CLINICAL CHEMISTRY 1 Types of Glassware:

PRELIM 1 Borosilicate glass (pyrex and kimax)

 It is used for heating and sterilization
Lesson 1: BASIC PRINCIPLES AND PRACTICE OF
purposes; MOST COMMONLY USED
CLINICAL CHEMISTRY.
 High resistance to thermal shock & chemical
 Instrumentation attack; Low Alkaline content
 Laboratory mathematics  Used for most beaker, flask, pipet can be
heated and autoclaved
Lesson 2: SPECIMEN COLLECTION & HANDLING  Strain point: 515° C (Pyrex)
 Types 2 Boron-free glassware / Soft glass
 Collection and Labelling  Alkali resistant, Poor heat resistance
 Handling, transport, processing, storage and  Used for highly alkaline solutions
presentation
3 Corex (Aluminosilicate glass)
Lesson 3: METHOD OF EVALUATION  Is a special alumina-silicate glass that has
 Basic Concepts been strengthened chemically than thermally
 Definition of terms  Six times stronger than borosilicate
 Statistics
4 Vycor (Corning)
 Quality control and quality assurance
 It is utilized for high thermal, heat shock
 Quality control charts
resistance
 Intra-laboratory QC monitoring  Acid resistant; it can heated to 900° C
 Analytical technique
5 Flint glass
 Made up of soda-lime glass and a mixture of
CLINICAL CHEMISTRY calcium, silicon and sodium oxides.
 It has poor resistance to high temperature –
 Comes from the Greek word “kline” (means bed)
easy to melt and used to make disposable
 Deals with science elements, compound,
glassware
chemical structure, and interaction of matter
 It is a basic science that utilizes the of chemistry 6 Low actinic
to study human beings in various stages of health  Contain materials that imparts amber/red
and disease color to the glass. It is used to decrease
 Involves with the quantitative measurement of exposure to light (eg. Bilirubin standards)
biochemical substances found in body fluids
essentially blood. This involves the knowledge
and understanding of the basic concepts and Pipet Classification
principles of their metabolism, laboratory
analysis, and pathophysiology. The course also I. Calibration Marks/Design:
deals with instrumentation, quality assurance and 1. To Deliver (TD) – it delivers the exact
safety are given due emphasis. amount it holds into a container.
2. To Contain (TC) – it holds the particular
volume but does not dispense the exact
BASIC PRINCIPLES AND PRACTICE OF volume.
CLINICAL CHEMISTRY II. Drainage Characteristics:
1. Blowout – it has continuous etched rings on
INSTRUMENTATION top of pipet; exact volume is obtained when
the drop is blown out.
2. Self-draining – absence of etched rings;
liquid is allowed to drain by gravity.
III. Types:
1. Transfer Pipet
 Volumetric Pipet – for nonviscous fluid; sel-
draining; small amount left in the tip should
CLINICAL LABORATORY SUPPLIES not be blown out.
  Ostwald Folin – for viscous fluid; with
etched ring.
AUTOMATION PARAMETERS/TERMINOLOGIES:
 Pasteur Pipet – transfer fluids without
consideration of a specific volume, no 1. Batch testing - All samples are loaded at the
calibration marks. same time, and a single test conducted on each
 Automatic macro- or micropipettes – sample.
MOST ROUTINELY USED pipet in 2. Parallel testing – more than one test is analyzed
today’s clin chem lab; may be fully concurrently on a given clinical specimen.
automated/self-operating, semiautomatic, or 3. Random access testing – any test can be
manually operated. performed on any sample in any sequence.
2. Graduated or Measuring Pipet 4. Sequential testing – multiple tests analyzed one
 Serological Pipet – with graduations to after another on a given specimen.
the tip; blowout pipet. 5. Open reagent system – a system other that
 Mohr Pipet – without graduations to the manufacturer’s reagents can be utilized for
tip; calibrated between 2 marks; self- measurement.
draining pipet. 6. Closed reagent system - a system where the
 Ball, Kolmer and Kahn Pipet, operator can only used the manufacturer’s
Bacteriologic Pipet reagents.
 Micropipet – eg. Lang-Levy Pipet, RBC
and WBC pipets, Kirk and Overflow
Pipet THREE BASIC APPROACHES TO
AUTOIMATION (ANALYZER)
1. Continuous Flow Analyzer
 Liquids are pumped through a system of
continuous tubing.
 Sample flow through a common reaction vessel
or pathway.
 A heating bath maintains the required
temperature of the reaction to allow complete
color development (same with discrete analyzers)
 Example: Simultaneous Multiple Analyzer
(SMA), Technicon
 Disadvantages: all tests are performed in parallel
MECHANISM OF AUTOMATIC MECHANICAL 2. Centrifugal Analyzer
MICROPIPET  It uses the force generated by centrifugation to
transfer specimen and reagents.
a. Air Displacement Pipet  Examples: Cobas-Bio (Roche) and IL Monarch
It relies on piston for suction creation to draw the  Major advantage: Batch analysis (discrete-batch
sample into a disposable tip. The piston does not type system)
come in contact with the liquid. 3. Discrete Analyzer
b. Positive Displacement Pipet  It is the most popular and versatile analyzer –
It operates by moving the piston in the pipet tip or measures only the tests requested on a sample.
barrel, much like a hypodermic syringe. It does
 It requires 2-6ul of the sample (minimum
not require a different tip for each use.
volume)
c. Dispenser/Dilution Pipet
 It is capable of running multiple-tests-one-
It obtains liquid from a common reservoir and
sample-at-a-time
dispensed it repeatedly. It combines sampling and
 Example: Vitros, Dimension Dade, Beckman
dispensing functions.
ASTRA System, Roche Cobas Integra
REMEMBER:  Major advantage: Random access capability –
allows STAT samples to be easily tested.
 Class A pipets don’t require calibration
 Distilled water is the calibrating medium for TD Advantages of Automation:
pipettes while mercury is for TC pipettes.
1. Increase the number of tests to be performed in a
 Glass Pipet is the MOST BASIC PIPET
given period.
 2. Minimizes variation of result from one ­ Bacterial/microbiological content, pH,
laboratorian to another. resistivity, SiO2, chemical oxygen
3. Eliminates the potential error in manual analyses demand, ammonia, ions, metals.
such as pipetting, calculation and transcription of  Filtration is the first step before the processes are
results. performed in reagent grade water preparation.
 Processes involved in the preparation of reagent
CHEMICALS USED INSIDE THE LABORATORY
grade water: distillation, ion exchange, reverse
Grades of Chemical/Chemicals used for reagent osmosis and UV oxidation.
preparation:
 The most commonly found organisms in water
GRADE CHARACTERISTICS after the purification process is complete are
1.Analytical Reagent - Very high purity Gram-negative bacteria.
Grade (AR) - Meets Specifications of American
Chemical Society. THREE GRADES OF REAGENT WATER
- Recommended for qualitative and 1. Type 1 Reagent Water
quantitative analyses / most  Is used for test methods requiring minimum
analytical lab procedures; essential
interference, for procedures that require
for accuracy.
maximum/highest water purity for accuracy
- Labels on these reagents either
state that the actual impurities for and precision. It should be used immediately
each chemical maximum allowable (storage is discouraged) after production.
impurities (percentage of impurities)  Use: Flame Photometry, AAS, blood gases
- Use: trace metal analysis and and pH, enzyme studies, electrolyte testing,
preparation of standard solutions. HPLC, trace metal and iron studies.
2.Ultrapure Reagents - These types of reagents have been 2. Type II Reagent Water
put through additional purification  It is acceptable for preparation of reagents
steps. and quality control materials. For
- Example: Spectrograde, nanograde, hematology, microbiology, immunology and
and HPLC grade/pure. chemistry.
- Use: gas chromatography, atomic 3. Type III Reagent Water
Absorption spectrophotometry
 Most commonly used for washing glass
(AAS), HPLC, fluorometry, and
wares.
Immunoassays.
 For urinalysis, parasitology and histology.
3.Chemically Pure (CP) - Limits of impurities not specified –it
or Pure Grade fails to reveal the tolerance limits of
Impurities. DISTILLED WATER
- Preparation of these chemicals is not
uniform. Purity is usually delivered  Is the condensate collected from steam and
by measurement of melting point. It created when water is boiled and vaporized
is not recommended for clinical labs  It has been purified to remove almost all
but may be for some lab applications organic materials.
when higher purity chemicals are not
available.
DEIONIZED WATER
4.Technical/Commercial - It is used primarily in manufacturing
Grade or for industrial use. It should never  It is prepared by using deionizer (anion and
be used in clinical laboratory testing. cation) and it is free from mineral salts;
5.United States - Is approved for human consumption removed by ion exchange processes.
Pharmacopoeia (USP) (not injurious to individuals) but  It has some or all ions removed, but organic
& National Formulary may not be applicable for laboratory material may still be present.
(NF) manufacturing analysis.  Is purified from previously treated water such
- Use: for drug manufacturing.
as prefiltered or distilled water.

