis- Glycogen is converted to glucose (starvation)
o Gluconeogenesis- Production of glucose from new or other sources, such as non-carbohydrate sources (amino acids, fatty acids)
 This process is not only done by the liver, but also by the kidneys.
o Glycolysis- Conversion of glucose and other hexoses into pyruvate and lactate (process where we get energy sources)
o Lipogenesis- Conversion of carbohydrates into fatty acids
o Lipolysis- Breakdown or decomposition of lipids or fats
 Roles of Hormones in Carbohydrate Metabolism
o Some components of the endocrine system that produces these hormones is found in the alimentary system.
o Pancreas (2 general functions)
 Exocrine function (Digestion): Pancreatic juice with digestive enzymes that neutralize the acidity in the stomach to digest the
ingested carbohydrates.
 Endocrine function (Hormones)
 Insulin- the only hormone that decreases glucose concentration and helps keep it constant
 Hypoglycemic hormone; it ↓blood glucose level by ↑cellular uptake of glucose molecule
 Actions:
a. Increases glycogenesis and glycolysis: Glucose→ Glycogen→ Pyruvate→ Acetyl-CoA
b. Increases lipogenesis c. Decreases glycogenolysis
 Glucagon- raises blood glucose level (effect opposite that of insulin)
 Hyperglycemic hormone; it ↑blood glucose level by ↓cellular uptake of glucose
 Actions:
a. Increases glycogenolysis: Glycogen→ Glucose
b. Increases gluconeogenesis: Fatty acids→ Acetyl-CoA→ Ketone, Proteins→ Amino acids
 Somatostatin- plays a role in overall endocrine regulation, including growth and neurotransmission; inhibits
insulin, glucagon and growth hormones
 Pancreatic polypeptide- self-regulates pancreatic, endocrine, and digestive secretion activities
 Other Hyperglycemic Hormones
 Epinephrine (produced from the adrenal medulla)
 Inhibits insulin secretion, increases glycogenolysis and lipolysis
 Insulin lowers the blood glucose level, and when inhibited, this will result to increased blood glucose level
 Released during stress (considered as fight or flight hormone)
 Growth hormone- decreases the entry of glucose into the cell; increases glycolysis
 It decreases the cellular uptake of glucose. If the glucose cannot enter the cell, the glucose will
remain in the blood causing an increased blood glucose level.
 Adrenocorticotropic Hormone (ACTH)- stimulates the adrenal cortex to produce cortisol to increase
blood glucose levels (also through the process of glycogenolysis and gluconeogenesis)
 Cortisol- a major glucocorticoid with a diurnal variation (increased at 8:00 am, decreased at 4:00 pm)
 When getting a blood sample from the patient to examine cortisol level, there should be some time intervals
 Thyroxine- increases glucose (through glycogenolysis, gluconeogenesis, and it increases intestinal absorption of glucose)
 Islets of Langerhans
 Mainly responsible for producing the hormones necessary for carbohydrate metabolism
 Green- produces insulin; Beta-cells of the Islets of Langerhans  Blue- nucleus
 Red- produces glucagon; Alpha-cells of the Islets of Langerhans
 Was incidentally discovered in 1869 by a German medical scientist Paul Langerhans. The name
was coined to him and he was just 21 years old when he discovered this.
 The foods that we eat usually contain carbohydrates, proteins, fats, salts, fibers, and toxins. If there
were no means to control the glucose in your blood, it would reach toxic levels and would eventually
kill you, and that is where the pancreas comes into the picture.
 When eating, the body breaks down the food taken in into glucose (a monosaccharide that is the
body’s main source of energy), and as the blood glucose rises, the body will send signal to the
 pancreas (specifically to the beta cells of the Islets of Langerhans, which will release insulin), and acting as a key the insulin will
bind to the cellular receptors, which will unlock the cell so that the glucose can pass into it. The glucose entry into the cell is
called as the cellular uptake of glucose and inside the cell, the glucose is used for energy right away.

Blood Glucose Regulation

 Blood glucose goes up and down throughout the day
o As your blood glucose rises (after a meal), the pancreas releases insulin to regulate the blood glucose level and to lower it returning
to its baseline level.
 Insulin is necessary so that the glucose can enter the cells of the body (adipose tissues, muscles, and liver cells) and this is in the
bloodstream wherein after eating, the blood glucose rises so in order to lower the blood glucose, it must be transferred to the cells of
the body to be used as an immediate energy source.
 When the insulin binds to the cellular receptors, there will be a conformational change that will happen
which will send signals to the nucleus of the cell to release the glucose transport channels (which
enable the entry of glucose from the bloodstream into the cell) and once inside the cell, the glucose can
now be used as an immediate energy source. With the help of pancreas and insulin, the blood glucose
level is regulated.
 Normal feedback loop of the body in regulating the blood glucose level
o When eating, the food is broken down into simple sugars and digested in the small intestine. The blood glucose level then increases,
which will eventually send signal to the pancreas to produce insulin, returning the blood glucose to normal. When the blood
glucose is normalized, the insulin will return to its baseline level.
o Hours after eating, the food is gone so the blood glucose decreases and during this time, the alpha cells of the Islets of Langerhans
will produce glucagon to normalize the blood glucose level, eventually returning back the glucagon to its baseline level.
 When feedback loops fail (if body produces very little amount of insulin only or if the pancreas cannot
produce insulin at all), a disease is developed called diabetes mellitus (DM).
o Type 1 DM (Juvenile onset Diabetes/Insulin-dependent DM)- Frequency: 10-15%
 Results from the body’s failure to produce insulin, and presently requires the person to inject
insulin.
 The pancreas cannot produce insulin, so glucose cannot be utilized as energy source.
 Destruction of beta cells due to a virus or autoimmunity attacking the pancreas.
 Onset is usually in early childhood.
 Usually with diabetic ketoacidosis (Since there is no insulin production, so glucose cannot be used as an energy source, so the
body will use the fats instead, and the fats are broken down, and the result is keto acid or the ketone bodies).
 Control by oral medication: No  Absolute insulin deficiency.
 Insulin injection: Yes
 Genetic basis: Possibly
o Type 2 DM (Adult onset Diabetes/Non-Insulin-dependent DM)- Frequency: 85-90% (most common form)
 Insulin resistance- cells fail to use insulin properly; sometimes combined with an insulin deficiency as well
 Pancreas doesn’t make enough insulin or the body does not use insulin correctly (insulin resistance)
 The pancreas can produce insulin, however the cells are resistant to the insulin production.
 The insulin resistance in the peripheral tissues or cells is frequently associated with aging, family history of diabetes, obesity, and
failure to exercise.
 Usually manifests symptoms such as hyperglycemia and hyperinsulinemia.
 Onset is usually in adulthood.
 Rare ketoacidosis  Relative insulin deficiency.
 Control by oral medication: Yes (at beginning)
 Insulin injection: Not in the beginning; however, as the disease progresses, insulin is frequently needed to control blood glucose
levels
 Genetic basis: Definitely
o Gestational DM- occurs during pregnancy
 Glucose intolerance during pregnancy  Due to metabolic and hormonal changes
 Pregnant women who have never had diabetes before developed a high blood glucose level during pregnancy.
o Type 3 DM- associated with Alzheimer’s Disease
 Corresponds to a chronic insulin resistance, but in this case there is an insulin deficiency state that is largely confined to the brain.
 The results from the studies of the Journal of Diabetes Science and Technology provides strong evidence in support of the
hypothesis that Alzheimer’s disease represents a form of diabetes mellitus, that selectively afflicts the brain cells.
 This study also showed that Type 2 DM was not deemed sufficient to cause Alzheimer’s disease, although it could possibly serve
as a co-factor in the pathogenesis of Alzheimer’s disease.

Signs and Symptoms of Diabetes Mellitus

 Type 1 Diabetes Mellitus
o Insulin is not produced either because of virus, or autoimmunity, or any other factors that might destroy the beta cells of the Islets
of Langerhans. There is a continued elevation of blood glucose level while the insulin level remains low (this is why a patient needs
to inject insulin because the pancreas cannot make its own insulin)
 o Appear suddenly o Weakness, tiredness
o Frequent urination (polyuria) o Feeling edgy
o Extreme hunger (polyphagia) o Nausea, vomiting
o Extreme thirst (polydipsia) o Blurred vision
o Extreme weight loss
o Treatment:
 Sugar controlled healthy diet  Regular testing of blood sugar level
 Exercise; daily insulin injection  Urine ketone test (if blood sugar level is too high)
 Type 2 Diabetes Mellitus
o The pancreas can produce insulin, but the cells of the body becomes resistant to it
o Sometimes, this is also due to fat deposits or obesity, but it can also happen in people with a healthy normal weight.
o Elevated levels not only of glucose but as well as of the insulin. As the blood glucose continues to rise, the insulin will be produced
from the pancreas as a response to increased blood glucose levels, but there will come a point in time where the pancreas will get
exhausted or be destroyed and waiver out, resulting to decreased insulin levels (patient needs insulin injection).
o Appear slowly and person may not feel any symptoms at o Increase appetite
all o Weight loss
o Increase thirst o Repeated or hard to heal infections of skin, gums,
o Increase urination vagina or bladder
o Feeling tired and edgy o Blurred vision
o Nausea o Tingling or loss of feeling in hand or foot
o Treatment:
 Sugar controlled healthy diet  Diabetic pills or insulin shots (some cases)
 Exercise  Regular testing of blood sugar levels
 Keep in normal weight range
 Symptoms
o Increase urination (Polyuria)- happens because as the glucose in the blood increases, the body tries to compensate it and decrease
its level by the kidney (eliminating excessive amounts of glucose in the blood through the urine output)
o Excessive thirst (Polydipsia)- as the kidney tries to filter the glucose in the blood, the water goes along with it and a lot of water is
lost, that is why the patient may feel thirsty most of the time
o Tired and excessive hunger (Polyphagia)- since the blood glucose cannot be used up by the cells of the body, the patient might
feel tired, lethargic, or unable to do daily routines because of lack of energy
o Thrush or genital itching- since the urine contains a lot of glucose, this creates a lot of bacteria to thrive in the genital region
o Slow healing of wounds- when there is a lot of glucose in the blood, the viscosity of the blood increases, and it also affects the flow
of oxygen all throughout our body, and also this is a suitable environment for the bacteria to grow
o Blurred vision- the glucose does not only build up or accumulate in the blood, but it can also stay and build up in the lens of the
eyes and causing the liquid of the eyes to become cloudy resulting to blurred vision or worse blindness
o Weight loss- very particular in T1DM because glucose cannot be utilized by the body as an immediate source of energy, so instead
the body will use fats, causing ketoacidosis, and that is why ketone bodies are seen in blood, leading to diabetic ketoacidosis
o Chronic complications of Diabetes: Blindness, Kidney disease, Nerve damage, Cardiovascular diseases, Stroke, Heart attack,
Loss of circulation in arms and legs, that could lead also to Amputation
 Lectured by: Alessandra Kamille P. Mallari, MD, DPSP