 Water is the most frequently used reagent in the

laboratory. Water is determined by (test for water Types of Solution:
purity):
1. Dilute Solution – it has the presence of relatively
little solute.
 2. Concentrated Solution – it has a large quantity between
of solute in solution. compartment of
3. Saturated Solution – a solution in which there is differing
an excess of undissolved solute particles. concentration.
4. Super Saturated Solution – it has a greater
concentration of undissolved solute particles that LABORATORY
does a saturated solution of the same substance. MATHEMATICS/CALCULATIONS
REFERENCE MATERIALS: PERCENT SOLUTION
 Is a calibration material that should meet the  is equal to parts per 100 or the amount of solute
identity, labelling and performance per 100 total units of solution
requirement of CLSI.  it is determined in the same manner regardless of
1. Primary Standard – is a highly purified whether it is w/w v/v or w/v units are used
chemical that can be measured directly to produce 1. Weight/Volume (w/v) % solutions = grams
a substance of exact known; could be measured solute + ml sol'n x 100
alone.  Is the most common type of solution prepared in
clinical laboratory. It refers to the number of
2. Secondary Standard – is a substance of lower
grams of solute per 100 ml of solution
purity whose concentration is determined by
comparison with a primary standard; can’t be Grams of solute = % solution desired x total volume désired
measured without primary standard. 100

2. Volume/Volume (v/v) % solutions = ml solute

COLLIGATIVE PROPERTIES
÷ mL sol’n x 100
 Physical properties of solution that depend on  It is used when both solute and solvent are liquid
the relative concentration of solute and it refers to the amount of solute in ml in 100mL
solvent but not on their identities. of solvent

COLLIGATIVE DEFINITION CHANGE mL of solute = % solution desired x total volume desired

PROPERTIES PER 100
OSMOLE OF 3. Weight/wright (w/w) % solutions = grams
SOLUTE solute ÷ grams solute x 100
Vapor Pressure Pressure at DECREASED
 refers to the number of grams of solute per 100
which liquid BY 0.3 mmHg
grams of solution.
solvent is in or torr
equilibrium Grams4.of solute = % solution desired x grams of the total solution
with water
vapor. 100
Freezing Point Temperature at DECREASED
which the vapor BY 1.86° C NOTE: When preparing concentrated acid solutions,
pressure of the always add acid to water
solid and liquid
phases of a
substance are
the same.
Boiling Point Temperature at INCREASED
which the vapor BY 0.52° C
pressure of the
solvent reaches
1 atm
Osmotic Pressure Pressure that INCREASED
opposes BY 1.7X10^4
osmosis when
the solvent
flows through a
semipermeable MOLARITY (M)
membrane to
 is the number of moles of solute per liter of
establish
solution (mol/L)
equilibrium
  1 mole of substance equals its gram molear
weight (gmw) RATIO
 Gram Molecular Weight (GMW) is obtained by  Amount of something in proportion to the amount
adding the atomic weights of the of something else Formula
component elements (ex. NaCl=58.44 g/mol  Volume of solute/volume of solvent
where Na: 22.99 Cl: 35.45)
TEMPERATURE
 C = (°F-32) x 5/9 or °C (°F-32) x 0.556
 °F = (°C x 9/5) +32 or °F = (°C x 1.8) +32
Molarity of sol’n (M) = Grams of solute____
 Kelvin (K)=°C+273.15
GMW x vol of solution (L)
Analytes-are simply just the test, that have been requested
by the physician to a px; common test in chemistry section
Mole (mol)-base SI unit for the amount of a substance
Problem Solving:
______Gram_____ 1.2 mL of solvent is added to 8mL of stock sol'n. Find the
Molecular weight dilution, ratio, total volume

Dilution: (solute/total vol of solution)

NORMALITY (N) Solute=8mL
 is the number of Equivalent weight per liter of Total volume = 8+2=10 mL
sol'n (Eq/L) Dilution: 8:10
 Often used in acid base calculations Ratio: solute to solvent
Normality (N) = Grams of solute EW =__ MW__ = 8.2 where solvent = 2mL
EWx vol (L) Valence
Total volume = 10 mL

MOLALITY (m)
Problem Solving:
 is the amount of solute per 1 kg of solvent
2.2 mL of stock sol'n added to 8 mL of solvent.
(mol/kg)
a. 2:10, 2:8
 MW is obtained by adding atomic weights the
b. 10:2, 8:2
given compound
c. 2:8, 2:10
Molality (m) = __ Grams of solute_____
MW solute x kg solvent Dilution: (solute/total vol of sol'n)
=2:10
where: solute- 2mL; 2+8= 10mL total vol of sol'n
MILLIEQUIVALENT Ratio: solute to solvent
 Is commonly used for reporting electrolyte.
= 2:8
mEq/L = __mg/dL x 10 x valence__
MW
Problem Solving:
How many moles of NaCl is needed to make 75 grams of
MILLIMOLES NaCl in 1L of sol'n?
 is molecular weight in millimoles (mmol/L)  MW of NaCl = 58 g/mol

mmol/L = _mg/dl x 10_ Gram

Moles =
MW Molecular weight

DILUTION 75 grams = 1.29 moles or 1.3 moles

 Indication of relative concentration 58 grams/mol
 Volume of solute/total volume of sol’n
 Formula: C1 x V1 = C2 x V2
 Formula: %1 x V1 = %2 x V2
Problem Solving:  Volume loss by sweating and changes in hormone
If a solution contains 111grams CaCl2 per liter, what is concentrations
the normality?  Increased in lactate, fatty acid, ammonia; long-
 MW of CaCl2 = 111g term increased in AST, LD and aldolase
Normality (N) = Grams of solute EW = MW  Increased in prolactin, testosterone and
EW vol (L) Valence luteinizing hormone (LH)
 Elevated levels of proteins in urine (proteinuria)
 Vigorous hand exercise (fist clenching)
= = ↑potassium, lactate and phosphorus; ↓ pH
.  Decreased plasma levels of follicle stimulating
hormone and luteinizing hormone in long-
=2 = 55.5
distance athletes.
2. Fasting
Problem Solving:  (Normal fasting) Glucose: 8 to 12 hours
How many grams are needed to make 1 liter of a/2 M (prolonged fasting causes falsely elevated)
(Molarity) solution of HCI?  (Normal fasting) Lipids: 12-14 hours
 MW of HCI = 36.5g  48 hours of fasting may increase serum bilirubin
 72 hours of fasting may result to increase of
Molarity of sole (M) = Grams of solute
plasma triglyceride while glucose decreases in
GMW vol of solution (L)
healthy woman to 45mg/dL
 Basal state collection: glucose, chole, TAG,
𝑥
2M = electrolytes
36.5 𝑥 1𝐿
-is early morning blood collection, 23 hours after
the last ingestion of food
x = 79g
3. Diet
 Metabolic products of food can increase in
venous blood (high protein diet-increases urea).
Problem Solving:
What amount of NaCl is needed to make 800 mL of 0.85%  High protein, low carbohydrate diets, such as the
solution? Atkins diet, greatly increased ketones in the urine
Percent Solution (w/v) = weight of solute x 100  Fat-rich food may increase potassium, ALP TAG
vol of soln and 5-hydroxyindole acetic acid (5-HIAA).
0.85% = x x 100  Serotonin-rich food (banana, pineapple, tomato
100 800 ml and avocado) increase the urinary excretion of 5-
100 HIAA.
800 (0.0085 = x) 800  Caffeine increases concentration of glucose; it
100 promotes the release of catecholamines from
6.8g = x adrenal medulla and brain tissue. NPS
 Increased in obese persons; LD, cortisol and
LESSON 2 SPECIMEN COLLECTION AND
glucose
HANDLING
4. Posture or position
PATIENT PREPARATION  Preferred position during phlebotomy: upright
position or supine (lying).
Prior to blood collection, patients must be given correct  Patient should be seated/supine for at least 15 to
instructions on how to prepare for each laboratory test. 20 minutes before blood collection to prevent
Utmost care must be observed to minimize factors that hemodilution or hemoconcentration.
may influence laboratory results.  Changing from supine to sitting or standing
position: causes constriction of the blood vessels
Factors contributing to the variation of results: and reduction of plasma volume-↑levels of
albumin, enzymes and Ca
1. Exercise  Sitting to supine: shifting of water and
 Volume shifts between the vascular and electrolytes into tissue causing
interstitial compartments hemoconcentration - ↑ levels of proteins. Lipids,
BUN, Fe and Ca
  Standing to supine: causes extravascular water Affected by gender (increased levels):
to transfer to the vascular system and dilutes non-
diffusable plasma constituents-↓of cholesterol,  Males – albumin, ALP, creatine, uric acid,
triglycerides and lipoproteins cholesterol, BUN
 Significant elevation of potassium after 30  Females – HDL, iron and cholesterol
minutes of standing is due to the release of
potassium from muscles. Affected by recent food ingestion:
 Prolonged bedrest: decreased plasma albumin
due to fluid retention  Increased levels – glucose, TAG, gastrin, free
5. Tourniquet application Ca2
 One-minute application is recommended.  Decreased levels – electrolytes (Cl, K), ALP,
 Prolonged tourniquet application results to AMS
hemoconcentration (venous stasis) and
SPECIMEN COLLECTION AND HANDLING
anaerobiosis.
 Increased levels: potassium, total proteins,  Proper patient identification is the first step in
enzymes, lactate, chole, NH3 sample collection – this is the prime factor in
6. Tobacco smoking (nicotine) order to attain accurate results in the clinical
 Increased in plasma catecholamines and cortisol laboratory
 Increased in glucose, growth hormone,  Observance on the confidentiality of results and
cholesterol, triglyceride, ammonia, urea, lactate, proper techniques in specimen collection must be
insulin and urinary 5-HIAA strictly followed
 Decreased plasma levels of vitamin B12 and
elevated thiocyanate. Patient Identification procedures:
7. Alcohol ingestion
 Increased level of urate, triglyceride and gamma 1. Conscious Inpatients/ Hospitalized patients
glutamyl transferase (GGT).  Verbally ask their full names using the
 It causes hypoglycemia (chronic alcoholism). identification bracelet (first and last names,
8. Stress (anxiety) hospital/unit number, room/bed number and
 It affects adrenal hormones secretion. physician’s name)
 Increased: catecholamines, cortisol, ACTH,  Do not state a patient name
prolactin, insulin, albumin, glucose and lactate 2. Sleeping patients
9. Drugs  They are identified in the same manner as
 Medications affecting plasma volume can affect conscious in-patients.
protein, BUN, iron and calcium concentrations  They must be awakened before blood collection.
 Vit. C positively/negatively affects analytic, 3. Unconscious, Mentally Incompetent Patients
methods based on reduction oxidation reactions  They are identified by asking the attending nurse
 Diuretics decease sodium and potassium. or relative; ID bracelet.
4. Infants and Children
Physiologic variation – refers to changes that occur
 A nurse or relative may identify the patient, or by
within the body such as cyclic changes (diurnal or
means of an identification bracelet.
circadian) or those resulting from exercise, diet, stress,
5. Outpatient/Ambulatory Patient
gender, age, drugs, posture or underlying medical
 Verbally ask their full names, address or birth
conditions.
date, and countercheck with driver’s license, or
Affected by diurnal variation: ID card with photo.
 If the patient has identification card or bracelet,
 Increased in AM: ACTH, aldosterone, cortisol same manner as with hospitalized patients.
and iron
 Decreased in PM: Acid Phosphatase (ACP), GH,
PTH, TSH

Affected by age (increased levels): albumin, ALP,

cholesterol and phosphorus
General Methods of Blood Collection:  Sites: antecubital
fossa region, veins on
 Average human the wrist and dorsal
body contains aspect of hands, veins
approximately 5 on the ankle
quarts (4.73 L) of  Median cubital vein
whole blood. is the best site for
 For adult males, venipuncture because it is the largest and the best
they approximately anchored vein.
5 to 6 liters of whole  An attempt must have been made to locate the
blood. median cubital vein on both arms before
 For adult females, considering an alternate vein.
they have
 Cephalic vein is the 2nd choice; basilic vein is the
approximately 4 to 5
3rd choice.
liters of whole blood.
 Basilic vein should not be chosen unless no other
 Whole blood is composed of approximately:
vein is more prominent due to its close proximity
a. 55% of plasma
to the brachial artery.
b. 45% of cells
 Veins on the dorsal part of hand or wrist area can
1. SKIN PUNCTURE
be chosen provided the antecubital veins are not
 A fingerstick to
acceptable.
obtain blood for
 Ankle vein should be used only if arm veins have
routine laboratory
been determined to be unsuitable.
analysis is usually
 If petechiae appear after veni, it indicates that
preferred for
small amounts of blood have escaped into skin
children older
epithelium.
than one year old.
 For blood gases measurement, venous blood is
 Length of the lancet: 1.75mm (preferred; to avoid
not the specimen of choice because it usually
penetrating the bone)
reflects the acid-base status of an extremity not
 The depth of the incision should be < 2.0 mm for
the body as a whole.
infants and children; and < 2.5 mm for adults, to
avoid contact with the bone. Sites to be avoided:
 The distance from the skin surface to bone or
cartilage in the middle finger is 1.5-2.4 mm. 1. Intravenous
 Wipe the first drop of blood and don’t squeeze it. lines in both
 During the blood collection, do not milk the site arms
to prevent hemolysis and excess tissue fluid 2. Burned or
 Preferred: 4th and middle finger (no the side, not scarred areas
in the middle) 3. Areas with
hematoma
Preferred Sites: 4. Thrombosed
veins
1. Lateral plantar heel surface – newborn 5. Mastectomy (on
2. Palmar surfaces of the fingers (3rd and 4th fingers) one or both
3. Plantar surface of the big toe arms)
4. Earlobes – least site 6. Edematous arms
2. VENIPUNCTURE 7. Arms with
 A process by which fistula or AV
blood is obtained from a shunt
patient’s vein. 8. Cast on arm
 Venous blood is the
deoxygenated blood
with a dark red color.
  23 gauge butterfly is most commonly used for
3. ARTERIAL PUNCTURE small and difficult veins.
 Arterial blood is the oxygenated blood with a  The gauge of the needle is inversely related to the
bright red color. size of the needle, the larger the gauge
 Use: for blood gas analysis and pH
measurement
 Sites: radial artery, brachial artery, femoral artery,
scalp artery and umbilical artery
 Blood sample is collected without a tourniquet
 The femoral artery is relatively large and easy to
puncture, but extra care must be given to older
individuals because the femoral artery can bleed
more than the radial or brachial.
 Arterial bleeding is the hardest to control and
usually requires special attention.
 Major complications: thrombosis, hemorrhage,
and possible infection
 Unacceptable sites: irritated, edematous, near a
wound, or in an area of an arteriovenous (AV)
shunt or fistula

Angle:

 Arterial puncture = 45-60 degrees (90 degrees for

femoral artery)
 Venipuncture = 15-30 degrees (bevel side up)

Tourniquet application:

 Apply tourniquet 3-4 inches above the site of

puncture
 If blood pressure cuff is used as a tourniquet, it is
inflated 60mmHg.
 Tourniquet is applied to obstruct the return of
venous blood to the heart and distend the veins

Disinfection of the site for puncture:

 No traces of alcohol should remain on the skin

because it may cause hemolysis, and may
contaminate glucose testing.
 Allow the area to dry, or wipe dry in an outward
circular motion with a gauze pad top prevent pain
and hemolysis due to residual alcohol. Do not
blow on the area as this will contaminate the site.