Hyperglycemia
 Diabetes
o A group of diseases in which blood glucose levels are o Leading cause of treated end-stage renal disease
elevated o most common cause of non-traumatic amputations.
o Most common set of disorder of carbohydrate metabolism
o Diabetic neuropathy occurs about 60-70% of people with diabetes. o Foremost cause of new blindness in adults.
o Most diabetes-related deaths however are related to the increased risk of developing atherosclerotic disease.
o People with diabetes are at least 2-4 times more likely to have heart and
cardiovascular diseases than those without diabetes.
o Diabetes is a very impactful and widely common disease today.
o According to WHO, diabetes has been in the rise, with about 3.7 million
deaths are attributed to diabetes and high glucose levels, and 1.5 million
of that is caused by diabetes.
o 422 million adults have diabetes and that is about 1 person in 11
developing diabetes.
o Down syndrome- primary genetic syndrome associated with diabetes
 Diabetes Mellitus
o Group of metabolic disorders sharing a common feature: hyperglycemia
o Caused by defects in insulin secretion or insulin action, or most commonly, both.
o The chronic hyperglycemia and attendant metabolic deregulation may be associated secondary
damage in multiple organ systems: Kidneys, Eyes, Nerves, and Blood vessels
o In the U.S., diabetes is the leading cause of end-stage renal disease, blindness and non-traumatic extremity amputations.
o Although all forms of diabetes have hyperglycemia as their common feature, the underlying abnormalities involve in its
development varies and can be divided into 2 major forms of diabetes:
 Type 1 diabetes- related to β-cell destruction that leads to absolute insulin deficiency
 Type 2 diabetes- a combination of insulin resistance and β-cell dysfunction; further classified based on underlying abnormality.
o Glucose Hemostasis (any derangement in these three processes will eventually lead to diabetes)
 Glucose Production in the Liver- Gluconeogenesis  Glucose Uptake and Utilization by Peripheral Tissues-
Glycolysis
 Actions of Hormones: Insulin and Glucagon
 Insulin and Glucagon are produced by the endocrine part of the pancreas. They have opposing effects on glucose hemostasis.
 Fasting State
 Low Insulin and High Glucagon  Decreased Glycogen
 Hepatic gluconeogenesis (formation of glucose in the liver) and glycogenolysis (glycogen breakdown in the liver) to maintain
normal glucose levels in the blood since in fasting, no glucose is taken from the diet, so in the blood, there are no glucose
levels.
 Prevents Hypoglycemia
 After a Meal
 High Insulin and Low Glucagon
 Since there are high glucose levels in the blood, insulin levels increases so that those glucose will be absorbed into the
tissues.
 Low glucagon levels since additional/backup glucose from the liver is not needed.
 Insulin promotes glucose uptake and utilization by the liver cells so that it can be formed to glycogen (storage form of
glucose)
 If it is not used by the body, the glucose will float around unused in the blood, resulting into high glucose levels
(hyperglycemia), while glucagon helps maintain hypoglycemia by acting up or mobilizing glycogen storage in the liver.
 Skeletal muscle is the major insulin responsive site.
 Prevents Hyperglycemia
 Insulin
 Most potent anabolic hormone, with multiple synthetic and growth promoting effects.
 Since it is a pro-synthesis hormone: glycogen synthesis, protein synthesis, lipid synthesis,
etc.
 Anabolic effect of insulin are attributed to the increased synthesis of reduced degradation of
glycogen, lipid, and proteins.
 Insulin does not only act on the striated muscle cells, but it will also act in the liver and
adipose tissue
 Skeletal muscle cell- promotes ↑glucose uptake, ↑glycogen
synthesis, and ↑protein synthesis
 Liver- promotes ↑glycogen synthesis, ↑lipogenesis, and
↓gluconeogenesis (genesis- producing)
  Adipose tissue- promotes ↑glucose uptake, ↑lipogenesis, and ↓lipolysis (lysis- breakdown)
 To increase the rate of glucose transport into the cell.
 Target: Striated muscle cell and Adipocytes
o Long before, Type 1 was called juvenile diabetes or insulin dependent diabetes, because they said this is more common in
younger population and are insulin-dependent. That term is no longer used because eventually even Type 2 diabetes will require
insulin.
o As lifestyle changes, not only adults develop Type 2 diabetes. More younger people are developing Type 2 diabetes.
o Type 1 Diabetes Mellitus
 Autoimmune disease in which islet destruction
 Caused primarily by immune effector cells (antibodies) reacting against endogenous β-cell antigens in the pancreas.
 An autoimmune disease that destroys the β-cells in the pancreas, which produces insulin.
 Commonly develops in childhood, becomes usually manifests at puberty, and it progresses with age.
 Risk Factors:
1. Genetic susceptibility: Abnormalities in the HLA cluster- usually 50% of the genetic susceptibility
2. Environmental factors: Largely unknown, contributes to only a part of diabetes risk
 Pathogenesis
 Fundamental immune abnormality in Type 1:
 Failure of self-tolerance in T cells specific for islet antigens→ β-cell destruction→ Diabetes
 Self-tolerance is the basis of majority of autoimmune diseases. In the blood, T cells are trained by the body not to identify or
to attack oneself. But in autoimmune diseases, those T cells become haywire and they start attacking the individual cells.
 In Type 1, our own T cells attack specific antigens or recognize specific antigens within the pancreatic β-cells, and start
attacking them, leading to β-cell destruction, that will cause
hyperglycemia, and eventually diabetes.
 While the clinical onset of type 1 is abrupt (occurs immediately), there is
usually a lengthy lag between the initiation of the immune process, and
the appearance of a symptomatic disease.
 It starts in childhood, but symptoms present during pre-adolescent or
adolescent, because it is an autoimmune disease, so it does not destroy β-
cells at one time, but it slowly destroys the β-cells so symptoms are
usually undetected early on in life and are divided into 3 stages:
 Stage 1: β-Cell autoimmunity, Normogylcemia, Presymptomatic- The immune system will recognize the β-cells in the
pancreas, however there is still normal blood glucose levels (pre-symptomatic stage, nothing happens), no derangement of
blood, and the functional β-cell mass (β-cells in the pancreas) are still 100% and still not destroying β-cells.
 Stage 2: β-Cell autoimmunity, Dysgylcemia, Presymptomatic- Derangement in blood glucose levels in the blood, decline
in the functional β-cell mass in the pancreas. The abnormalities in blood are correlated with the functional β-cell in the
pancreas. The more β-cells are destroyed, the higher the derangement in blood becomes.
 Stage 3: β-Cell autoimmunity, Dysgylcemia, Symptomatic- Patients are symptomatic in this stage because there is severe
loss of β-cells in the pancreas. Manifestations of the disease typically appear when more than 90% of β-cells are destroyed.
 The β-cells can still control the blood glucose levels if you still have 20% of your β-cells.
o Type 2 Diabetes Mellitus
 Involves the interplay of genetic factors and environmental factors, and a pro-inflammatory state.
 Unlike type 1, there is no evidence of an autoimmune basis.
 Genetic Factor
 Susceptibility contributes to the pathogenesis is evidenced by concordance between twins.
 There is some familial correlation in the development of diabetes, but it is not the only basis.
 Patients who have “ kalahi ng diabetes ” will have an increased risk but it does not mean that the person will develop diabetes.
 Environmental Factor (most important factor)
 Most important environmental factor for type 2: Obesity (central or visceral obesity)
 Obesity contributes the cardinal metabolic abnormality in diabetes and to insulin resistance early in the disease.
 Sedentary lifestyle
 Must be prevented; typified by a lack of exercise which is another risk factor for diabetes that is independent of obesity
 Metabolic syndrome- combination of obesity, hyperglycemia, increased serum cholesterol, triglyceride, and hypertension
 Interrelated with diabetes and increases risk of atherosclerotic disease, that will lead to myocardial infarction.
 Pathogenesis
 Insulin Resistance
 Decreased response of peripheral tissues, especially skeletal muscle, adipose tissue, and liver to
insulin.
 Insulin is still produced but the cells that will accept or utilize glucose are not using glucose properly.
 Failure of the target tissues to respond normally to insulin.
 The liver, skeletal muscle, and adipose are the major tissues where insulin acts on, and this is also the
key players involved in the insulin resistance. When there are high glucose levels circulating in the
blood, for it to be absorbed by skeletal muscle and adipose tissue, insulin is needed.
  When there is insulin resistance, those glucose in the blood are not utilized or used by the those target organs, therefore there
will be increased glucose levels in the blood. The glucose will remain in the blood.
 High fasting blood glucose
 In insulin resistance, there is failure to inhibit endogenous glucose production.
 Insulin promotes gluconeogenesis in the liver, so without insulin prompting the liver to stop producing glucose, the liver
itself does not stop producing its own glucose, thereby increasing fasting blood glucose levels (since in the fasting state, the
body is not supposed to produce glucose, but is supposed to produce glycogen and glycogenolysis)

 High postprandial glucose

 There is failure of glucose uptake and glycogen production in the skeletal muscle following a meal, which will contribute
to high postprandial blood glucose levels.
 High circulating free fatty acids
 There will be failure to inhibit activation of hormone-sensitive lipases in the adipose tissue that will lead to excess
triglyceride breakdown in adipocytes and high level of circulating free fatty acid.
 How obesity propagate or facilitate insulin resistance
 In obesity, the increased number of vasculature and adipocytes, because there are increased number of fat cells in the body,
therefore, there are increased blood vessels in the body. In obese patients, there is increased adipokines, free fatty acids,
and inflammation in the blood that will also contribute to insulin resistance.
 β-cell Dysfunction
 Inadequate insulin secretion in the face of insulin resistance and hyperglycemia (develops later on).
 While insulin resistance by itself can lead to impaired glucose tolerance, β-cell dysfunction is a requirement for the
development of overt diabetes (diabetes itself cannot ensue without β-cell dysfunction). These two abnormalities are important
to each other.
 β-cell dysfunction increases early on the disease process, because patients with sporadic or type 2 diabetes, a compensatory
measure to counter insulin resistance is seen to maintain euglycemia.
 When there is insulin resistance, there is increased glucose in the blood.
 The pancreas will increase production of insulin since the body is sensing increased glucose
levels, since glucose is not used and nobody is stopping the liver to produce glucose. The main
goal or first reaction of the body is to increase insulin, thinking that there must be low insulin
levels so insulin must be produced to maintain the normal blood glucose level. This is
exhibited or seen in the blood as normal to impaired glucose tolerance.
 However, as the disease progresses, β-cells will become exhausted, since they are always
producing more than their own capacity. They fail to adapt to the long-term demands posed by
insulin resistance.
 Eventually, the hyperinsulinic state will give way to a state of relative insufficient deficiency,
and insulin levels then become deficient to maintain normal glucose levels in the blood.
 Eventually, insulin will decrease in number because they cannot maintain high glucose levels
for long. When this happens, diabetes mellitus now develops.
o Sequence of Metabolic Derangements underlying the clinical manifestations of diabetes (For type 1 and type 2)
 Insulin deficiency (type 1) and insulin resistance (type 2) will inhibit uptake of glucose in the adipose tissue, muscle, and uptake
of glucose and utilization in the liver.
 Since glucose is not being used, the glucose will spill over into the blood, leading to hyperglycemia.
 Adipose tissue: Since it does not have glucose to use, there is increased lipolysis, which will also increase free fatty acids in the
blood, and causes polyphagia (falsely senses that the person is starving).
 Muscle: Since there is no glucose available, this will lead to increased protein catabolism (e.g. amino acids), and this also
increases lipolysis and catabolism of protein, which will lead to polyphagia (body will sense and will want to eat more because
the cells in the organs are not being fed properly)
 Liver: Since nothing stops the liver to produce its own glucose, and there will be glucagon excess, which causes uncontrolled
gluconeogenesis, that will also contribute to hyperglycemia. Due to increased free fatty acid in the blood, this will contribute to
ketogenesis in the liver, that will eventually lead to ketoacidosis.
 Kidney: Hyperglycemia in the blood and increased levels of ketones will affect the kidney, and they pull with them water, so
there will be high ketones and high glucose in the urine, which will result to polyurea (the patient will keep on urinating because
of the high levels of ketones and glucose and increased loss of water), and also there will be volume depletion, which results to
polydipsia (patient becomes thirsty).
 Combining volume depletion with increased ketones in the blood will cause a development of diabetic coma.
o Clinical Manifestation
 Seen in obese patients older than 40 years of age (type 2, not the rule anymore because type 2 diabetes is common in young
patients nowadays), or in adolescent (type 1).
 First clinical manifestations: Unexplained fatigue, dizziness, or blurred vision
 Most frequently the diagnosis of routine blood testing in asymptomatic persons.
 Diabetes is one of the silent killers, because patient will feel fine and will not have any main problem when there is
hyperglycemia.
 Since most of the patients are asymptomatic, patients usually go undiagnosed for years or decades before they see a problem.
  End organ problem is the end stage of diabetes
 Acute Metabolic Complications of Diabetes
 Hypoglycemia (most common)- usually due to overtreatment or the clinician gives a wrong or inappropriate diabetic drug
 Diabetic ketoacidosis- due to increased circulating free fatty acid, not as severe/common than in type 1
 Increased fatty acid in the blood because the adipose does not have glucose, so it thinks that it is starving. It breaks down
adipose tissue to produce energy, producing a free fatty acid.
 Chronic Complications
 Diabetic Macrovascular Disease- increased risk of myocardial infarction, stroke, and lower extremity ischemia
 Involves larger blood vessels  Diabetic foot gangrene- infection in the foot
 Number one cause of amputation in the US is due to diabetes.
 In diabetes, eventually there will be improper wound healing, decreased blood flow to an organ, especially in the foot.
 There will be decrease sensations in the foot because of diabetic neuropathy (main pathology)
 In diabetic neuropathy, the nerves does not function properly anymore and no sensation can be felt in the foot.
 When diabetic individuals are wounded→ they will not notice the wound→ wound will grow until the whole foot becomes
infected→ Diabetic gangrene (infection in the foot)→ Amputation
 Older individuals are not advised to wear closed shoes, because if they get wounded in the feet, they will not feel it.
 Increased risk in myocardial infarction and stroke because there is increase in lipogenesis (increase in triglycerides) in the
blood that will also contribute to the atherosclerotic plaque.
 Diabetic Microvascular Disease- seen in the retina, kidneys, and peripheral nerves,
resulting in diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy
 Diabetic neuropathy- high glucose levels will destroy the glomerulus in the kidneys,
resulting in the derangement in urine function
 Example of long-term complications of diabetes:
 Brain- microangiopathy, cerebral vascular infarcts, and hemorrhages
 Eyes- retinopathy, cataracts, and glaucoma (in the US, the #1 leading cause of blindness
is diabetes)
 Contributes to hypertension, which will contribute to myocardial infarction, cerebral
cardiovascular infarcts, and hemorrhages.
 Heart- atherosclerosis will contribute to myocardial infarction
 Kidneys- nephrosclerosis (scarring in the kidney), contributed by atherosclerosis; infections in the kidney
 Foot- develop into gangrene (due to poor vascular circulation due to atherosclerosis, which leads to diabetic gangrene of the
foot, and eventually amputation)
o Diagnosis
 Fasting Plasma Glucose, 2-Hour Plasma Glucose Level, and HbA1C
 Prediabetes: HbA1C of 5.7-6.4%
 Impaired fasting glucose: Abnormality in the fasting plasma glucose
 Impaired glucose tolerance: Abnormality in the glucose tolerance test
 Combining these 3 will have diabetes
o Gestational Diabetes (GDM)
 Normally associated with increased insulin resistance, particularly in the late 2nd and 3rd trimesters.
 Euglycemia is maintained by increased insulin secretion, with GDM developing in women who fail to augment insulin
sufficiency.
 Main pathogenesis: Normally, pregnant people will have increased insulin resistance. The normal reaction of the body is supposed
to increase that insulin production, but if the person cannot increase or meet that demand, gestational diabetes ensue.
 Increased risk: Still birth, Congenital malformations (fetus), Excessive birth weight
(macrosomia), and Long-term sequelae (for the infant, causing obesity and diabetes in the future)
 Risk factors: Family history (1st degree relative with DM), Obesity, Advanced maternal age,
Glycosuria (+ glucose in urine), and Adverse outcome in the previous pregnancy (stillbirth,
macrosomia)
 Screening and Diagnosis of Gestational Diabetes Mellitus
 Screening
1. Perform at between 27 and 28 weeks’ gestation on all average and very high-risk pregnant
women not identified as having glucose intolerance.
2. Give 50 g oral glucose load without regard to time of day or time of last meal.
3. Measure venous plasma glucose at 1 hour
4. If glucose is >140 mg/dL (7.8 mmol/L) (cutoff of 130 mg/dL (7.2 mmol/L) for some), perform
glucose tolerance test.
 Diagnosis (in any abnormality, patient will be diagnosed with GDM)
1. Perform in the morning after an overnight fast of at least 8
hours
2. Measure fasting venous plasma glucose.
3. Give 75 or 100 g of glucose orally.
4. Measure plasma glucose hourly for 3 hours (or for 2 hours if 75 g of glucose is given).
 5. At least two values must meet or exceed the following:
6. If results are normal in a clinically suspect situation, repeat during the third trimester.