Needle specifications:

 21 gauge needle is considered the standard for

venipuncture
 23 gauge is used for children
 23 or 25 gauge is for winged infusion set
(butterfly)
 Phlebotomy Complications
1. Vascular complications Bleeding from the site of the venipuncture and hematoma formations are the
most common vascular complications.
2. Infections The second most common complication of venipuncture is infection.
3. Anemia Iatrogenic anemia is also known as nosocomial anemia, physician-induced
anemia, or anemia resulting from blood loss for testing. This can be a
particular problem with pediatric patients.
4. Neurologic Post-phlebotomy patients can exhibit some neurologic complications,
complications including seizure or pain.
5. Cardiovascular Cardiovascular complications include orthostatic hypotension, syncope,
complication shock and cardiac arrest
6. Dermatologic The most common dermatologic consequence of phlebotomy is an allergic
complications reaction to iodine in the case of blood donors

Reasons for rapid separation of blood (after centrifugation):

 Ideally, all measurement should be performed within 45 minutes to 1 hour after collection
1. To prevent glycolysis
2. To prevent lipolysis
3. Certain substances are very unstable
4. To prevent shift of electrolytes
5. To prevent hemolysis
Effect of hemolysis
 ↑enzymes (LD, ACP, ALT, AST), electrolytes (Mg, K, P), total protein, albumin, chole, iron,
bilirubin levels
 Inhibits lipase enzyme, interferes with color reactions

SPECIAL TEST REQUIREMENTS

REQUIREMENTS EXAMPLES COMMENT

Fasting Fasting blood sugar (FBS), Nothing to eat or drink (except water) for at
triglycerides, lipid panel, gastrin, least 8 hours
insulin
Chilling ACTH, acetone, ammonia, gastrin, Place in slurry of crushed ice and water.
glucagon, lactic acid, pyruvate, Don’t use ice cubes alone because RBCs may
PTH, renin lyse
Warming Cold agglutinins, cryoglobulins Use 37˚ C heat block, heel warmer, or hold in
hand.
Protection from light/ Bilirubin, carotene, erythrocyte Wrap in aluminum foil/ amber bottle
Photosensitive analyte protoporphyrin, vitamin A, vitamin
B12
Chain of custody/ Chain Any test used as evidence in legal Documentation of every step from patient
of evidence proceedings (e.g. blood alcohol, identification, handling, processing, testing,
drug screens, DNA analysis) and reporting results
CHEMISTRY PANEL

PANEL TEST
BASIC METABOLIC PANEL Na+, K+ chloride, CO2, Glucose, creatinine, BUN, Calcium
ions
COMPREHENSIVE METABOLIC PANEL Na+, K+ chloride, CO2, Glucose, creatinine, BUN, albumin,
total protein, ALP, AST, bilirubin, Calcium ions (Ca2+)
ELECTROLYTE PANEL Na+, K+, Cl-, CO2 or Bicarbonate (HCO3-)
HEPATIC FUNCTION PANEL Albumin, ALT, AST, ALP, bilirubin (total & direct), total
protein
LIPID PANEL Total cholesterol, HDL cholesterol, LDL cholesterol,
triglycerides
RENAL FUNCTION PANEL Na+, K+, CO2, glucose, creatinine, BUN, Calcium ions,
albumin, phosphate

Ten Common Errors in Specimen Collection

1. Misidentification of patient
2. Mislabeling of specimen
3. Short draws/wrong anticoagulant ratio
4. Mixing problems/clots
5. Wrong tubes/wrong anticoagulant
6. Hemolysis/ lipemia
7. Hemoconcentration from prolonged tourniquet time
8. Exposure to light/extreme temperatures (photosensitive)
9. Improperly timed specimens /delayed delivery to laboratory
10. Processing errors: Incomplete centrifugation, incorrect log-in, improper storage
Commonly encountered pre-analytical errors, improper filling of sample tubes, wrong choice of tubes or containers
and selecting the incorrect test.

Reasons for Spx Rejection:

1. Hemolysis/ lipemia
2. Clots in an anticoagulated tube
3. Non-fasting specimen (if required)
4. Wrong blood collection tube
5. Short draws
6. Improper transport (temperature)
7. Discrepancies between requisition and
specimen label
8. Unlabeled or mislabeled specimen
9. Contaminated specimen/leaking container
Types of samples

 Hemolyzed: rupture of RBCs so, HB released from

RBCs
 Icteric: serum appears yellow due to high-bilirubin
 Lipemic: serum appears milky or turbid due to high
lipid
ANTICOAGULANTS

1. Oxalate  It combines with calcium to form an insoluble salt.

8-10x INVERSION  It interferes with Na, K, and most BUN (urease) measurements.

2. Citrate  It combines with calcium in a non-ionized form.

3-4x INVERISON  1 part to 9 parts of blood (1:9 anticoagulant to blood ratio); light blue
citrated= 1:10 dilution
 Use: plt aggregation, coagulation studies
3. Ethylenediaminetetraacetic  Commonly used anticoagulant for hematology
acid (EDTA)  It combines with calcium in a process called chelation or sequestration
which prevents coagulation
 Use: carcinoembryonic antigen (CEA), TDM, lead testing, CBC, usually
serological and hematological test

ORDER OF DRAW
VENIPUNCTURE (EVACUATED AND SYRINGE)
SKIN PUNCTURE
“STOP LIGHT RED, STAY PUT, GREEN LIGHT GO”
1. Blood gases
 S- sterile tubes, blood culture tube (yellow)
2. Slides, unless made from EDTA
 L- light blue/citrated tube
microcollection tube
 R- red tube w/o clot activator (plain)
3. EDTA microcollection tube
 S- serum separator w/ clot activator (red top)
4. Other microcollection tubes with
 P- plasma separator
anticoagulants (i.e., green or gray)
 G- green tube or Heparin tubes w/ or w/o gel
5. Serum microcollection tubes
 L- lavender tube or EDTA tube
 G- gray top tube or Fluoride

LABORATORY SAFETY

Class of Fire Type of Hazard Type of Extinguisher

1. Type A Ordinary Combustibles Water, dry chemical and loaded steam
-cloth, paper, rubbish, plastics and wood
2. Type B Flammable liquids Dry chemical, carbon dioxide halon foam
-grease, gasoline, paints, and oil
3. Type C Electrical equipment and motor switches Carbon dioxide, dry chemical and halon
4. Type D Flammable metals Metal X
-mercury, magnesium, sodium and lithium (should be fought fire fighters only)
5. Type E Detonation (arsenal fire) None; usually allowed to burn out and nearby
material protected
 Commonly used PPEs:
DEGREE OF HAZARD
a. Gloves
“No SMS Ex”
b. Face mask
0 : No or minimal c. Gowns
d. Cap
1 : slight
e. Shields
2 : moderate f. Closed shoes

3 : serious Never reused gloves, always

dispose gloves in every patient
4 : extreme

Notes to Remember:

Disinfection
 1:10 dilution of chlorine is used to disinfect items contaminated (1 part of chlorine 9 parts of water)
 Bench tops: 1:100 dilution of chlorine bleach
 Bleach should be in contact with the area for at least 20 minutes before wiping
 10% solution of common household bleach inactivates hepatitis B virus in 10 minutes and HIV in 2 mins

Chemical Precaution
 If a spill occurs, first step should be to assist or evacuate personnel
 Arrangements for the storage of chemicals will depend on the quantities of chemicals needed and the nature or type
of chemicals
 Poisonous vapors: chloroform, methanol, carbon tetrachloride, bromide, formaldehyde, mercury
 Flammable and combustible solvents: acetone, alcohol, methanol, toluene, xylene, benzene, isopropanol
 Strong acids or bases should be neutralized before disposable (acid to water)
 Benzidine is a known carcinogen
 All contaminated PPE must be removed and properly disposed off before leaving the laboratory and must not be
taken home or outside the laboratory.
 Spx should remain “capped during” centrifugation because biologic spx produce finely dispersed aerosols that are
high risk source of infection
 The speed of centrifuge should be checked every 3 months by a tachometer.
Lesson 3: METHOD OF EVALUATION TERM DESCRIPTION
Quality assessment or Process by which lab ensures
• Basic Concepts
quality assurance (QA) quality results by closely
• Definition of terms monitoring pre-analytical,
• Statistics analytical, & post-analytical stages
• Quality control and quality assurance of testing.
• Quality control charts Primary goal: ensure quality
• Intra-laboratory QC monitoring services and products to
• Analytical techniques customers.
Pre-analytical QA Everything that precedes test
CENTRIFUGE performance, e.g., test ordering,
patient preparation, patient ID,
• Centrifugation is a process in which centrifugal specimen collection, specimen
force is used to separate solid matter from a transport, specimen processing.
liquid suspension. Analytical QA Everything related to assay, e.g.,
• Centrifugal force depends on three variables: test analysis, QC, reagents,
mass, speed, and radius. calibration, preventive
• The speed is expressed in revolutions per minute maintenance.
(rpm) determined by tachometer. Post-analytical QA Everything that comes after test
• The centrifugal force generated is expressed in analysis
terms of relative centrifugal force (RCF) or Examples: verification of
calculations & reference ranges,
gravities (g).
review of results, notification of
• Radius (r) – distance in cm from center of critical values, result reporting,
rotation to bottom of tube when rotating. test interpretation by physician,
follow-up patient care.
Quality system All of the lab’s policies, processes,
procedures, & resources needed
to achieve quality testing

International Organization for Standardization (ISO)

ANGLE-HEAD/FIXED • the world’s largest developer and publisher of

ANGLE CENTRIFUGE international standards.
• ISO 15189:2007 is for use by medical laboratories
• For rapid centrifugation in developing their quality management systems
• Tubes are at fixed angle (25° - 40°) when rotating. and assessing their own competence, and for use
• Capable of higher speeds. by accreditation bodies in confirming or
• Decantation is not recommended. recognizing the competence of medical
• Produces a slanted sediment surface that isn’t laboratories.
tightly packed.
The Joint Commission (TJC)- formerly the Joint
Commission on Accreditation of Healthcare
Organizations
HORIZONTAL-HEAD CENTRIFUGE (SWINGING- BUCKET)
• Requires hospital laboratories to be accredited
• Tubes are in horizontal position when rotating
by TJC itself, the Commission on Office
and vertical in rest.
Laboratory Accreditation (COLA), or the College
• Recommended for serum
of American Pathologists (CAP).
separator tubes.
• Announced that a periodic performance review
• Produces a tightly packed,
(PPR) will be required for the laboratory
flat sediment surface.
accreditation program.
ULTRA CENTRIFUGE

• High-speed. Capable of 100,000 rpm.

QUALITY CONTROL
• Refrigerated to reduce heat
• QC in the laboratory
*Always make sure centrifuge is balanced. Don’t open
involves the
while spinning. Keep tubes capped.
systematic
QUALITY ASSESSMENT monitoring of analytic
processes to detect
analytic errors that
 occur during analysis and to ultimately • Testing control material not built into test
prevent the reporting in incorrect patient system.
test results. • Term also used for QC that extends beyond
• Is a system of ensuring accuracy and lab, e.g., participation in proficiency testing
precision in the laboratory by including program (periodically/once a yr sending
quality control reagents in every series of samples of unknown concentrations to
measurements. participating laboratories)
• Is a process of ensuring that analytical • Important in maintaining long-term
results are correct by testing known samples accuracy of the analytical method
that resemble patient samples. • Gold std: College of American Pathologists
• It involves the process of monitoring the (CAP) Proficiency Program /NEQUAS in
characteristics of the analytical processes Philippines
and detects analytical errors during testing,
RESEARCH INSTITUTE FOR TROPICAL MEDICINE
and ultimately prevent the reporting of
inaccurate patient test results. • National Reference Laboratory for Dengue,
• One of the components of quality assurance Influenza, Tuberculosis and other Mycobacteria,
system Malaria and other parasites, Bacterial enteric
• Should be done everyday to have reliable diseases, Measles and other Viral exanthems,
results Mycology, Enteroviruses, Antimicrobial
resistance and Emerging Diseases
• NRL for confirmatory testing of blood units.

EAST AVENUE MEDICAL CENTER

• National Reference Laboratory for Environmental

and Occupational Health; Toxicology and
Micronutrient Assay

SAN LAZARO HOSPITAL

• National Reference Laboratory for HIV/AIDS,

Example of Quality Control
Hepatitis, Syphilis and other Sexually Transmitted
Normal (non-pathologic) and Abnormal (pathologic) Infections (STIs).

NATIONAL KIDNEY AND TRANSPLANT INSTITUTE

CONTROL
• National Reference Laboratory for Hematology
• Sample that is chemically & physically similar to including Immunopathology and Anatomic
unknown specimen & is tested in exactly the Pathology
same manner.
LUNG CENTER OF THE PHILIPPINES
• Monitors precision of test system.
• For non-waived quantitative tests, CLIA requires • National Reference Laboratory for Biochemistry
at least 2 levels of controls each day test is
performed. PHILIPPINE HEART CENTER
• For qualitative tests, positive & negative controls • National Reference Laboratory for Anatomic
must be included with each run. Pathology for Cardiovascular Diseases
KINDS OF QUALITY CONTROL
Objectives of Quality Control:
1. Intralab or Internal QC 1. To check the stability of the machine.
• QC within the laboratory 2. To check the quality of reagents.
• Part of analytical phase of quality assurance 3. To check technical (operator) errors.
• Process of monitoring results from control
Characteristics of an Ideal QC Material:
samples to verify reliability of patient results
• System of ensuring daily monitoring of 1. Resembles human sample and be available for
accuracy and precision in the laboratory by (min.) 1 year w/ the same lot no.
including quality control reagents in every 2. Inexpensive and stable for long periods.
series of measurements 3. No communicable diseases.
• Run/done daily or end shift 4. No matrix effects/known matrix effects
2. External QC or Interlab QC 5. With known analyte concentrations
• Proficiency testing programs to participating 6. Convenient packaging for easy dispensing and
laboratories storage.
Human control materials are preferred but because of of 120 healthy Ranges may need
limited sources and biohazard considerations, bovine subjects & to be adjusted to
control materials are used (except for immunochemistry, determining fit lab’s patient
dye-binding and certain bilirubin assays) range in which population.
95% fall.
➢ Restoring of lyophilized (freeze dry) control
materials must be properly done to avoid (Note: 5% of
incorrect control values healthy
➢ Stabilized frozen controls do not require population falls
reconstitution but may have different outside of
characterizations compared to actual patient reference range.)
specimens Labs may use
manufacturer’s
reference ranges
MATRIX EFFECTS or published
reference ranges,
➢ Are results of improper product manufacturing if appropriate for
➢ Use of unpurified human and nonhuman analyte their patient
additives and altered protein components. population.
ANALYTICAL Same as detection Determined by
PARAMETER DESCRIPTION VERIFICATION SENSITIVITY limit. Lowest manufacturer.
ACCURACY How close Lab tests samples concentration of For unmodified.
measurement is of known values substance that FDA-approved
to true value. (controls, can be detected tests, verification
Is the nearness or calibrators, by test method. isn’t required.
closeness of the proficiency Increased
assayed value to samples, sensitivity means
the true or target previously tested decreased false
value. patient negs. Desirable in
specimens) to see screening tests.
how close results ANALYTICAL Ability of method Determined by
are to known SPECIFICITY to measure only manufacturer.
value the analyte it’s For unmodified.
PRECISION Reproducibility. Lab repeatedly supposed to FDA-approved
How close results tests same measure & not tests, verification
are when same samples (on same other related isn’t required.
sample is tested day & different substances.
multiple times is days) to see how Increased
the ability of an close the results specificity means
analytical method are. decreased false
to give repeated positives and
results on the decreased cross-
same sample that reactivity.
agree with one Desirable in
another. confirmatory
(Note: A tests.
procedure can be
precise but not
accurate.) CHARACTERISTIC EXPLANATION
REPORTABLE Range of values Lab tests samples True positive (TP) Pos result in patient who
RANGE over which lab can with known has the disease
verify accuracy of values at highest False positive (FP) Pos result in patient who
test system. Also & lowest levels doesn’t have the disease
known as claimed to be True negative (TN) Neg result in patient who
linearity. accurate by doesn’t have the disease
manufacturer. False negative (FN) Neg result in patient who
REFERENCE Formerly called if manufacturers does have disease
INTERVAL/ normal value. or published Diagnostic sensitivity % of population with the
REFERENCE Can vary for reference ranges disease that test pos
VALUE different patient are used, lab must TP/(TP + FN) x 100
populations (age, test specimens Diagnostic specificity % of population without
gender, race). from normal the disease that test neg
Established by subjects to verify TN/(TN + FP) x 100
testing minimum ranges.
Positive predictive value % of time that a pos result • Instability of instrument
(PPV) is correct • Variation in handling techniques: pipetting,
TP/ (TP + FP) X 100 mixing, timing
Negative predictive value % of time that a neg result • Variation in operators
(NPV) is correct
• Anticoagulant or drug interference
TN/ (TN + FN) X 100
* Values are determined by manufacturer. Information Indicated by control value significantly
helps physicians interpret results. different from others on Levey-Jennings
• The Sensitivity of a test is defined as the chart, or violation of the 13S or R4S Westgard
proportion of cases with a specific disease or rules. Usually a 1-time error, & controls &
condition that give a assessment positive test samples can be rerun with success
result 2. Systematic Error (predictable, determinate,
• Specificity of a test is defined as the proportion INACCURACY)
of cases with absence of the specific disease or Is an error that influences observations
condition that gives a negative test result consistently in one direction (constant
• Positive predictive value (PPV) – for a test difference).
indicates the number of patients with an It is detected as either positive or negative bias
abnormal test result who have the disease, often related to calibration problems,
compared with all patients with an abnormal deterioration of reagents and control materials,
result. improperly made standard solutions,
• Negative predictive value (NPV) for a test contaminated solutions, unstable and
indicates the number of patients with a normal inadequate reagent blanks, leaky ion selective
test results who do not have the disease, electrode (ISE), failing instrumentation and
compared with all patients with a normal poorly written procedures.
(negative) result. Are like built in errors and not more on human
VARIATIONS errors.
Parameters: Mean
• Are errors encountered in the collection,
preparation and measurement of samples, 2. Systematic Error (predictable, determinate,
including transcription and releasing of INACCURACY)
laboratory results. • Dirty photometer faulty/leaky ISE
• Aging reagents, aging calibrators
Types of Error:
• Instrument components, wear and tear of
1. Random Error (indeterminate, Unpredictable, instrument
IMPRECISION) • Optical changes
• Fluctuations in line voltage
Is present in all measurements; it is due to chance.
• Reagent lot variability
Is a type of error which varies from sample to sample. • Calibration differences
• Technologist interactions
Is the basis for varying differences between repeated
measurements – variations in technique. • Poorly written procedures