Hypoglycemia
 Abnormality in the glucose utilization and glucose production.
 Imbalance in the utilization of the brain, RBCs, muscle, kidney, production of the liver, and diet.
 Rate of utilization is more than the production.
 Neurogenic Symptoms
o Tremulousness, palpitations, and anxiety are catecholamine mediated.
o Diaphoresis (sweating), hunger, and paresthesias are related to acetylcholine release.
 Neuroglycopenic Symptoms
o Dizziness, tingling, difficulty concentrating, blurred vision, confusion, behavioral changes, seizure
 Whipple Triad
o A triad to diagnose hypoglycemic disorders, and is composed of:
1. Low plasma glucose
2. Symptoms related to low plasma glucose
3. Correction of the symptoms when treating hypoglycemia
o Important tool in assessing patients with episodes of suggestive low plasma glucose.
o Seen in pathologic disease states and hospitalized patients, basis used to monitor if patient is hypoglycemic or not.
o Low plasma glucose and relief of symptoms with correction
 Severe Medical Illness
o Widespread hepatic disease and severe cardiac failure- Due to impaired gluconeogenesis→ hypoglycemia
o Low muscle mass, spinal muscular atrophy (bedridden)- Due to prolonged fasting, poor alanine availability→ hypoglycemia
o End stage renal disease- Defective gluconeogenesis, poor nutritional status
 Drug-Induced Hypoglycemia
o Several drugs can cause hypoglycemia- manifest as altered mental status (primary causes are Insulin and Sulfonylureas)
o Most cases of drug-induced hypoglycemia: have been described in patients with diabetes mellitus who are on glucose-lowering
medications (due to improper medication or over
administration)
 Alcohol-Induced
o Inhibit hepatic gluconeogenesis and increase glycogen
phosphorylase
o Seen in alcohol intake of 50-300g of alcohol without food
intake (6-36 hours)
 Endogenous Hyperinsulinism
1. Insulin-secreting beta cell tumors (insulinoma) or disease states
where there is increased insulin
2. Congenital hyperinsulinism
3. Autoantibodies to insulin
 Autoimmune-related Hypoglycemia
o Autoimmune insulin syndrome (AIS)
 Antibodies vs endogenous insulin or insulin receptor
(antibody attacks endogenous insulin or insulin receptors)
 Recent ingestion of sulfhydryl-containing medications
(methimazole, penicillamine, captopril, imipenem,
hydralazine, procainamide, isoniazid, penicillin G)
 Alimentary Hypoglycemia
o Occurs within 4 hours after eating
o At risk: Patients who had vagotomy or gastric surgery→
caused by elevated peptides (GLP-1)→ endogenous
hyperinsulinemia

Inborn Errors of Carbohydrate Metabolism

 Glycogen storage diseases or glycogenosis result from the
defects in glycogen metabolism.
o Inherited deficiencies of enzymes that control the synthesis or
breakdown of glycogen.
o Liver and muscle are most commonly affected
 Hepatic glycogenosis- hypoglycemia and hepatomegaly
 Muscle glycogenosis- muscle cramps, exercise intolerance,
fatigue, and weakness
o Divided into 9 types, defect will be a particular enzyme
 Rare disorders
 Lectured by: Aldrin Jeff B. Autencio, MSMT

Objectives:
 Know the different lipid chemistry  Enumerate and understand the different lipid and protein
 Understand the lipoprotein physiology and metabolism analysis

Lipids and Lipoproteins

 Have become increasingly important in clinical practice.
 Known to be the central to the (source of) energy metabolism of the human body
 High fat consumption is associated between the blood lipid levels in blood, which results in the association with
atherosclerosis and coronary heart disease (CHD).

Lipids (Fats)
 Functions:
o Composed of carbon-hydrogen bonds, which are a rich source of energy and an efficient way for the body to store excess
calories.
o Because of their unique physical properties, lipids are an integral part of cell membranes, therefore they play an important
structural role in cells.
 The lipids transported by lipoproteins, namely triglycerides, phospholipids, cholesterol, and cholesteryl esters, are also
known to be found in cells.
 Fatty Acids
o In plasma, most of the fatty acids are bound to albumin.
o Described to have simple linear chains of carbon-hydrogen bonds that terminate with a carboxyl group
o In plasma, only a relatively small amount of fatty acids exist in the free or unesterified form, because most of the fatty
acids are bound to albumin.
o Majority of plasma fatty acids are instead found as a constituent of triglycerides or phospholipids.
o Classified according to length:
 Short-chain (4-6 carbon atoms)  Medium-chain (8-12 carbon atoms)
 Long-chain (>12 carbon atoms)- in diet, the most fatty acids obtained are from the long chain fatty acids
o Classified according to Carbon-Carbon double bonds:
 Saturated (no double-bonds)  Polyunsaturated (two or more double-bonds)
 Monounsaturated (one double-bond)
 Triglycerides
Predominantly found in plasma, containing three fatty acid molecules attached to one molecule of glycerol by ester bonds.
o
If containing saturated fatty acids: pack together more closely and tend to be solid at room temperature
o
If containing cis unsaturated fatty acids: form oils at room temperature
o
Most triglycerides are derived from plant sources such as corn, seeds (e.g. sunflower seeds): are rich in polyunsaturated
o
fatty acids
o Triglycerides from animal sources: are mostly saturated fatty acids, which solidify at room temperature
o Have no charged groups or polar hydrophilic groups, therefore triglycerides are hydrophobic in nature, and virtually
water insoluble.
o Classified as a neutral lipid (has no charge)
 Phospholipids
o Are similar in structure to triglycerides except that they only have two esterified fatty acids.
o Various types of phospholipids are named based on the type of phospholipid head group present.
 Example: Phosphatidylcholine contains a choline head group and is the most common phospholipid found on lipoproteins
and in cell membranes.
o Known to be amphipathic lipid molecules (found on the surface of lipid layers)
o The polar hydrophilic head group faces outward, toward the aqueous environment.
o The fatty acid chains face inward, away from the water in a perpendicular orientation with respect to the lipid surface.
 Cholesterol
o Is an unsaturated steroid alcohol containing four rings (A, B, C, and D), and it has a single carbon-hydrogen (C-H) side
chain tail similar to a fatty acid in its physical properties.
o Is described to be an amphipathic lipid (just like phospholipids they are found on the surface of lipid layers)
o The only hydrophilic part of cholesterol is the hydroxyl group in the A-ring.
o Not readily catabolized by most cells therefore, does not serve as a source of fuel. However, cholesterol can, be converted in
the liver to primary bile acids, therefore promoting fat absorption in the intestine by acting as detergents.
 o A small amount of cholesterol can be converted by some tissues, which produces the hormones, such as glucocorticoids,
mineralocorticoids and estrogen.
o Though amphipathic in nature can be converted for vitamin D synthesis.
o Can also exist in an esterified form, called cholesteryl ester.
 Cholesteryl ester is hydrophobic (water-fearing) and a neutral lipid (do not contain any charge).
 They are located in the center of lipid drops and lipoproteins, along with triglycerides.

Lipoproteins
 As the name implies, lipoproteins are composed of both lipids and proteins, called apolipoproteins.
 The amphipathic cholesterol and phospholipid molecules are primarily found on the surface of lipoproteins as a single
monolayer, whereas the hydrophobic and neutral triglyceride and cholesteryl ester molecules are found in the central or core
region.
 Apolipoproteins (Lipids and Proteins)
o Help maintain the structural integrity of lipoproteins
o Serve as ligands for cell receptors
o Serve as activators and inhibitors of the various enzymes that modify lipoprotein particles
o Located on the surface of lipoprotein particles.
o Apolipoprotein (apo) A-I
 Major protein on high density lipoprotein (HDL/good cholesterol).
 Frequently used as an index of the amount of the antiatherogenic HDL present in plasma.
o Apo B
 Principal protein on low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicrons.
 Exists in two forms: Apo B-100 (LDL and VLDL) and Apo B-48 (Chylomicrons).
o Apo E
 Serves as a ligand for the LDL receptor and the chylomicron remnant receptor.
 There are three major isoforms: Apo E2, Apo E3, and Apo E4.
 There is an association with hyperlipoproteinemia with patients containing Apo E2 isoform.
 Patients containing Apo E4 is greatly associated with the increased risk for developing Alzheimer’s disease.
 Chylomicrons
o Are the largest and the least dense of the lipoprotein particles, having diameters as large as 1200 nm.
o Hallmark: Described to have the “creamy layer on top of stored plasma”
 When storing plasma and a creamy layer on top was found after storing, this is probably due to chylomicrons, since they
are the least dense, they float at the topmost layer.
o Produced by the intestine.
o Principal role: Delivery of dietary lipids to hepatic and peripheral cells
o Since chylomicrons are large in size, they reflect light and account for the turbidity of postprandial plasma. However, since
they are considered light, they also readily float on top of stored plasma (hallmark of chylomicrons)
o Once chylomicrons enter the circulation, triglycerides and cholesterol esters are rapidly hydrolyzed by lipases (enzymes
responsible for the hydrolysis process). They are then transformed into chylomicron remnant particles, which are
recognized by proteoglycans and remnant receptors in the liver, facilitating their uptake.
 Very Low-Density Lipoproteins (VLDL)- Bad Cholesterol
o Produced in the liver. Major carriers of endogenous triglycerides and transfer these triglycerides from the liver to
peripheral tissue.
o Contains Apo B-100, Apo E, and Apo C
o Rich in triglycerides
o Increases during excess dietary intake of carbohydrates, saturated fatty acids, and trans fatty acids.
 Low-Density Lipoproteins (LDL)- Bad Cholesterol
o Contains Apo B-100
o Is more cholesterol-rich than other Apo B-containing lipoproteins and is readily taken up by the cells
o LDL form as a consequence of the lipolysis of VLDL.
o Since LDL particles are significantly smaller than VLDL and chylomicrons, LDL can infiltrate into
the extracellular space of the vessel wall, which they can be oxidized and taken up by macrophages,
and when taken up by the macrophages, the cell is described to be as foam cell.
 Macrophages that take up too much lipid become filled with intracellular lipid drops, and turns into foam cells.
 The cell is the predominant cell type of fatty streaks, an early precursor of atherosclerotic plaques.
 Lipoprotein(a)
o Are LDL-like particles that contain one molecule of Apo (a) linked to Apo B-100 by a disulfide bond.
o It has been proposed that lipoprotein(a) may compete with plasminogen for binding sites, thereby promoting clotting, which
is a key contributor to both myocardial infarction and stroke.
o Elevated levels of lipoprotein(a) confers an increased risk for premature coronary heart disease (CHD) and stroke.
 High-Density Lipoprotein (HDL)- Good Cholesterol
The smallest and most dense lipoprotein particle (therefore it is found at the bottom).
o
Synthesized by both the liver and intestine.
o
Since it is the good cholesterol, HDL has the ability to remove cholesterol from cells, called reverse cholesterol transport.
o
 This is one of the main mechanisms proposed to explain the anti-atherogenic property of good cholesterol.
 Lipoprotein Physiology and Metabolism
o All pathways depends on the Apo B-containing lipoprotein particles
o Lipid Absorption Pathway
 Since fats are water-insoluble, special mechanisms are required to facilitate its intestinal absorption.
 During the process of digestion, pancreatic lipase (enzyme in the pancreas) cleaves the fatty acids, and converts dietary
lipids into more polar compound. Triglycerides thus is transformed into monoglycerides and diglycerides, cholesterol
esters transformed into free cholesterol, and phospholipids transformed into lysophospholipids.
 When these analytes are transformed, they are now ready to be absorbed because they are in a more polar compound.
o Exogenous Pathway
 The newly synthesized chylomicrons in the intestine are initially secreted in the lymphatic ducts and eventually enters the
circulation of the thoracic duct.
 The hormones epinephrine and cortisol play a key role in the mobilization and hydrolysis of triglycerides from adipocytes,
whereas insulin prevents lipolysis by adipocytes and promotes fat storage and glucose utilization.
o Endogenous Pathway
 VLDL particles, once secreted into the circulation, undergo a lipolytic process similar to that of
chylomicrons.
 Most triglycerides in the liver that are packed into VLDL are derived from the diet after recirculation
from the adipose tissue.
o Reverse Cholesterol Transport Pathway
 This is the proposed mechanism to explain the anti-atherogenic property of HDL.
 Adult Reference Ranges for Lipids
Analyte Reference Range Analyte Reference Range
Total Cholesterol 140-200 mg/dL LDL (Bad) Cholesterol 50-130 mg/dL
HDL (Good) Cholesterol 40-75 mg/dL Triglyceride 60-150 mg/dL
o The incidence of heart disease is strongly associated then to serum cholesterol concentration.

Lipid and Lipoprotein Analyses

 Cholesterol Measurement
o Early analytic methods involve strong acids and other chemicals to produce color change. Strong acids can be used but is
considered to be non-specific and a need for organic solvents to improve the reaction.
o Cholesterol is measured because lipids and lipoproteins are important indicators of CHD risk, and also to ensure that the
patient do not fall on heart attack.
o The lipid workup traditionally has begun with a measurement of total serum cholesterol.
o Reference method
 Uses hexane extraction after hydrolysis with alcoholic KOH, followed by reaction with Liebermann-Burchard color
reagent.
o Definitive method: Isotope Dilution Mass Spectrometry (IDMS)
o Enzymatic method
 Has the advantage of being highly specific in reacting to chosen analyte
 Drawback: Tedious to perform
 Uses the enzymes cholesteryl esterase, cholesterol oxidase, and peroxidase, producing an end colored product, which is
then quantitated spectrophotometrically at 500 nm.
 Triglyceride Measurement
o Triglycerides are measured because it is also commonly used in the estimation of LDL cholesterol by the Friedewald
equation.
o Measurement of serum triglycerides in conjunction with cholesterol, is useful in detecting certain genetic and other types of
metabolic disorders as well as in characterizing risk of coronary virus diseases.
o Enzymatic method (most common)
 Uses the enzymes bacterial lipase, glycerokinase, glycerophosphate oxidase,
and peroxidase, to which colored product is measured spectrophotometrically
using a spectrophotometer.
o Reference method: involves alkaline hydrolysis, solvent extraction, and a color reaction with chromotropic acid.
 This assay is tedious to perform and is poorly characterized.
 Lipoprotein Measurement
o Various methods have been used for the separation and quantitation of serum lipoproteins.
 o Ultracentrifugation
Separates lipoproteins according to their densities, which allows better fractionation of lipoproteins

It takes advantage of physical properties of the lipoproteins, such as density, size, charge, and apolipoprotein content.

Range in density observed among the lipoprotein classes is a function of the relative lipid and protein content and enables

fractionation by density.
o Electrophoretic Separation: Takes advantage of the differences in charge and size.
o Chemical Precipitation: Depend on the particle size, charge, and differences in the apolipoprotein content.
o Chromatographic Assays: Takes advantage of size differences in molecular sieving methods or composition in affinity
methods
 High-Density Lipoprotein Methods
o Chemical Precipitation
 Involving a two-step procedure with manual pre-treatment
 Heparin and manganese precipitates Apo B-containing lipoproteins but possess cross-reactivity
 Sodium phosphotungstate is added to increase the sensitivity of the test (recommended)
 Use of dextran sulfate with magnesium is preferred to have better reaction of the analyte with the reagent.
o Reference method
 Involves ultracentrifugation to remove VLDL, heparin manganese precipitation from the 1.006 g/mL infranate is
needed to remove LDL, and analysis of supernatant cholesterol by the Abell-Kendall assay.
 This is a three-step procedure developed at the CDC, but not routinely performed because it is rather expensive and
tedious.
 Low-Density Lipoprotein Methods
o The most common research method for LDL cholesterol quantitation and the basis for the reference method has been
designated beta-quantification, in which beta designation refers to the electrophoretic term for LDL.
o A doctor orders LDL determination, because this is considered to be the primary basis for treatment decisions.
o Friedewald calculation
 A more common approach, bypassing ultracentrifugation and using reagents.
 The denominator 5 is used to extract the value of LDL
 HDL cholesterol is quantified either after precipitation or using one of the direct methods, and total cholesterol and
triglycerides are measured in the serum. VLDL cholesterol is estimated as the triglyceride level divided by 5 (when using
mg/dL units).
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 Apolipoprotein Methods
o Uses immunoassays:
 Turbidimetric Assay  Radio Immunodiffusion (RID)
 Nephelometric Assay  Radioimmunoassay (RIA)
 Enzyme-linked Immunosorbent Assay (ELISA)
 Specimen Collection and Handling
o 12 hours fasting is required (depending on laboratory protocol, other laboratories may require only 9 hours, while others
may require 10 to 12 hours)
o Non-fasting of the patient may cause: Increased triglyceride levels and Turbid serum
 Lectured by: Alessandra Kamille P. Mallari, MD, DPSP
Dyslipidemia
 Diseases associated with abnormal lipid concentrations  Defined by the clinical characteristics of patients and laboratory
test
 They can be caused directly by:
o Genetic abnormalities o May develop secondarily as a consequence of other
diseases
o Environmental/lifestyle imbalances
 Many, but not all, are associated with chronic heart disease, or atherosclerosis.

Atherosclerosis
 Majority of dyslipidemias are associated with atherosclerosis.
 In developed countries, atherosclerosis- single leading cause of death and disability.
 Increased awareness of this disease, and importance of diet and exercise in preventing chronic heart
disease- resulted in an overall decrease in the average serum cholesterol and in lower prevalence of this heart
disease. However, it still exceeds all other causes of deaths combined.
 Although many women develop atherosclerosis as men, women typically develop 10 years later than men.
 The relationship between heart disease and dyslipidemias stems from the deposition of lipids, mainly in the
form of esterified cholesterol, in artery walls called atherosclerosis.
o Lipid deposition results in fatty streaks along the vessel wall due to the excess fat in the macrophages in the subendothelial cells.
o Eventually when these fatty streaks develops and forms within the vessel wall, it may cause
occlusion of that blood vessel, blood clot may also form limiting the blood flow.
 It is important in the development of coronary, cerebral, and peripheral vascular disease.
o Causes more morbidity and mortality in the Western world than any other disorder
 Coronary Heart Disease- important manifestation of this disorder (e.g. myocardial infarction)
 General overview of the basic structure of Atherosclerotic plaque
o Fibrous cap- smooth muscle cells, macrophages, foam cells, lymphocytes, collage, elastin, proteoglycans, and neovascularization
o Necrotic center- cellular debris, cholesterol crystals, foam cells, and calcium
 Risk Factors
o The development and the likelihood of atherosclerosis is determined by the combination of acquire,
inherited, gender, or age associated risk factor.
o Nonmodifiable (Constitutional)- Inherent, we are born with this
 Genetics- family history is the most important independent risk factor for atherosclerosis
 Patients who have family members or close relatives who have atherosclerosis are more likely to
develop than in patients who does not have an immediate relative with atherosclerosis.
 Age- a dominant influence since plaque formation is a progressive process, clinically manifests between 40 to 60 years old
 Coronary heart disease caused by atherosclerosis is called as a “silent killer” as this is very hard to control or to educate people
because people usually do not have symptoms.
 When eating a very fatty meat or high cholesterol meal, the patient will feel nothing wrong, but inside the blood vessel walls,
there is already initial formation of atherosclerotic plaque, and this plaque will develop throughout the person’s lifetime.
 Gender- affects formation of atherosclerosis
 Pre-menopausal women are relatively protected. Patients who are still menstruating has a less chance of developing an
atherosclerotic plaque than women who are postmenopausal.
o Modifiable
 Hyperlipidemia (hypercholesterolemia)- major risk factor in the development of atherosclerosis
 Even in the absence of other risk factors (only hypercholesterolemia), the risk for atherosclerosis is high
 Patients will have High LDL and Low HDL levels
 Hypertension- up to 60% increased risk for atherosclerosis
 Cigarette Smoking- especially in prolonged use  Diabetes Mellitus- markedly increased risk
o Other Risk Factors
 Inflammation  Lipoprotein a [Lp(a)]
 Hyperhomocysteinemia- increased level of homocysteine in the blood  Factors affecting hemostasis
 Metabolic syndrome
 Important and emerging disorder that is associated with central obesity
 Characterized by insulin resistance, hypertension, dyslipidemia,
hypercoagulability, and proinflammatory state
 Increased risk for the development of chronic heart disease due to the
formation of atherosclerosis.
 Relationship of Dyslipidemia and Atherosclerosis
o Dyslipidemia (particularly high LDL levels, low HDL levels, and formation of
abnormal lipoprotein(a)) is a major factor in its development.
 o Risk factors: Hyperlipidemia, hypertension, smoking, toxins, hemodynamic abnormalities, immune reactions, inflammation or
infection due to viruses.
 Endothelial Injury or Dysfunction- first step in the formation of an atherosclerotic plaque
 All of these risk factors will contribute to either endothelial injury or endothelial dysfunction.
o Cholesterol and Cholesterol Esters- dominant lipid in an atheromatous plaque
o With chronic hyperlipidemia, lipoproteins or HDLs will accumulate within the intima of the endothelium.
o Increased LDL in the blood will accumulate within the blood vessel intima, where they may aggregate and become oxidized.
o Modified (Oxidized) LDL are accumulated or eaten up by the macrophages.
 Since these cannot be completely degraded, the chronic ingestion of modified LDL will result into formation of foam cells.
 Foam cells are endothelial cells (e.g. macrophages) that accumulate cholesterol, such as LDL in the cytoplasm.
 Smooth muscle cells can also transform into foam cells when there is high levels of oxidized LDL in the environment.
 These are toxic substances that cannot be degraded, so this adds injury to the cells, thereby releasing growth factors that will
lead to a vicious inflammatory cycle that will add to the progression of an atheromatous plaque.
 Vicious Cycle includes:
 Extracellular Matrix Synthesis- intima thickens
 Proliferation of smooth muscle cells- become disorganized and will contribute to the thickening of the intima
 Within that part of the thickened intima, extracellular lipids (that are also excess LDL) will accumulate within this area
 Recruitment and migration of smooth muscle cells that will add up.
 Result: Thickened intima of the endothelium composed of foam cells, disorganized smooth muscle cells, excess LDL in that
area, as well as necrotic debris, which will in turn still recruit more additional cells that will go to that area (macrophages, foam
cells producing growth factors, and all other cells are called up to help then get rid of excess cholesterol).
 Eventually progress and develop an atherosclerotic plaque
 Gross appearance of a blood vessel and of an aorta/large vessel containing numerous atherosclerotic plaques
 Yellowish areas have thickened and have concentrated→ cholesterol, foam cells
 Early lesions containing lipid filled macrophages are called fatty streaks.
 As these develop (as the disease progresses), they will harden and become calcified.
 Pathogenesis
o Clinical importance of atherosclerosis has simulated enormous interest in understanding the mechanisms
that underlie its evolution and complications.
o Atherosclerosis progresses in the following sequence:
1. Chronic endothelial injury
 Hyperlipidemia, Hypertension, Smoking, Homocysteine, Hemodynamic factors, Toxins, Viruses, and
Immune reactions
2. Endothelial dysfunction
 Increased permeability, leukocyte adhesion, monocyte adhesion, and emigration of WBCs.
 Since there is dysfunction and abnormalities within the blood vessel wall, it will call up all of the WBCs
into the area.
 The cells will promote a pro-inflammatory state (growth factors released and helps activate cells and
becomes macrophages)
3. Macrophage activation, smooth muscle recruitment
 Macrophages will eventually become foam cells
 Recruitment of smooth muscle cells will add up to the initial injury, causing thickening of the intima
(recruitment was haphazard so it was not arranged like a normal endothelial cell)
4. Macrophages and smooth muscle cells engulf lipid
 When macrophages are activated, they engulf excess cholesterol and transform them as foam cells.
 Seen on gross as yellowish fatty streaks (yellowish because of cholesterol)
5. Smooth muscle proliferation, collagen and other extracellular matrix deposition, extracellular lipid
 Lipid accumulation (both extra- and intracellular) happens
 Process still continues as it becomes older (there is smooth muscle proliferation, collagen and other extracellular matrix
deposition in areas wherein there is cholesterol deposition, and macrophages)
 Macrophages’ main function is to engulf any foreign substances. When they engulf the modified LDL into their cytoplasm, they
cannot degrade it, so eventually they will die and remain in that area contributing to the lipid debris or necrotic debris.
 If necrotic debris are not cleared by macrophages, they will calcify and harden and becomes fibrofatty atheroma.
 Main consequence of an atheromatous plaque: When it becomes a thick calcified atheroma, it will block the blood vessel wall.
 When it is occluded, blood cannot flow. When blood cannot flow, derangement to that organ will ensue.
 Consequences
o Initially there will only be a fatty streak. After all of the recruitment of smooth muscle
cells and macrophages, there is intimal thickening, and eventually plaque expansion.
o The plaque can have a thin cap, thick cap, and fibrotic plaque (20-30 years after)
 Thin cap- easily ruptures and when this ruptures, it produces a sudden cardiac death
 Thick cap- erosion may ensue and thrombosis will happen (coagulation factors will
be activated, blood clots will be created, and blood clots will flow into the blood
vessel, into the vascular system, and it may lodge into smaller arteries, and will then occlude that particular artery).
  If there is blood clot secondary to the disturbance of atheromatous plaque:
 Myocardial infarction- if it lodges to the heart  Cerebral infarction/Stroke- if it occurs in the brain
 Aortic aneurysm and Peripheral vascular disease- when blood vessel becomes injured, it may also become thin
 Fibrotic plaque- if the plaque hardens, it will occlude the passage of blood in that blood vessel and will create a critical stenosis
and sudden cardiac death
o Target Arteries: Aorta, carotid (feeds the brain), iliac, coronary (feeds the heart), and popliteal arteries (lower extremities)
 Target arteries will correspond to the most common consequences of atherosclerosis.

Metabolic Syndrome
 This physiologic syndrome is characterized by a constellation of known and emerging risk factors for Coronary Heart Disease.
 First described as “Syndrome X” in the 1980s
 Metabolic syndrome is diagnosed if the person has:
o Abdominal obesity (measure the waistline) o Insulin Resistance (diabetic; with or without glucose intolerance)
o Atherogenic dyslipidemia o Prothrombotic or proinflammatory
 Elevated triglycerides (TG) states
 Small LDL particles and low HDL-C
o Raised Blood pressure (hypertensive)

Dyslipidemias (Abnormalities in Lipid Levels)

 Lipoprotein and lipid levels are used to predict heart disease. They are measured to know if that person has an increased risk for the
development of coronary heart disease or if that patient needs any
intervention to reduce or eliminate the risk for development of heart
problems.
 Early in the days of clinical laboratory, the Fredrickson
Classification was used to characterize lipid disorders.
o This classification system uses the gel electrophoresis and
refrigerator test (a standing plasma test for chylomicrons to
correlate clinical disease syndromes with laboratory phenotypes).
Divided them into five types based on the results.
o This is largely abandoned, except for the nomenclature for few
pathognomonic syndromes, such as type 1 and type 3 lipidemia.
 Disorders of lipoprotein metabolism are categorized in the basis of the laboratory findings.
 High Cholesterol with High LDL
o These disorders share one feature, hyperbetalipoproteinemia
(Fredrickson Type 2A)- Elevated LDL-C and Normal TG
o It is associated with a high cardiac risk
o This is a commonly encountered laboratory presentation.
o Caused by either familial or non-familial hypercholesterolemia
o Polygenic (Non-familial) Hypercholesterolemia
 General term for hypercholesterolemia
 Due to multifactorial risk factors
 85% of high cholesterol with high LDL fall into this category
o Familial Hypercholesterolemia
 Inherited genetic defect- resulting to the loss of LDL receptor activity.
 LDL receptor gene is found in chromosome 19
 The resulting defective receptors cannot bind or clear LDL from the circulation.
 Classified into 5 mutational classes:
 Abnormality in the LDL receptor can be due to: Abnormal synthesis, Abnormal transport,
Abnormal binding, Abnormal clustering, or Abnormal recycling
 Suspected in:
 Adults (≥20 Years): LDL-C is of >190 mg/dL or non-HDL-C of >220 mg/dL
 Children and Adolescents (≤20 Years): LDL-C of >160 mg/dL or non-HDL-C of >190 mg/dL
 Signs of the disease include: Corneal arcus, Tendinous xanthomata, and Xanthelasma
 Treatment
 Adult with familial hypercholesterolemia (FH)- higher potency statins (atorvastatin, rosuvastatin, pitavastatin, simvastatin)
titrated to achieve an LDL-C reduction of 50% or more from baseline.
o Familial Defective ApoB
 Autosomal dominant disorder of apoB gene and chromosome 2.
 Interferes with the recognition of apoB-100 by the LDL receptor (abnormality in the LDLR)
 High Triglycerides with Normal Cholesterol
o Related to elevations of TG-rich particles (VLDL and Chylomicrons), considered Frederickson types 1 and 4.
o Commonly encountered laboratory presentation is usually due to hyperprebetalipoproteinemia (VLDL)
 May be due to secondary causes- excess alcohol or a high-carbohydrate diet
 o Usually, diabetic patients are seen with this type of profile.
o Since only triglyceride-rich particles are unaffected, LDL and LDL-C are typically normal.

o Diabetic Dyslipidemia
Atherogenic dyslipidemia (causes atherosclerosis formation) in person with type 2 DM.