It is due to instrument, operator and environmental Affects all results. Indicated by trend or shift on
conditions (variations in techniques) such as Levey-Jennings chart, or violation of 22S, 41S,
pipetting error, mislabeling of samples, temperature or 10x Westgard rules.
fluctuation, and improper mixing of sample and
Requires investigation to determine cause.
reagent.

Parameters: Standard Deviation, Coefficient of

Variation VARIATIONS
Types of Error: Types of Error:
1. Random Error (indeterminate, Unpredictable, a. Constant Error – it refers to a difference between
IMPRECISION) the target value and the assayed value. It is
• Reagent dispensing, calibrator reconstitution independent of sample concentration. It exists
• Sample evaporation when there is a continual difference between the
• Temperature of analyzer comparative method and the test method
• Electro-optical mechanism regardless of the concentration.
• Environmental conditions
 b. Proportional/Slope/Percent Error – it results in CALIBRATION VERIFICATION
greater deviation from the target value due to
• Testing materials of known concentrations
higher sample concentration. It exists when the
(calibrators, controls, proficiency testing
difference between the test method and the
samples, patient specimens with known values)
comparative method values is proportional to
to ensure accuracy of results throughout
the analyte concentration.
reportable range. Test 3 levels-high, midpoint, &
3. Clerical Error – is the highest frequency of clerical low. Required every 6 months, when lot of
errors occurs with the use of handwritten labels and reagents changes, following preventive
request forms. maintenance or repair, & when controls are out
of range.
Pre-analytical Errors:
STATISTICS
1. Improper patient preparation
2. Mislabeled specimen MEASURES OF Statistical parameters
3. Incorrect order of draw DISPERSION describing spread of data
4. Incorrect patient identification about mean, e.g., standard
5. Wrong specimen container deviation, coefficient of
6. Incorrect anticoagulant to blood ratio variation, range.
7. Improper mixing of sample and additives Measurements of precision.
8. Incorrect specimen preservation RANGE Difference between highest &
lowest values in data set.
9. Incorrect used of tubes for blood collection
Simplest expression of spread
10. Mishandled specimen (transport and storage)
MEAN Sum of all observations
11. Missed or incorrectly interpreted laboratory
divided by number of
requests observations. Average of all
Analytical Errors: observations.
STANDARD Statistical expression of
1. Laboratory staff competence DEVIATION dispersion of values around
2. Assay and instrument selection mean. Requires a minimum of
3. Assay validation (including linearity, accuracy, 20 values.
̅ )𝟐
∑(𝒙 − 𝒙
precision, analytical limits, specificity) 𝑺=√ Where: x = individual value, X
𝒏−𝟏 = mean, n = number of values
4. Internal quality control
5. External quality assessment COEFFICIENT Expresses standard deviation
VARIATION as percentage.
Post-analytical Errors: Index of precision
The higher the CV, the lower
1. Unavailable or delayed laboratory results the precision. CV % = (SD+
2. Incomplete laboratory results mean) x 100
3. Wrong transcription of the patient’s data and
laboratory results
NOTES TO REMEMBER:

STATISTICS 1. PURPOSE OF STATISTICAL ANALYSIS: It

determines the types and quantity of error that
➢ Is the science of gathering, analyzing, a method has, and for us to decide whether the
interpreting and presenting data. test is still valid or unacceptable to make clinical
CALIBRATION decisions
2. The acceptable range is 95% confidence limit
• Process of testing and adjusting analyzer’s which is equivalent to ± 2SD
readout to establish correlation between 3. Method evaluation and statistical analysis are
measured & actual concentrations. essential, but not sufficient to decide if a test is
valid
CALIBRATORS/STANDARD
QUALITY CONTROL CHART
• Used for calibration
• Reference material with known concentration of • Is used to observe values of control materials
analyte. Concentrations programmed into over time to determine reliability of the
analyzer’s computer for use in calculating analytical method.
concentration of unknowns. Formerly called • Is utilized to observe and detect analytic errors
standard. Does not have to be similar to the pt such as inaccuracy and imprecision.
spx. 1. Gaussian Curve (Bell-shaped curve)
• It occurs when the data set can be accurately
described by the SD and the mean
 •A population probability distribution that is but maintain a constant level (e.g. an increase
symmetric about the mean shift)
• It is obtained by plotting the values from • May be observed with the sudden malfunction of
multiple analyses of a sample. an instrument
• It occurs when data elements are centered • Main cause: Improper calibration
around the mean with most elements close
TREND (DRIFT)
to the mean.
2. Cumulative Sum Graph (Cusum) • Gradual change in control sample result or
• Calculates difference between QC results gradual loss of reliability in test system
and the target means. • Values for the control that continue to either
• Gives earliest indication of systematic errors increase or decrease over a period of 6
(Shifts and not Trends) consecutive runs
• It calculates the difference between QC • Progressive problem with the testing system
results and the target means. or control sample, such as deterioration of
• It is very sensitive to small, persistent errors reagents or control spx
that commonly occur in the modern, low • Main cause: Deterioration of reagents
calibration-frequency analyzer.
3. Youden/Twin Plot
• used to compare results obtained on a high
and low control serum from different labs
• it displays the results of the analyses by
plotting the mean values for one specimen
on the ordinate (y-axis) and the other
specimen on the abscissa (x-axis).
4. Shewhart levey-Jennings Chart (Dot Chart)
• It is the most widely used QC chart in the
clinical laboratory. It allows the laboratorians
to apply multiple rules without the aid of a
computer.
• It easily identifies random and systematic
WESTGARD MULTIRULES
errors.
• A graphic representation of the acceptable ➢ 12s – it is used as a rejection or warning rule when
limits of variation in the results of an one control result exceeds the mean ±2SD; for
analytical method and easily identifies screening purposes.
random and systematic errors. ➢ 13S – one control result exceeds the mean ±3SD;
it is effective in determining random error.
INTERPRETATION OF QUALITY CONTROL DATA
➢ 22S– the last 2 control results (or 2 results from
➢ CONTROL LIMITS the same run) exceed either the mean ±2SD;
Range within which control values must fall for respond most often to systematic errors.
assay to be considered valid. ➢ 41S – the last four (or any four) consecutive
Many labs used mean ± SD. 1 determination in control results exceed either mean ± 1SD;
20 will fall outside ±SD. respond to systematic errors.
1 outlier = normal every 20 days but is a warning ➢ R4S – the range or difference between the highest
This is anticipated part of normal variation. and lowest control result within an analytical run
➢ OUTLIER exceeds 4SD; respond to random errors or
A control result outside established limits (not increased imprecision.
inside the mean). ➢ 10x – ten consecutive results are on the same
May be due to chance or may indicate problem side of the target mean; systematic error.
in test system. If it occurs more than once in 20
RULE EXPLANATION TYPE OF ERROR COMMENTS
successive runs, investigation must be carried
12s 1 control >±2s Initiates
out. from mean. testing of
SHIFT Warning flag of other rules.
possible change If no
• Abrupt, sudden and sustained change in one in accuracy or violation of
direction in control sample values precision. other rules,
• 6 or more consecutive daily values that distribute run is
themselves on one side of the mean value line, considered
in control.
13S 1 control > ±3s Random Rejection 41s – reject when 4 consecutive control measurements
from mean rule exceed the same mean plus 1s or the same mean minus
22S 2 consecutive Systematic Rejection 1s control limit.
controls > 2s rule
from mean on
same side
R4S 2 consecutive Random Rejection
controls differ rule
by >4s
41S 4 consecutive Systematic Rejection
controls >1s rule
from mean on
same side
10x 10 consecutive Systematic Rejection
controls on rule
same side of 8 x – reject when 8 consecutive control measurements all
mean
on one side of the mean.