 Laboratory findings: High TG, Low HDL, Small and dense LDL in the blood (normal)
 Treatment of LDL-C is the primary target of therapy.
 Moderate-intensity statin should be given in: Diabetics (40-75 years old) and Moderately high LDL-C levels (70-189 mg/dL)
o Familial Hypertriglyceridemia: Isolated Hypertriglyceridemia (Type 4 Hyperlipidemia)
 Relatively common, autosomal dominant disorders (occurs in about 5-10% of individuals)
 Defined by familial occurrence of isolated high VLDL and high TG values (200-500 mg/dL)
 TG and chylomicrons are decreased, VLDL is increased, abnormal ApoB production→ fluffy triglyceride-rich VLDL
 Consequence:
 Increased prevalence of metabolic syndrome  Increased cardiovascular disease (CVD) risk
o Lipoprotein Lipase Deficiency (Hyperlipoproteinemia Type 1 or Hyperchylomicronemia)
 Rare autosomal recessive disorder  Presents in childhood- complain of colicky abdominal pain and
pancreatitis
 Defective or absent LPL- inability to clear the chylomicrons in the blood
o ApoC-II Deficiency
 ApoC-II is an activating cofactor for LPL  Affected: Children and young adults
 Absence of apoC-II= functional LPL deficiency (presents similarly to LPL deficiency)
 Recurrent bouts of abdominal pain and pancreatitis.
 High Triglycerides with High Cholesterol
o Related to elevations of LDL and triglycerides (Fredrickson types 2B and 3)
o These disorders are associated with increased cardiac risk due to the elevated LDL.
o Familial Combined Hyperlipidemia (Type 2B)
 Relatively common disorder- involves simple hypercholesterolemia, simple hypertriglyceridemia, or a mixed defect
 Phenotypic heterogeneity and no definitive biochemical marker (either high cholesterol, high TG, or mixed)
o Acquired Combined Hyperlipidemia
 Common in patients with metabolic syndrome.
 High levels LDL→ Increased levels VLDL in blood→ hypercholesterolemia and hypertriglyceridemia
 There is saturation of mature LDLs in the blood (LDL is very increased that VLDL, cholesterol, and TG are also increased)
o Dysbetalipoproteinemia (Type 3)
 Usually affects adults, and is more common in men than in women.
 Laboratory Findings:
 Elevated cholesterol and triglycerides
 Pathognomonic finding in gel electrophoresis: Broad abnormal band between VLDL and LDL
abnormal migrating beta-lipoprotein (β-VLDL, rises in the β region of electrophoresis)
 VLDL, VLDL-C, and Triglyceride ratio is more than 0.3 (more triglyceride than VLDL).
 Clinical Manifestation
 Palmar xanthoma- yellowish discoloration of the palms of the patient because of decreased levels of the cholesterol
 Tuberoeruptive xanthoma- usually seen in the elbows, knees, and buttocks (manifestations of high TG and high cholesterol)
 Premature atherosclerosis- highly prevalent
o Hepatic Lipase Deficiency
 Caused by mutation of the HL gene  Rare familial disorder
 Laboratory Findings:
 Total cholesterol (TC): 250-1500 mg/dL  HDL-G: Normal or increased
 Total triglycerides (TG): 400-8000 mg/dL  TC:TG ratio is normal (both increase in same amount)
 Clinical Findings (similar to Type 3): Physical stigmata include Palmar xanthoma and Tuberoeruptive xanthoma.
 Increased risk of atherosclerosis.
o Cholesterol 7-Alpha Hydroxylase Deficiency
 Recessive disorder of the CYP7A1 gene
 Encodes an enzyme that is involved in the first step of the classical pathway for bile acid biosynthesis (high TG and TC).
 Low Triglycerides and Cholesterol
o Uncommon disorders- primarily associated with defective apoB synthesis or metabolism abnormalities
o Low or absent levels of apo-B lipoproteins (Chylomicrons, VLDL, and LDL) (low TG and TC).
o Fat-soluble vitamin deficiencies are common.
o Abetalipoproteinemia (Bassen-Kornzweig Syndrome)
 Rare autosomal recessive disorder.
 Due to a mutation in the MTTP gene (chromosome 4)- Microsomal Triglyceride Protein (MTP)
 MTP incorporates lipids into the nascent apoB protein and prevents degradation.
 Lack or abnormal ApoB protein that will lead to its destruction.
  Laboratory Findings:
 Decreased apoB-48 and apoB-100  Decreased TG, TC (<50 mg/dL)
 Clinical Manifestations:
 Fat soluble vitamin deficiencies
 Caused by malabsorption of vitamins A, K, and E- have transport systems independent of lipoproteins
o Chylomicron Retention Disease (Anderson’s Disease)
 Associated with SARA2 gene (seen in the long arm of chromosome 5) which encodes Sar1 GTPase protein- involved in
intracellular regulation of new apoB-48 containing lipoproteins
 Usually seen in childhood- manifests with fat malabsorption and low plasma lipid
 Only apoB-48 deficiency
 Characterized by:
 Hypercholesterolemia  Failure to thrive (seen in children)
 Chronic diarrhea  Deficiency in fat-soluble vitamins
 Isolated Low HDL-C
o HDL in the blood helps clean up excess lipid in the peripheral system back into the liver.
 Without HDL, these excess lipids in the peripheral circulation will not be recovered, so they will remain there.
o Low HDL levels are associated with CHD, presumably because insufficient HDL is available to participate in reverse cholesterol
transport, the process by which cholesterol is eliminated from peripheral tissues.
o Familial Hypoalphalipoproteinemia
 Common autosomal dominant disorder (seen in 1 in 400 people).
 HDL-C is <30 mg/dL (men) and <40 mg/dL (women)
 Half of the families have hepatic lipase or apoA-I/C-II/A-IV gene defect.
 Premature Chronic Heart Disease (CHD) is present (due to low HDL)
 Manifestation of chronic heart disease in patients who are much younger than the usual population.
 There are problems with the heart (e.g. with history of myocardial infarction, stroke, peripheral vascular diseases) in patients
who are very young.
 Criteria for Diagnosis:
 Low HDL cholesterol, normal VLDL cholesterol and LDL cholesterol levels
 Absence of diseases or factors that will lead to secondary effects of hypoalphalipoproteinemia
 The presence of a similar lipoprotein pattern in a first-degree relative (for it to become familial)
o ApoA-I Deficiency and ApoC-III Deficiency
 Rare autosomal recessive disease  Due to mutation in apoA-I gene
 Reduction in the formation of HDL (reduced HDL production)
 Laboratory Findings:
 HDL-C <5 mg/dL  Corneal opacification
 Premature CHD
o Tangier Disease
 Rare autosomal recessive disorder
 Complete absence of HDL due to mutation in the ABCA1 gene (in chromosome 9).
 Due to absence of HDL, there is inability to effectively transfer cholesterol and phospholipids from nascent apoA1.
 Buildup of cholesterol (primarily in reticuloendothelial cells)
 Usually since there is much cholesterol in the peripheral circulation, macrophages will ingest them. and those macrophages (or
WBCs) will return to the reticuloendothelial system (e.g. tonsils). Yellow discoloration is due to accumulation of cholesterol.
 Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Identify the parts and functions of the liver
 Determine important cell types associated with the liver and their functions
 Familiarize the blood flow mechanism in the liver

Gross Anatomy (Macroscopic Examination)

 Weighs about 1.2-1.5 kg (about the size of a football), can increase to over 10 kg especially in chronic cirrhosis
 Located at the right-hand part of the abdomen, behind the lower ribs (means of protecting the liver and other internal organs,
e.g. heart)
 Functionally divided into two lobes, and the external division is marked on the front of the liver by the falciform ligament
o Right lobe is 6 times larger than the left lobe, however there is no known functional difference between the lobes, and the
communications flows freely between all areas of the liver.
o Falciform ligament attaches the liver to the front body wall, and it separates the liver into the left medial lobe and the right
lateral lobe
o The right lobe is separated from the other lobes by the gallbladder fossa and the fossa for the inferior vena cava.
 Fossa is a shallow depression/pit/shallow part/hollow part of an organ.
o The left lobe includes the quadrate lobe and the caudate lobe.

Functions of the Liver

 Metabolism- the liver is capable of metabolizing proteins, carbohydrates, and fats (catabolism/anabolism)
 Storage- carbohydrates (glycogen), proteins (lipoprotein), fats (TAG); Lipoproteins are types of proteins that carry lipids
 Synthesis- main substances produced by the liver are proteins (albumin), and blood clotting factors
 Bile production- the liver is the one that produces bile because bile is stored in the gallbladder
o Bile- a digestive fluid which is produced in the liver, but it is stored in the gallbladder
 Detoxification- the liver plays several roles in detoxification
1. It filters blood to remove large toxins
2. The bile synthesized by the liver is full of cholesterol and other fat soluble toxins (the liver removes toxins from the body
through bile)
3. It disassembles unwanted chemicals so that they could be removed out from the body
o Cytochrome p450- this enzyme decreases drug efficacy (drugs that we take are foreign to our body, and the liver will break
down the drug so it will not hurt our body, and specific dose will be given to account for the amount to be detoxified.

Blood Supply to the Liver

 About 1500 mL of blood/min passes the liver (the approximate amount of blood that is detoxified and used up
in the liver.
 Blood enters the liver via:
o Hepatic artery: Supplies blood to the liver from the heart (oxygen-rich); 25% of the total blood supply
o Portal vein: Carries the blood to the liver from the gastrointestinal tract (nutrient-rich); 75% of the total blood supply
 Blood leaves the liver via:
o Hepatic vein: since the blood has been used up by the liver, it is already nutrient and oxygen
depleted
o Hepatic ducts: may carry the bile to the gallbladder
 The Portal Circulation
o The venous blood coming from gastrointestinal tract drains into the superior
and inferior mesenteric veins. These two vessels are then joined by the splenic
vein, just posterior to the neck of the pancreas, and the three of them will form a
portal vein. It will split to form the right and left branches, each supplying
about half of the liver with blood coming from the gastrointestinal tract.
o Portal venous blood contains all the products of digestion absorbed from the gastrointestinal tract.
All useful and non-useful products are processed in the liver before being either released back into the hepatic veins which
join the inferior vena cava or stored in the liver for later use.
 The Portal Triad
o Portal vein- supplies the blood to the liver and that is coming from the gastrointestinal tract
 Nutrient-rich blood coming from the bowel flows into the liver through the portal vein
o Hepatic artery- supplies the liver with blood coming from the heart
 Oxygen-rich blood flows into the liver through the hepatic artery
o Common bile duct- the bile is excreted out from the liver
  Bile flows out of the liver through the bile duct
 The Biliary Tree (serves as the Excretory System of the Liver)
o Mainly, it excretes bile
o The bile is produced from the liver and it is stored in the gallbladder (bile sac)
o Main function of Bile: The digestion of the food that we eat and mainly, it emulsifies the fats (it will
break down fats into fatty acids, which can then be taken into the body by the digestive tract, aids in
the digestion process)
o Bile contains cholesterol, bile pigments, bile acids, and bile salts.
o Once the bile is synthesized in the liver, it will pass through the hepatic duct (right and left hepatic duct) towards the
common hepatic duct, and it will go to the cystic duct, and the bile will be stored in the gallbladder.
o Cholecystokinin (CKK) comes from the Greek word chole- means bile; cysto- means sac; kinin- means to move
 This hormone will help the gallbladder to move/contract to squeeze out the bile if needed to digest the food.
 Cholecystectomy- the removal of the gallbladder
o Once bile is needed for the digestion of food, the gallbladder (with the help of the hormone cholecystokinin) will squeeze out
the bile.
o Once the bile is excreted from the gallbladder, it will pass through the common bile duct (last part of the biliary tract) and it
reaches the gastrointestinal tract and released into the duodenum (first part of the intestine), where the bile is secreted.
o The bile salt will emulsify the fats in the ileum, and this is the part where the absorption of fats and bile salts occur. The bile
salts are reabsorbed back to the liver to be used.

Microscopic Anatomy
 The liver is divided into microscopic units called the lobules
 Lobules
o Functional units of the liver
o Microscopic, six-sided structure with a central vein
o Each corner contains the portal triad
 Portal vein  Bile duct
 Hepatic artery
 Major Cell Types
o Hepatocytes (80%)
 Mainly responsible for the regenerative properties of the liver
 The liver can regenerate
 In cases of liver injury or liver damage, all the cells in the liver can change and divide until the normal size of the liver is
restored
 If the injury to the liver is very serious that the liver could no longer regenerate, then a person might need a liver
transplant
o Kupffer Cells
 Specialized macrophages (phagocytes) that line the sinusoids of the liver.
 Main function: To engulf bacteria and other microorganisms or foreign debris, toxins, and other
substances flowing through the sinusoids (mainly responsible for eliminating any microorganisms or
toxins and substances that could harm the liver, means of protection)
 Lectured by: Alessandra Kamille P. Mallari, MD, DPSP

Liver
 The largest organ in the body. It is composed of 3 systems:
o Hepatocytes- concerned with metabolic reactions, macromolecular (especially protein synthesis), and the degradation
and metabolism of synbiotics, such as drugs
o Biliary system- involved in the metabolism of bilirubin and bile acids
o Reticuloendothelial system- concerned with the immune system and the production of heme and globin metabolites,
also part of the bilirubin synthesis
 The function of each of these systems can be measured conveniently and virtually non-invasively by determining the serum levels
and specific analytes in the so-called liver function test profile.