This rule is used as a warning rule to trigger careful

inspection of the control data by the following rejection
rules.

10 x – reject when 10 consecutive control measurements

22S – Reject when 2 consecutive control measuerments fall on one side of the mean.
exceed the same mean plus 2s or the same mean minus
2s control limit.

12 x – reject when 12 consecutive control measurements

R4S - reject when 1 control measuement in a group
fall on one side of the mean.
exceeds the mean plus 2s and another exceeds the mean
minus 2s.
OTHER COMPONENTS OF QA PROGRAM The ultimate goal of proficiency testing is to
ensure our clinicians that patient results are
➢ CORRELATION STUDY
accurate.
Study to verify accuracy of new method. Proficiency testing allows each laboratory to
compare and evaluate test results or outcomes
Split patient samples are analyzed by existing method with those laboratories that use the same
& new method. methods (reagents and equipment).
Requires a minimum of 40 patient samples ➢ STANDARD OPERATIONG PROCEDURE (SOP)
representing wide range of concentrations. Set of instructions for methods used in the
laboratory. Also known as procedure manual.
Reference values (esistng method) are plotted on x
axis, values from new method on y axis.
Analytical Methods
Perfect correlation is straight line passing through Electromagnetic radiation has wave-like (energy) and
zero at 45° angle. particle-like (proton) properties.
The correlation coefficient (r) can derive • Radiant energy is characterized as a
mathematically. spectrum from short wavelength to long
Values range from -1 to +1. wavelength: cosmic, gamma rays, X-rays,
ultraviolet, visible, infrared, microwave,
0 = no correlation between methods. radio wave.
+ 1 = perfect direct correlation. • Wavelength (A) – is the distance traveled by
one complete wave cycle (distance between
≥0.95 = excellent correlation. two successive crests) measured in
nanometer (nm).
• The shorter the wavelength, the greater the
➢ PREVENTIVE MAINTENANCE
energy contained in the light, and the greater
Schedule of maintenance to keep equipment in
the number of photons.
peak operating condition. Maintenance must be
documented and must follow manufacturer’s • Ultraviolet (UV) light <400 nm has very short
specifications & frequencies. wavelengths.
➢ FUNCTION CHECKS
Procedures specified by manufacturer to Wavelength is inversely proportional to
evaluate critical operating characteristics of test frequency and energy; the shorter the
system, e.g., stray light, background counts. Must wavelength the higher the frequency and
be withing manufacturer’s established limits energy and vice versa.
before patient testing is conducted.
Documentation required. • Infrared (IR) light >700 nm has very long
➢ DELTA CHECKS wavelengths.
Comparison of patient data with previous • When all visible wavelengths of light 400-700
results. Detects specimen mix-up & other errors. nm are combined white light results.
When limit is exceeded, must determine if due to • Visible color: wavelength of light transmitted
medical change in patient or lab error. (not absorbed) by an object.
➢ CRITICAL VALUES • Frequency – number of vibrations of wave
Test results that indicate a potentially life- motion per sec.
threatening situation. List typically includes • Absorption or emission of energy forms a
glucose, Na, K, total CO2, Ca2+, Mg2+, line spectrum that is characteristic of a
phosphorus, total bili (neonates), blood gases. molecule and can help identify a molecule.
➢ PERSONNEL COMPETENCY ASSESSMENT
Normal wavelength – wavelength in nanometers
CLIA requires documentation of competency
at peek transmittance.
assessment on hire, at 6 months, & then
annually. Wavelength accuracy – wavelength indicated on
➢ PROFICIENCY TESTING (PT) the control dial and is the actual wavelength of
Testing of unknowns submitted by outside light passed by the monochromator.
agency, e.g., CAP.
Unknown must not receive preferential
treatment. Results reported to agency, which TYPES OF SPECTROSCOPY/SPECTROMETRY:
compares them to results from other labs.
CLIA requires all labs performing nonwaived tests 1 Emission Spectroscopy – measures the light
(moderate or high complexity) to participate in emitted. Examples: Fluorometry, Flame
PT. photometry, Atomic Emission Spectroscopy
 2 Absorption Spectroscopy • Xenon Discharge Lamp –
Examples: UV/Visible Spectroscopy, IR continuous source of radiation in
Spectroscopy, Nuclear Magnetic Resonance both the UV and visible region.
Spectroscopy (NMRI), Atomic Absorption b. Line source - emits limited radiation and
Spectroscopy wavelength.
• Mercury and sodium Vapor lamp
– UV and visible regions in
I. PHOTOMETRY Spectro.
1. Spectrophotometry – optical instrument that • Hollow cathode lamp – UV and
measures light transmitted by a solution in order visible in AAS
to determine the concentration of the light-
VISIBLE Tungsten light bulb
absorbing analyte; It mathematically establishes Mercury/Sodium vapor
the relationship between concentration and Hollow cathode
absorbance. Amount of light absorbed is directly UV Mercury/Sodium vapor
proportion of analyte. Xenon lamp
➢ PRINCIPLE: BEER’S LAW Deuterium lamp
Hydrogen lamp
Concentration of unknown is directly
Hollow cathode
proportional to the absorbance/optical density INFRARED Merst glower
and inversely proportional to the transmitted REGION (IR) Globar (Silicone carbide)
light (% Transmittance). 2 Entrance slit – is fixed in position and size to minimize
It mathematically establishes the relationship unwanted or stay light and prevents scattered light
between concentration and absorbance. into the monochromator system.