Disorders in Metabolic Functions

 Jaundice
o Comes from the French word John, which means yellow, and is one of the oldest known
pathologic conditions supported to having been described by Hippocrates.
o Yellow discoloration of the skin, eyes, and mucous membranes most often resulting from the
retention of bilirubin.
o It may be due to the retention of other substances.
 Icterus- Discoloration of the serum or plasma due to elevated bilirubin.
 Metabolism of Bilirubin
o Bilirubin is a byproduct of heme, which comes from the RBCs. When the RBCs are senescent or
to any causes of RBC destruction, its components are recycled in the body (the iron and protein
will be recycled, and heme will be degraded).
o Bilirubin initially is unconjugated in the body, and will circulate in the body, bound to albumin.
When this is unconjugated and it is bound to albumin, this is not water-soluble, and it does not
pass freely into the urine or kidney.
o The liver will then act on the unconjugated bilirubin using the UDP-glucuronyl transferase, and becomes conjugated bilirubin.
o Conjugated bilirubin is water-soluble and can pass freely into the urine, which is filtered by the kidneys.
o Majority of the conjugated bilirubin goes into the intestine, and it is further degraded by GI bacteria, becoming urobilinogen.
o Urobilinogen gives feces its color. After the GI bacteria will act on conjugated bilirubin, urobilinogen is produced and is seen in
feces.
o Other urobilinogen are recycled back into the body, into the extrahepatic circulation, wherein 20% of urobilinogen is absorbed
and recirculated to the liver and re-excreted in the feces, some of them go to the systemic circulation, and enters the circulation, and
it will go to the urine, and this also gives urine is yellowish color (majority goes to the feces and urine).

Disorders in Liver Function associated with patients presenting with

jaundice
 Jaundice is classified based on its inherent defect and abnormality.
 Classification: prehepatic, hepatic, and post hepatic jaundice
o This classification is very important in terms for the clinician or
anybody assessing the patient, because the diseases causing jaundice
can be narrowed down (many diseases can cause jaundice).
 Prehepatic Jaundice
o Most commonly (but not exclusive to this type of disorder), it is usually caused by hemolytic anemia.
o Pre-hepatic- problems comes before the liver
o Example: In hemolytic anemia, there is increased destruction of RBCs. When there is increased destruction in RBCs, a lot of heme
and bilirubin are produced. These bilirubin are unconjugated bilirubin, which will circulate freely into the plasma. Because it is an
abnormal event, usually at the start of the disease, the liver cannot cope with the increased amounts of unconjugated bilirubin and
the liver cannot process all of the unconjugated bilirubin from the system, so it will not be conjugated.
o Majority of the bilirubin that is circulating or seen are the unconjugated form of bilirubin.
o Usually, the liver is normal (no problem with the liver), but the main problem is that there is abundant/increased amounts of heme
being seen in the blood. Since they are unconjugated, they are not filtered into the urine.
 Hepatic Jaundice
o Usually, the main problem is the liver. Any problems that will affect the metabolic function of the liver will cause hepatic jaundice.
o These diseases may be due to any intrinsic liver defect, or diseases such as Crigler-Najjar syndrome, Dubin-Johnson syndrome,
Gilbert disease, and Neonatal disease of the newborn. There is problem inherently in the cells of the liver, or it may be due to any
hepatocellular injury or destruction (e.g. hepatitis, drug-induced hepatitis).
o There is destruction or inflammation of the liver secondary to virus or drugs that will damage the liver, or it can be due to any
space-occupying lesions, such as tumors or parasitic infections, that will cause cysts formation in the liver. Anything that involves
the liver may cause hepatic jaundice.
 o Liver conjugates the bilirubin, so what is seen is a mixed conjugated and unconjugated hyperbilirubinemia. It will depend on the
amount of damage the liver suffered. There will still be an increase in bilirubin in the blood, but is mixed.
 If 90% of the liver tissue is destroyed, the circulating bilirubin will be of the unconjugated form.
 If there is little damage, both conjugated and unconjugated hyperbilirubinemia are seen (depends if it can be filtered in urine or
not).
 Post hepatic Jaundice
o From the liver, all of its products will go to a canalicular system that will eventually pass into the gallbladder, and into the intestine.
o If there are problems in the canalicular system, that will produce post hepatic jaundice.
o Type of bilirubin seen is the conjugated bilirubin. There is no problems in the liver, because the liver will normally keep on
conjugating bilirubin, but the problem is it cannot go through the intestine or to the urine (cannot pass through the canalicular
system).
o Depending on etiology of post hepatic jaundice, the liver may be normal or abnormal, because sometimes, there are liver diseases
that will cause post hepatic jaundice, since it affects the canalicular system more than the intrinsic system of the liver (in biliary
obstructive diseases, like gallstones). Anything that will obstruct the bile duct system after the liver will cause a post hepatic
jaundice.
o Since there is obstruction in the bile canalicular system and increase in conjugated bilirubin, urobilinogen will be absent, so clay-
colored or acholic stools will be seen (since no urobilinogen is produced), and also in urine.

Intrinsic Inherited Disorders of the Liver

 Gilbert Syndrome
o Benign hereditary disorder that has a defective conjugation (cannot conjugate bilirubin).
o Most common problem: mutation in UGT1A1 o One of the most common causes of hepatic jaundice.
o No genetic consequences o C: intermittent unconjugated hyperbilirubinemia without hemolysis.
o Usually presents in adolescent or early adulthood. o Total Bilirubin: 20-50 umol/L
 Crigler-Najjar Syndrome
o Inherited disorder o C: non-hemolytic, unconjugated hyperbilirubinemia
o Defect in the gene involved in bilirubin conjugation
 The problem here is with bilirubin conjugation. The liver cannot conjugate bilirubin due to a mutation in the gene. Since it cannot
conjugate, the circulating bilirubin will be of the unconjugated form (similar with Gilbert syndrome).
o Type 1: complete absence of bilirubin conjugation o Type 2: severe deficiency of the enzyme
o More serious disorder (treatment must be done) but not as common as Gilbert syndrome.
 Dubin Johnson Syndrome
o Inherited obstructive disorder
o Deficiency of the canalicular multi-drug resistance/multi-specific organic anionic transporter protein (problem in protein that is
involved in a transport of the conjugated bilirubin from cell to cell)
o The circulating bilirubin present is of the conjugated form (acts more like a post hepatic jaundice). The bilirubin is conjugated by
the liver, but because of this defect, the cell itself cannot process/excrete/transport the conjugated bilirubin into the biliary system,
resulting to a conjugated hyperbilirubinemia.
o C: conjugated hyperbilirubinemia circulates as delta bilirubin
 The type of bilirubin that is produced is a conjugated bilirubin that is bound to albumin (referred to as delta bilirubin).
 Sometimes can be hard to distinguish from other diseases, but one feature is the appearance of dark-stained granules that is seen
in liver biopsy, and was initially thought as pigmented lysosomes. On endoscopy, the liver may appear black on gross inspection.
o Relatively mild with good prognosis o Total Bilirubin: 2-5 mg/dL (50% conjugated)
 Rotor Syndrome
o Inherited obstructive disorder o Benign with good prognosis
o Unknown defect reduction in the concentration or activity of intracellular binding protein
o Clinically similar to Dubin Johnson syndrome
 Wherein there is reduction in the concentration or activity of the intracellular binding protein, except that it has an unknown defect
and the gene that is involved is not identified (cannot excrete the bilirubin produced the liver into the biliary system)
 Physiologic Jaundice of Newborn
o Deficiency in the enzyme glucuronyl transferase, one of the last liver functions to be activated in prenatal life.
 Very low in activity in newborns, and almost absent in babies who are prematurely born (reason why infants are yellow)
o More commonly seen in premature newborns o Bilirubin levels of the baby is closely monitored.
o C: unconjugated hyperbilirubinemia (severity of disease is dependent on the level of bilirubin that builds up in the neonates’ body).
 Since babies cannot process bilirubin, rapid buildup of bilirubin may happen→ deposits
into the nuclei of the brain and nerve cells, causing kernicterus
 This causes severe neurologic impairment in newborns (problems with motor, speech,
anywhere that bilirubin will build up)
o Treatment: Mothers are advised to expose the baby into sunlight every morning. In ICU
patients, they are placed in under UV light.

Assessment of Liver Function/Liver Function Tests

 Bilirubin
 o Diazotized Sulfanilic Acid (main method)
Principle: Forms conjugated azo compound with bilirubin, producing color reaction; measured by spectrophotometry in 540nm

Direct Bilirubin (Conjugated)- Conjugated bilirubin will directly react with the diazotized sulfanilic acid.

Indirect Bilirubin (Unconjugated)- Needs accelerant (e.g. caffeine or methanol) to react with the diazotized sulfanilic acid.

Clinically, it is called conjugated/unconjugated bilirubin; but in the laboratory, it is called direct/indirect bilirubin.

Before, it was thought that total bilirubin= conjugated + unconjugated when using this method.

Direct bilirubin is only 70-80% of the total bilirubin, and the rest are of delta bilirubin and unconjugated bilirubin.

Specimen Collection and Storage

 Total Bilirubin (diazotized sulfanilic acid)- serum or  Malloy-Evelyn procedure- serum
plasma
o Malloy-Evelyn Procedure
 Principle: Bilirubin reacted with a diazo reagent, with a pH of 1.2 that will produce a red-purple reaction that can be detected in
560 nm wavelength.
 Accelerator used: methanol
o Jendrassik-Grok Method (Total and Conjugated Bilirubin Det.)
 Principle: Diazo reagent (sulfanilic acid HCl) is used→ after conjugation with bilirubin, it produces azobilirubin (purple)→
added with an alkalinized alkaline tartrate solution→ azobilirubin (purple)→ read at 600nm
 Total and conjugated bilirubin can be detected in this method.
 Indirect bilirubin- calculated by subtracting conjugated bilirubin and total bilirubin concentration
 Fractions: 1st aliquot: diazo reagent; 2nd aliquot: diazo reagent + accelerator (caffeine benzoate)
o False Decrease
 Prolonged exposure to light (direct bilirubin decreases 30-50% per hour)
 Infants with physiologic jaundice of newborn are exposed to sunlight to
destroy or degrade the circulating bilirubin. Infants in the ICU are placed into UV lamp with a similar principle.
 Use of wetting agents or incorrect pH buffer (indirect bilirubin)  Lipemia
o Falsely Increase
 Hemolysis- Any problems with extraction that will cause hemolysis will cause an abnormally elevated bilirubin
o Reference values for both total bilirubin are age and gender dependent.
o Stable for 2 days room temperature, 1 week at 1°C, and indefinitely 20°C.
 Urobilinogen
o Principle: Detected using the p-dimethyl aminobenzaldehyde (Erlich’s reagent)→ red color (detected by spectrophotometry)
 Ascorbic acid is used as reducing agent (patients tested for urobilinogen are usually advised not to take Vitamin C or any
Ascorbic acid products, which may interfere with false increased urobilinogen).
o Fresh 2-hour urine
o 0.1-1.0 Erlich units every 2 hours or 0.5-4.0 Erlich units per day o 1 Erlich = 1 mg urobilinogen
o Increased In: Hemolytic disease (abnormal amounts of heme), Defective liver cell function (hepatitis)
o Absent/Decreased In: Obstruction, Hepatocellular Carcinoma (tumor is a space-occupying lesion which will obstruct)
 Ammonia
o Enzymatic assays- incorporates glutamate dehydrogenase
 In this reaction, ammonia and alpha-ketoglutarate are acted upon by glutamate dehydrogenase, forming glutamate.
 Indicator: NADPH is then reduced into NADP  Detected using spectrophotometry at 340 nm
o Dry slide method
 Uses alkaline pH buffers  Converts ammonium ions to ammonium gas
 Indicator: Bromphenol blue→ measured spectrophotometrically
o Uses arterial blood
o Very unstable analyte, sample should be kept in ice water until plasma is separated
o Increased ammonia in the circulation→ causes damage to the CNS→ causing hepatic encephalopathy
 Commonly seen in patients with: Cirrhosis, Intrahepatic portal-systemic shunting, or Liver failure
 In liver failure, there is increased ammonia, which causes severe neurologic and CNS damage that causes hepatic
encephalopathy.
 Congenital deficiency of enzymes in the urea cycle  Cirrhosis- More than 80% of the liver is destroyed
 Acute fulminant hepatic failure (Reye’s syndrome)
o Reye Syndrome
 Group of disorder caused by infectious, metabolic, toxic or drug-induced disease
 Occurs exclusively in children  Unknown etiology; often preceded by a viral
syndrome
 Acute illness that causes noninflammatory encephalopathy and fatty degeneration of the liver
 Clinical Manifestation: Profuse vomiting with varying degrees of neurologic impairment
 In Reye syndrome, encephalopathy happens, there is inflammation by the brain and degeneration of liver.
 Degeneration of the liver causes: Mild hyperbilirubinemia, More than 3 times increase in ammonia,
AST, and ALT

Tests of Liver Injury (enzymes found in the liver)

 As metabolically complex cells, hepatocytes contain high levels of a number of enzymes.
 With liver injury, these enzymes will then leak into plasma, and can be useful for the diagnosis and monitoring of liver injury.
Within the hepatocytes, the commonly measured enzymes are found in specific locations (cytoplasm, mitochondria, or canalicular
system).
 The type of liver injury will then determine the pattern of enzyme change.
 Cytoplasmic Enzymes: LDH, AST, ALT  Mitochondrial Enzyme: Isoenzyme of AST
 Canalicular Enzymes: ALP, GGT
 Certain drug reactions and certain diseases that target a specific part of the cell/liver will cause different types of increase in the
number of enzymes.
 Liver enzymes play an important role in the assessment of liver function because injury to the liver resulting in the cytolysis or
destruction of the cell will cause release into the enzymes.
 Aminotransferase
o AST/SGOT (Aspartate aminotransferase) and ALT/SGPT (Alanine aminotransferase)
 Responsible for the conversion of aspartate and alanine to oxaloacetate and pyruvate, respectively.
 AST converts aspartate into oxaloacetate, and ALT converts alanine to pyruvate in an enzymatic reaction
 ALT/SGPT- are found in liver (primarily), skeletal muscle, and kidney
 Overall, ALT activity is more specific for detecting liver diseases in non-alcoholic asymptomatic patients.
 AST/SGOT- more widely spread, may be seen in heart, skeletal muscle, and liver
 In diagnosing certain diseases, a clinician/doctor must be aware of the locations on where the enzymes occur so that the range of
differential diagnosis will not be limited into a particular organ.
 Example: Patient had an accident, and there was severe skeletal muscle injury and kidney problems, so ALT was elevated. That
ALT elevation may lean towards a skeletal muscle or kidney in origin (based in the history).
 Rise rapidly  Remains elevated for 2-6 weeks
 Highest increased in: Viral hepatitis, drug and toxin-induced liver disease
 Since they are located in the cytoplasm of the cell, so any damage or destruction of
the hepatocytes will cause an increase in these enzymes because they will leak out
after destruction of the cell.
 Mild increased in: Obstructive liver damage
o Detection (uses an enzymatic assay)
 Uses alanine and aspartate for ALT and AST respectively→ glutamate→ glutamate dehydrogenase→ α-ketoglutarate
 Wherein oxidation of NAD→ NADH measured at 340 nm
 AST: aspartate→ oxaloacetate (malate dehydrogenase)→ malate  Reduction of NADH→ NAD at 340nm
 ALT: alanine→ pyruvate (pyruvate dehydrogenase)→ acetyl coenzyme  Reduction of NADH→ NAD at 340nm
A
 Alkaline Phosphatase (ALP)
o The ALP family of enzymes are zinc metalloenzymes that are widely distributed in all tissues.
o Highest activity is seen in the liver, bone, intestine, kidney, and placenta.
o Differentiates hepatobiliary disease from bone disease. o Liver- ALP localized in the microvilli of the bile
canaliculi
o Marker for extrahepatic biliary obstruction.
 Example: A patient who presented with jaundice. The patient took an ALP determination and found out that there is an increase in
ALP, that means that the jaundice is secondary to an extrahepatic biliary obstruction or any problems in the bile canaliculi system.
o Increase in: Stone with obstruction in the common bile duct (CBD), or any intrahepatic cholestasis
o Markedly increased in: Extrahepatic obstruction o Moderately increased in: Hepatitis and cirrhosis
o Also elevated in any primary bone-related disorder (one must be mindful of the clinical history of the patient)
 Lactate Dehydrogenase (LDH)
o Cytosolic glycolytic enzyme o Catalyzes the reversible oxidation of LD to pyruvate
o 5 major LD isoenzyme exist (LD1 to LD5): LD1 and LD2- heart muscle; LD4 and LD5- liver and skeletal muscle
o Historically, the LDH are measured based on their isoenzymes. Nowadays, it can also be used in diagnosing myocardial infarction.
o Wide distribution
o General, nonspecific marker of cellular injury (because of its wide range of location in the body).
o Moderately increase: acute viral hepatitis, cirrhosis o Mild increase: Biliary tract disease
 γ-Glutamyltransferase (GGT)
o Membrane-localized enzyme, regulates transport of amino o Found in: kidney, liver, pancreas, intestine, and
acid. prostate
o Main use: Differentiate the cause of elevated ALP o Half-life is about 10 days, but may remain as long as 28
days
o Usually in patients with liver disorders, the whole liver function liver profile has to be taken to determine if the disease in question
is coming from the liver (not one enzyme only). GGT will help confirm an elevated ALT that the disease is secondary to a liver
problem and not a bone problem (since ALT can also be seen in primary bone disorders).
o Elevated in: Chronic cholestasis secondary to primary biliary cirrhosis or sclerosing cholangitis
o 60-70% elevated in: chronic alcohol abuse (remains elevated 1 month after abstinence)
 5’-Nucleotidase (5NT)
 o Catalyze the hydrolysis of nucleoside-5’5 phosphate ester
o Although 5NT is found in a variety of cells, serum levels become significantly elevated in hepatobiliary diseases.
o No bone source- very useful in differentiating elevated ALT to the liver from other conditions where ALT may also be increased
 α-Fetoprotein (AFP)
o A protein (not an enzyme) synthesized in the embryonic hepatocytes, yolk sac cells; peak production in 2nd trimester
o Unknown function (but known to have an immunologic property in them that will help the infant/fetus in their immune system)
o Elevated in:
 Neural tube defects (e.g. spina bifida)- AFP is usually measured in pregnant women to monitor AFP levels because increased AFP
in a pregnant mom, it can be concluded to add additional work up for the diagnosis of neural tube defects
 Acute hepatic injury- usually increased due to regenerating hepatocytes; the cells in the liver usually regenerate; since they come
from embryonic hepatocytes, any acute hepatic injury that will cause regeneration of the cells will cause increase in AFP
 Germ cell tumors- yolk sac tumors and Sertoli Leydig cell tumors
o Marker for: Hepatocellular carcinoma (elevated in >90% of hepatocellular carcinoma)

Synthetic Functions
 Protein Synthesis
o Liver is a site of synthesis of most plasma proteins, except immunoglobulins and von Willebrand factor
o Inherently, any defect or disorders in the liver will also affect this function.
o Synthesis of more than 90% of all protein and 100% of albumin occurs in the liver, thus extensive destruction of the liver tissue
will result in low serum levels of total protein and albumin.
o Decreased in: Cirrhosis, hepatic destruction, and portal hypertension; may also be seen in patients with kidney problems (e.g.
glomerulonephritis) wherein there is increase in the excretion of protein into the urine
o 2 vital measurements: Total protein and Albumin
o Albumin- Usually seen in the blood for 20 days (half-life) and decrease in serum levels occur more slowly
 Any disorders of the liver, when albumin is measured, if it is in acute phase (days after the disorder), decrease in albumin is not
yet seen by then, and is only seen again 2-3 weeks after the first insult to the liver.
 The more acute reflection of liver injury may be seen in determining levels of Factor VII (coagulation factors) and transthyretin,
since they have a shorter half-life.
o Factor VII- Usually seen in the blood for 4-6 days (half-life)
o Transthyretin- Usually seen in 1-2 days
o Determination:
 Biuret Method
 Principle: Reflects the ability of the peptide backbone C=O groups of the
protein to form color complexes with copper, that absorb strongly at 540nm.
 Dye-Binding Method
 Principle: A protein forms a complex with the dyes
 Dyes that can be used:
 Coomassie blue
 Bromcresol green- used in determination of albumin exclusively
 Bromcresol purple- used in determination of albumin and globulins (serum albumin levels may be slightly lower when
determined with bromcresol purple)
 Serum Protein Electrophoresis
o Reference Range:
 Total Protein: 6-7.8 g/dL range; at least 60% is albumin
 Albumin: 3.5-5 g/dL
 Lectured by: Lovelyn Mae E. Cuison, RMT, MSMT
Objectives:
 Describe the location of the heart in the body  Trace the pathway of blood through the heart
 Identify the major anatomical areas of the heart

Anatomy
 Hollow muscular organ (has an empty space inside)  Approximately the size of a clenched fist
 Weight: 325 grams (male) and 275 grams (female)
 Length: 12 cm
o The heart of a well-trained athlete, especially if specializing in aerobic sports, doing a lot of exercises, or having an active
lifestyle, is considerably larger because cardiac muscles respond to exercise in a manner similar to that of skeletal muscles.
o The exercise could add up to the numbers of the protein myofilaments, and that could also increase the size of the
individual cells of the heart, without increasing their numbers (hypertrophy)
o Enlarged hearts are not always a result of exercise because they can also result
from pathologies like in hypertrophic cardiomyopathy or other conditions that
might be a sign of a disease involving the heart
 The apex of the heart is directed towards the left hip.
 Enclosed by a sac called pericardium
 Location of the Heart
o Located within the thoracic cavity medially between the lungs in a space
known as mediastinum, and within the mediastinum, the heart is separated
from the other mediastinal structures by a tough membrane or sac known as the
pericardium or pericardial sac, and it sits in its own space called the
pericardial cavity.
o The dorsal surface of the heart lies near the bodies of the vertebrae and the anterior surface sits deep to the sternum and the
coastal cartilages.
o The great veins (superior and inferior vena cava), great arteries (aorta), and the pulmonary trunk are attached to the superior
surface of the heart, called base. The base is located at the level of the 3rd coastal cartilage.
o The inferior tip of the heart, known as the apex, lies just to the left of the sternum between the
junction of the 4th and 5th ribs near their articulation within the coastal cartilages.
 Coverings of the Heart
o Pericardium- double layered fibrous membrane, double-walled sac
 Fibrous pericardium (superficial layer)
 Anchors the heart or the surrounding structures of the heart
 Prevents overfilling of heart with blood
 Superficial layer of the heart which protects the heart from overstretching because it contains
tough and elastic sac of fibrous connective tissues
 Serous pericardium
 Parietal (outer layer) pericardium- lines the internal surface of the fibrous pericardium
 Visceral (inner layer) pericardium- lines the surface of the heart itself (also known as
epicardium)
 Layers of the Heart Wall
o Epicardium (outer)- a thin transparent layer and it helps lubricate the heart and is composed
of delicate connective tissues where the coronary arteries and veins can be found
o Myocardium (middle)- largest and is mostly cardiac muscles and it makes up the majority of
the cardiac wall, and this layer is the one responsible for the pumping action of the heart
o Endocardium (inner)- consists of simple squamous endothelium layer and it provides a
smooth lining for the chambers of the heart, and it covers the valves of the heart
 Main function: Responsible for keeping the blood from sticking to the inside of the heart
and forming potentially deadly blood clots
 Cardiac Chambers (4 chambers of the heart)
o Receiving chambers- right atrium and left atrium
o Discharging chambers- right ventricle and left ventricle
o Chambers are separated by septum
o Two (2) atria- right and left
 Superior, smaller than ventricles
 Have thinner and less muscular walls than the ventricles because they do not do the
actual pumping of the heart (only receiving chambers)
 Primary receiving chambers (connected to the veins that carry blood to the heart, superior and inferior vena cava)
 Blood flows into the atria

o Two (2) ventricles- right and the left
 Pumps blood (they do the actual pumping of the heart, larger and stronger)
 Send blood out of the heart
 Connected to the arteries that carry blood away from the heart (e.g. pulmonary artery and aorta)
 That is where the ventricles are connected
o Septum- separates the left and the right side (chambers) of the heart
 Interatrial septum- separates the two upper chambers (right atrium and left atrium)
 Interventricular septum- separates the two lower chambers (right ventricle and left ventricle)
 Valves of the Heart
o Valves usually function in order to prevent the regurgitation/backflow of the blood in the heart
o Atrioventricular valves- connects each atrium to its ventricles
 Tricuspid valve- connects upper and lower chambers (right side, connects the right atrium to the right ventricle)
 Named such because it contains 3 cusps/flops
 Bicuspid/Mitral valve- connects the upper and lower chambers (left side, connects the left atrium to the left ventricle)
 Only contains 2 cusps/flops
 Atrioventricular valves are attached on the ventricular side of the heart to a tough string called as chordae tendineae
o Semilunar valves- are shaped like crescent moon; located between the ventricles and the arteries that carry blood away from
the heart
 Pulmonary valve- prevents the back flow of blood from the pulmonary trunk into the right ventricle (right side of heart)
 Aortic valve- prevents the aorta from regurgitating blood back into the left ventricle (left side of heart)
 Do not have chordae tendineae (heart strings)

Blood Circulation
 The blood from the systemic circulation will enter the heart. Because it has been
used up by the organs and the other parts of the body, the blood entering the heart is already oxygen-
poor (deoxygenated). The deoxygenated blood will travel through the inferior or superior vena
cava.
 Deoxygenated blood enters the right atrium, and passes through the tricuspid valve, and it
enters the right ventricle. It moves through the pulmonary valve, and enters the
pulmonary trunk/arteries, where it will be carried towards the lungs.
 In the lungs, the blood will gain oxygen, and once they are already oxygenated, they will be
returned to the heart via the pulmonary veins. The blood then enters the left atrium, and it
travels through the mitral/bicuspid valve, and enters the left ventricle. The blood is
pumped and moves through the aortic valve, through the aorta, and through the systemic
circulation.
 The blood going out of the heart is already oxygenated.
 Lectured by: Alessandra Kamille P. Mallari, MD, DPSP
Acute Coronary Syndrome
 The term acute coronary syndrome (ACS) is a general term, includes: angina, reversible tissue injury, unstable angina,
myocardial infarction (MI), and extensive tissue necrosis
 Most common (widely known) symptoms: chest pain, referred pain (pain referred to the arm, jaw, neck, back, or abdomen), nausea,
vomiting, dyspnea, diaphoresis, and lightheadedness (layman’s term: heart attack)
 Major cause of ACS is atherosclerosis.
o Atherosclerosis- essential to the development of acute coronary syndrome
o An inflammatory disorder, not a cholesterol issue, as there are many mechanisms that lead to the cellular injury (e.g. bacterial
infection, hyperlipidemia, glycosylated products in diabetes mellitus, and pro-inflammatory cytokines, among others)
o Basic concept: Cholesterol buildup→ Plaque formation→ Thickening and hardening of the blood vessel walls→ Narrowing of
arteries→ causes a Reduced blood supply or Ischemia (central pathophysiology of ACS)
 Predisposing Factors: Age, Sex, Family history, Dyslipidemia, Smoking, Hypertension, Sedentary lifestyle, and Diabetes mellitus
o Age- described to be present early in life; ACS common in adults > 40 years old
o Dyslipidemia- high Triglycerides and LDL with low HDL levels are the most detrimental, that can
lead to formation of atherosclerosis, that will lead to acute coronary syndrome
 Lowering LDL- one of the mainstay therapy/targets; majority of maintenance treatments for
patients who have ACS usually take anti-LDL or medicines that will reduce the LDL levels in the
blood
o Family history- atherosclerosis is often found in members of the same family; it is difficult to
distinguish genetic from lifestyle factors in predicting patients who had coronary artery disease
o Smoking- research has shown that there is a relationship between the number of cigarettes smoked and the risk of coronary artery
disease in men, it is not well defined in women
o Diabetes mellitus and vascular disease- there is a strong relationship/link between the development of atherosclerosis and ACS
o Sedentary lifestyle and history of hypertension- risk factors for the development of atherosclerosis
 Ischemia
o End-result/consequence of atherosclerosis
o Usually due to narrowing and stiffening of the blood vessels, that lead to reduction in blood flow to the heart, that becomes
ischemia
o 3 things that may result from ischemia in the heart: Chronic heart failure, Angina pectoris, and Myocardial infarction
 Angina pectoris- term used to describe chest pain due to coronary artery disease; often a symptom of ischemia; occurs when the
heart is not supplied with an adequate amount of blood due to blockage in one or more arteries that supply the blood
o Uncomfortable pressure, squeezing in the center of the chest.
o Myocardial infarction and angina pectoris typically occur in a step-wise fashion.
 Step-wise: Stable angina→ Unstable angina→ Blockage in angina pectoris→ Myocardial infarction (if blockage is not relieved)
o Angina pectoris has similar pathophysiology with myocardial infarction. In angina pectoris, the blockage is immediately relieved,
and no cellular death happens (if blockage is not resolved, it will become a myocardial infarction), but in myocardial infarction,
there is blockage and ischemia that is never relieved, resulting in tissue necrosis or cellular death.
o Stable Angina- presents as pain and discomfort in the chest, only when the patient is engaged in moderate activity, such as running
or walking; most patients usually experience chest pain/angina pectoris when they do any strenuous activity, but once the activity is
removed, the pain will then subside
o Unstable Angina- presents with pain and discomfort unpredictably at rest, and then it will be gone; no triggering factor
 Diagnosis of ACS
o Although the diagnosis is often straightforward, the challenge is to achieve a very high level of accuracy.
o A misdiagnosis of ACS in the emergency department (occurs in about 2% in cases) carries an increased risk of mortality, and is the
leading cause of malpractice payouts in the US.
o On the other hand, if the doctors are unable to justify the diagnosis of the patient, but they admit the patient and treat them for ACS
but the patient is not ACS, hundreds of thousands of money are wasted due to inaccurate diagnosis.
o 3 things: Chest pain and shortness of breath, Biochemical markers, 12-Lead ECG
 Usually in the diagnosis of ACS, the earliest decision making (which ideally should take place within 10 minutes upon the
patient’s arrival in the ER) is based on history and physical examination, and 12-Lead ECG.
 The ECG may establish a diagnosis of an ST-elevated myocardial infarction, at which point the patient is now a candidate for
immediate intervention. During this time, biomarker results are not necessary in these emergency interventions, and are likely to
be negative at this early in time.
 In ER: Patient arrives with chest pains, shortness of breath, pressure chest pain (a chest pain that radiates
in the back, arms, or shoulder), diaphoretic (sweating)→ Physician will take history (what did the patient
do prior to the chest pain), and is done while the patient is being placed on the 12-Lead ECG
 ST-elevation in the ECG + history of the patient of chest pain, shortness of breath, any predisposing factor= ER doctor can
conclude that it is most likely a heart attack/myocardial infarction→ Treatment will be done
 Most common treatment for myocardial infarction during this stage: relieve the blockage (by heparin,
etc.)
  Biomarkers does not have an important role at this time because:
1. Still very early after the onset of symptoms
2. Since conclusion can be made from the patient history and ECG
 Patient with shortness of breath, and other predisposing factors + Normal ECG (normal sinus rhythm)→ Biomarkers
 Cardiac Biomarkers are usually needed if the initial ECG is negative for ST elevation, and patient has predisposing factors.
 Biomarkers
 Cardiac Troponin (cTn)- preferred biomarker (usually, it is not present in a normal serum)
 Does not rise until a few hours after.
 Example: 1-2 hours after chest pain, the patient goes to the ER and gets a biomarker (troponin), it will be negative even if the
patient has myocardial infarction (timing of the test is important in these cases).
 Patients initially tested negative- repeat testing is warranted (if the symptoms persist)
 Testing of cTn happens in 0, 4, and 8 hours or 0, 3, and 6 hours after presentation of symptoms.
 During this time, the physician will now monitor if there is an increase in cTn of the patient (especially if ECG is negative).
 Testing happens at this timing because cTn rises usually 3-4 hours (depending on the cTn) after onset of symptoms. If it is
myocardial infarction, it will slowly rise during that time. If the disease is progressing, it will continue to rise.
 The 3rd repeat is important because if the damage to the heart is large, or if there is evolving or evolution of the ischemia, it
will correlate with the increase in cTn (e.g. values will double after 8 hours).
 When modest elevation of troponin is seen, repeat testing is important to establish baseline levels
 The 1st testing is important because it gives a gauge/baseline for reference in the future/subsequent testing.
 Patient experiences chest pain and shortness of breath + 12-Lead ECG normal + Biomarkers repeatedly normal (negative)
results→ Rule out NSTEMI (Non-ST elevation Myocardial Infarction)- other diagnosis must be sought for chest pain (e.g.
Angina pectoris)
 If patient’s symptoms resolve, has consistently negative ECG findings, and no increase in the biomarker:
 Suggested follow-up test: Testing for CHD (Chronic Heart Disease) is done or do a stress test
 Stress test- shows if the patient has a stable angina (which may be the cause of the chest pain)
 Not all chest pains are MI. Infections like costochondritis, heartburn, or any other infection can cause chest pain.
 Myocardial Infarction
o Defined as myocardial necrosis due to prolonged ischemia (endpoint)
o When there is narrowing of the arteries of the blood vessels secondary to atherosclerosis, it will cause ischemia. Without
treatment/intervention, ischemia will lead to necrosis.
o Categorized by the size of the infarct:
 Microscopic (focal necrosis)  Moderate (10-30% of the left ventricular myocardium)
 Small (10% of the left ventricular myocardium)  Large (30% of the left ventricular myocardium)
o Depending on the site of ischemia, or depending on what type of blood vessel it is affecting (e.g. if it is a large vessel that affects
majority or large portion of the heart, it will be a large myocardial infarction).
o Diagnosis (Biomarkers for the detection of myocardial necrosis like cardiac troponins I and T)
 More specific and sensitive for myocardial necrosis.
 Traditionally, a combination of cardiac markers has been recommended in the evaluation of many types of heart conditions,
because there is a lack of a single and ideal diagnostic test.
 The ideal biomarker must have:
 High specificity for myocardial damage in the presence of skeletal muscle injury.
 High sensitivity to detect very minor heart damage.
 Capability to differentiate reversible from irreversible cardiac damage.
 Ability to be used as a monitor of prognosis and therapy.
 Availability of rapid, easy-to-perform, and cost-effective quantitative assays.
 Absent or not detectable in patients without myocardial damage.
 Recommendation/guidelines from the National Academy of Clinical Biochemistry
 Use of 2 biomarkers to diagnose acute myocardial infarction:
1. Marker increased early after onset of symptoms (within 6 hours).
2. A definitive marker with a high sensitivity and specificity of myocardial damage that increases within 6-9 hours, and remains
elevated for several days.
 It is important that a cardiac biomarker should be detected within 6 hours, because there is a golden rule. With ischemia (the
oxygen to that particular area is cut off), the longer the oxygen is cut, the more cells will die. The faster the obstruction is
removed, the better the prognosis. In myocardial infarction, time is of the essence (usually chest pain, diaphoretic, or high
suspicion for a myocardial infarction is an emergency situation).
 If left untreated, the heart might stop, and there may be heart failure and death.
 Cardiac Enzymes
 Historically have been used to detect myocardial damage.
 AST and LDH are no longer used in diagnosis of MI. CK-MB remains the only enzyme routinely used for the diagnosis of MI.
 CK-MB
 Rise: 4-6 hours  Peaks: 12-24 hours  Normal within: 2-3 days
 Relative index: >3 is indicative of AMI.
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 Cardiac Proteins
 Myoglobin (1st one to rise and the 1st one to disappear)
 Rise: 1-4 hours  Peaks: 6-9 hours  Normal within: 18-24 hours
 Myoglobin concentration remains within the reference range within 8 hours after onset of chest pain- AMI can be ruled out
Example: Chest pain within the 9th or 10th hour that a myoglobin value was obtained, but it is normal (no changes, did not
rise), AMI can then be ruled out.
 Not cardiac-specific- increased in renal failure, trauma, and diseases involving skeletal muscle
 Small size and rapid clearance- persistently normal myoglobin will rule out reinfarction (in recurrent chest pain after AMI)
Similar to CK-MB in that it can also assess reinfarction in patients, but is not commonly used in the clinical lab
Example: Patient came in with chest pain→ Myoglobin was taken→ Normal→ Rule out AMI
 When repeated after few hours→ Myoglobin goes up→ AMI cannot be ruled out
Example: Patient came in diagnosed with AMI→ (+) ECG and has chest pain
 After a few hours or a day→ Chest pain→ Myoglobin was taken→ Normal→ No recurrent infection (reinfarction)
 Troponin
 Rise: 4-10 hours  Peaks: 12-48 hours  Normal within: 4-10 days
 Preferred biomarker because it is the sustained elevation of troponin that enables them to serve as a definitive marker for
AMI, because usually it will peak within 24 hours, and would last for several days.
 High sensitivity and specificity for myocardial damage because unlike CK-MB, serum troponins are not found in serum of
healthy individuals.
 Measured at 6-9 hours and 12-24 hours after onset of symptoms
 Useful in patients who do not seek ER within the 2-3 days.
 Cardiac Troponin T (cTnT)
Rise: 4-10 hours Peaks: 48 hours Normal within: 7-10 days
 Cardiac Troponin I (cTnI)
Rise: 4-6 hours Normal within: 6 days
Peaks: 12-18 hours (may show biphasic release) and at 48 hours
Useful in patients monitored after reperfusion therapy. Cardiac-specific
 Other Markers of Inflammation
 These biomarkers does not play a role in the diagnosis of MI. They will prognosticate or
identify patients who have higher risk than others to develop acute coronary syndrome.
 High-sensitivity C-Reactive Protein (hsCRP)
 In the development of atherosclerosis, inflammation plays a big role.
 The atherosclerotic plaque formation in acute coronary syndrome is usually centralized
due to inflammatory disorders or abnormalities. Because of their implication in these
processes, inflammatory cells, cytokines, and other biomolecules have been considered a
potential marker for the assessment of the risk for the development of such events.
 Most extensively studied marker of inflammation.
 Considered to be a general non-specific marker inflammation
 Increased baseline levels of hs-CRP are correlated with higher risk of future cardiovascular
(CV) morbidity and mortality among those with or without clinical evidence of CVD.
 Activates the classic complement pathway
 Stimulates phagocytosis
 Endothelial dysfunction via increased nitric oxide metabolism
 Increased LDL deposition in plaque by CRP-stimulated macrophages
 Clinical Uses
Screening for cardiovascular risk in otherwise “healthy” individuals
Predictive value of CRP levels for disease severity in pre-existing coronary artery diseases
 Elevated levels are predictive of: Long-term risks of first MI, Ischemic stroke (in an otherwise healthy individual)
 Limitations of CRP
Low specificity- If the patient has any other disorder that would affect the immune system or inflammatory agents in the
body, this might also be increased. Example: Lupus patients will also have increased CRP, so this
should be taken with context to the disease that is monitored.
No evidence that lowering CRP levels decreases cardiovascular risk.
 FDA staff guidelines 2005 had given clinical cut off value as less than 1 mg/L as safe level.
 Homocysteine
 Intermediary amino acid formed by conversion of methionine to cysteine.
 Recognized as an independent risk factor for the development of atherosclerotic vascular disease and venous thrombosis.
 Homocysteine is implicated directly in vascular injury including: Intimal thickening, Disruption of elastic laminin, Smooth
muscle hypertrophy, and Platelet aggregation
 Proposed mechanisms by which it induces vascular injury via leukocyte recruitment, foam cell formation, and inhibition of
nitric oxide synthesis.
 Elevated levels of homocysteine appear to be an independent risk factor, though less important than the classic CV risk factors.
 If homocysteine levels in the blood are increased, they may be predisposed to the development of ACS, because
homocysteine is usually seen in the walls of the blood vessels.
When they are increased numbers, a part of the blood vessel somewhere is exposed.
 Normal Levels: 3.7-13.9 umol/L

 Novel Cardiac Markers

 miRNA
 miRNA are found and involved in almost every biological process, from cellular differentiation and proliferation to cell
death and apoptosis.
 Can be detected in circulating blood and these miRNA are present on remarkably stable form that even withstand repetitive
freezing/thawing cycles, and protected against RNAses.
 miRNA that regulates cardiovascular system can be divided into 4 groups:
1. miRNA regulating endothelium function and angiogenesis: miR126, miR17-92 cluster, miR130
2. Cardiac myocyte specific miRNA: miR208a
3. Cardiac and skeletal muscle miRNA: miR1, miR133a, miR499
4. Smooth muscle miRNA: miR143, miR145
 miRNA hold promise as very specific and accurate marker of cardiac dysfunction.
 Markers for Myocardial Ischemia
 Ischemia Modified Albumin (IMA)
 A non-specific marker; also reported to be elevated in pulmonary infarction, critical limb ischemia, and cerebrovascular
disorders.
 Rises within minutes to 6 hours from onset of ischemia.
 Remains elevated up to 12 hours after cessation of ischemia
 Endothelial/extracellular hypoxia, acidosis, and ROS causes increase in IMA.
 Clinical Uses of IMA
Used as diagnostic criteria for myocardial necrosis that develops after CABG (Coronary artery bypass graft surgery)
operation
Used to rule out ischemia rather than diagnosing the occurrence of ischemia (ischemia can be ruled out if it is normal, but it
cannot diagnose ischemia when it is elevated).
Helpful in differentiating pain of Angina from Myocardial Ischemia (if normal: NOT myocardial ischemia)
 Heart Type Fatty Acid Binding Protein
 Found in the cytoplasm of myocardial cells.
 Involved in active fatty acid metabolism where it transports fatty acid from the cell membrane to mitochondria for oxidation.
 Appears as early as 1-3 hours after onset and peaking within 6 hours. Returns to normal levels within 12-24 hours.
 Normal Levels: 1.6-19 ng/mL
 20 times more specific to cardiac muscle than myoglobin.
 Recommended to be measured with troponins to identify MI and ACS in patients presenting with chest pain.
 H-FABP Prognostication
 The high risk associated with increased H-FABP is dependent upon its concentration.
 Patients who were cTnI (-) but H-FABP (+) have more risk of morbidity and mortality after 1 year follow-up than those cTnI
(+) but H-FABP (-) patients.
 These are all being currently studied, and has not been used in practice.