Absorbance = abc = 2-log%T • Stray Light – any wavelengths outside the band
Where: a = molar absorptivity transmitted by the monochromator and does not
b = length of light originate from the polychromatic light source
c = concentration of soln. - Most common cause of loss of linearity at
➢ Remember: UV 700nm I Visible 400-700nm I high analyte concentration
Infrared >700nm 3 Monochromator – dispenses the light into isolated
1. Single Beam Spectrophotometer wavelengths.
- simplest type of absorption spectrometer makes one a. Glass filters and interference filters are used in
measurement at a time at one specified wavelength. photometers.
The absorption maximum of the analyte must be - simple, least expensive, not precise;
known in advance when using this technique. cheapest
2. Double Beam Spectrophotometer - It produces monochromatic light based on
- splits the monochromatic light into 2 components: 1 the principle of constructive interference of
beam passes through the sample, the other passes waves.
through the reference solution or blank that corrects b. Diffraction gratings and prisms are used in
for variation in light source intensity. spectrophotometers.
- absorbance of the sample can be recorded directly. - Diffraction gratings are most commonly
used; wavelengths are bent as they pass a
2 types of Double Beam Spectro: sharp prism can be rotated allowing only the
1 Double-beam in space: 2 photodetectors desired wavelength to pass through the exit
slit.
2 Double-beam in time: uses 1 photodetector and
a copper or rotating sector mirror. 4 Exit Slit – selects the bandpass of the selected
PARTS OF SPECTROPHOTOMETER: wavelength to pass through the cuvet onto the
detector.
1 Light source/exciter lamp - produces an intense,
Bandpass/spectral bandwidth
reproducible, constant beam of polychromatic
light. - total range of wavelengths transmitted
between 2 points where the light transmitted is
2 Types:
½ the peek (maximum) transmittance.
a. Continuum source - emits radiation that
- the narrower the bandpass, the greater the
changes in intensity; widely used
resolution and spectral purity of the instrument.
• Tungsten light bulb – most
common, used in visible and 5 Cuvet – holds the solution being examined; sample
near infrared regions. cell, analytical cell, absorption cell; container of
• Deuterium lamp – used in the analytes
UV region.
 - Have a path length of 1 cm; some have 10cm ➢ Used for: measurement of excited ions.
path length to increase sensitivity by 10x ➢ Principle: Excitation of electrons from lower to
higher energy state
Kinds of Cuvet:
➢ Flame serves as the light source and the cuvet
➢ Alumina/silica glass: most commonly used; ➢ Method: Indirect Internal Standard Method
transmit light at wavelengths of ≥ 220nm (350- ➢ Internal std: Lithium/Cesium - corrects variation
2000nm); near IR and near UV in flame and atomizer characteristics.
➢ Quartz/plastic: used for measurement or METAL ION COLOR OF FLAME
solution requiring visible and UV spectra Potassium Violet/Lilac
➢ Borosilicate glass: for alkaline solution or visible Sodium Yellow
spectra (380-700nm) Lithium Rubidium/Red
➢ Soft glass: for acidic solution Magnesium Blue
Calcium Brick-Red
6 Photo detector – converts transmitted light into Copper Green blue
photoelectric energy; The more light transmitted, Barium Green
the more energy, and the greater the electrical
signal that is measured. 3. ATOMIC ABSORPTION SPECTROPHOTOMETRY
a. Barrier layer cell/Photovoltaic/Photocell ➢ Measures light absorbed by atoms in ground
- Simplest, lest expensive, temperature sensitive state dissociated by heat or unexcited atoms
- Used in filter photometers with a wide bandpass ➢ Used for: unexcited trace elements (Ca & Mg)
- Requires external voltage source ➢ Principle: Dissociated of chemical bonds by heat
- Maximum sensitivity at 550nm (visible region) into unionized, unexcited ground state
b. Phototube ➢ Light source: Hollow-cathode lamp (HCL)
- Contains cathode & anode enclosed in glass case ➢ No internal standard needed
- Photosensitive material give off electron when ➢ Atomizer (nebulizer/granite furnace): convert
light strike it ions to atoms
- Requires external voltage (the only one) ➢ Chopper: modulate the light source
c. Photomultiplier (PMT) ➢ Lanthanum and strontium chloride reduce
- Most commonly used; UV and visible region interference by phosphate
- Excellent sensitivity; detect very low level of light ➢ This technique is more sensitive that the flame
- Should never be exposed to room light method.
d. Photodiode II. TURBIDIMETRY
- Excellent linearity • Principle: measures amount of light blocked
- Most useful as a simultaneous multichannel (reduction of light) by particle formation in a
detector turbid solution
• It depends on specimen concentration and
7 Meter/Readout device – displays output of the
particle size
detection system. Example: galvanometer, ammeter,
light-emitting diode (LED) display. • Light transmitted is inversely proportional to
concentration
• Measurement done by visible
photometers/spectrophotometers
• Used for: protein measurement (CSF, urine),
detect bacterial growth in broth cultures,
1. BLANKING TECHNIQUE broth antimicrobial tests, detect clot
➢ AKA “dual-wavelength method” corrects for formation.
artifactual absorbance readings caused by the III. VOLUMETRIC (TITRIMETRIC)
components of the system. • Principle: unknown sample is made to react
➢ Reagent blank - corrects for the absorbance of with known solution on the presence of an
the reagent color. indicator.
➢ Patient blank – measures absorbance of sample • Examples: Schales and Schales method
and reagent without the end-product. Corrects (Chloride Test) EDTA Titration method
for optical interference from the sample (e.g (Calcium Test)
lipids, hemoglobin at 410nm) IV. NEPHELOMETRY
➢ In some cases of turbidity, blanking may not be • Principle: determines amount of light
effective; Ultracentrifugation may be necessary scattered by particulate matter in a turbid
to clear the serum/plasma of chylomicrons. solution
2. FLAME EMISSION PHOTOMETRY • Light scatter depends on wavelength and
➢ Measures light emitted by a single atom burned particle size (forward scatter for larger
in a flame. particles)
 • Used for: measurement of antigen-antibody areas). It is ideal for separating proteins of identical size
complexes but different net charges. Proteins move in the electric
V. ELECTROPHORESIS field until they reach a Ph equal to their isoelectric point.
• Not a measuring device but separation
Advantages of Isoelectric focusing;
techniques (for proteins)
1. Ability to resolve mixture of proteins
• Is the migration of charged particles in an 2. Detect isoenzymes of ACP, CK and ALP in serum
electric field 3. Identify genetic variants of alpha-1 antitrypsin
• Used clinically to separate and identify 4. Detect CSF oligoclonal banding
proteins, including serum, urine and
cerebrospinal fluid (CSF) proteins, Capillary Electrophoresis
lipoproteins, isoenzymes, and so on. ➢ Sample molecules separated by electro-osmotic
• Component electric power, support medium, flow (EOF) based on difference in solute size
buffer, sample and detecting system ➢ Uses nanoliters of specimen
• Buffer: Barbital (pH 8.6) for alkaline ➢ Applications: separation, quantitation and
electrophoresis, citrate (pH 3 to 6.2) for acid determination of molecular weights of proteins
electrophoresis and peptides; analysis o PCR products; analysis of
• Electrophoretic mobility is directly organic and inorganic substances and drugs.
proportional to net charge and inversely VI. CHROMATOGRAPHY
proportional to molecular size and viscosity • Not measuring device but separating
of support medium. technique
TERMINOLOGIES: • Is a technique where solutes in a sample are
separated for identification based on
• Electroendosmosis/endosmosis – physical differences that allow their
movement of buffer ions and solvent differential distribution between a mobile
relative to the fixed support phase and a stationary phase.
• Iontophoresis – migration of small • Mobile Phase: may be an inert gas or a liquid;
charged ions solvent
• Zone electrophoresis - migration of • Stationary Phase: may be silica gel bound to
charged macromolecules the surface of a glass plate or plastic sheet;
may be silica or a polymer that is coated or
Supporting media:
bonded within a column; solvent & solute
1. Cellulose Acetate – separates by molecular 2 forms:
size (serum protein electrophoresis) 1. Planar
2. Agarose Gel – separates by electric charge; it a. Paper Chromatography - used for
does not bind protein fractionation of sugar and amino acid
3. Polyacrylamide Gel – separates on the basis Sorbent (stationary phase): Whatman
of charge and molecular size; separates paper
proteins into 20 fractions; used to study b. Thin Layer Chromatography (TLC) –
isoenzymes Semiquantitative drug screening test for
drugs of abuse (spx urine)
Stains (Electrophoresis): Factors affecting rate of
• Extraction of the drug is pH-
1. Amido Black migration:
dependent
2. Ponceau S ➢ Net electric
3. Oil Red O charge of the • Biological samples: serum /
4. Sudan Black molecule plasma (TDM), urine (prohibited
5. Fat Red 7B ➢ Size and shape of drugs) and gastric fluid
6. Coomassie Blue molecule • Each drug has a characteristic
7. Gold/Silver stain – ➢ Electric field Retention factor (Rf) value and it
very sensitive to strength – nature must match the Rf of the drug
nanogram quantities of supporting standard
of proteins; not medium • Rf values are affected by
standard stain ➢ Temperature of chamber saturation,
operation
temperature, humidity, and
composition of the solvent.
Densitometry – measures the absorbance of stain
2. Column
(concentration of the dye and protein fraction) using a
a. Gas Chromatography (GC)
densitometer
• Used for separation of steroids,
Isoelectric focusing – separates molecules by migration barbiturates, blood, alcohol and
through a Ph gradient (acidic anode and basic cathode lipids
 • Used for compounds that are IX. OSMOMETRY
naturally volatile or can be easily • Principle: based on measuring changes in
converted into a volatile form the colligative properties of solutions that
• MS can be used as detector for occur due to variations in particle
definitive identification concentration
• Osmotic particles: glucose, urea nitrogen
Mass Spectroscopy – based on the fragmentation
and sodium
and ionization of molecules using a suitable source of
• When osmolality increases:
energy.
Osmotic pressure: increased by 1.7 x 10^4
GC-MS – Gold standard for drug testing. Also used for Boiling point: Increased by 0.52°C
xenobiotics, anabolic steroids and pesticides. Vapor pressure: Decreased by 0.3 mmHg
Freezing point: Decreased by 1.86°C
Tandem Mass Spectroscopy (MS/MS) – can detect X. ELECTROCHEMISTRY TECHNIQUES
20 inborn error of metabolism from a single blood
spot The measurement of current or voltage generated by
the activity of a specific ion
b. Liquid Chromatography (LC) – for non-volatile
substances (e.g proteins, hormones, CHO, 1. Potentiometry Technique – measurement of
glycoted hgb) differences in voltage potential at a constant
current
• High Performance Liquid Chromatography a. Reference electrode: calomel and silver-
(HPLC) silver chloride
➢ Uses pressure for fast separation, b. Uses: pH and pCO2
controlled temperature, in-line
detectors, and gradient elution Ion Selective Electrode (ISE)
technique. - Measures the electrolyte dissolved
➢ Uses: fractionation of drugs, hormone, in the fluid phase of the sample in
lipids, carbohydrates and proteins, mmol/L of plasma water
separation and quantitation of various
hemoglobin associated with specific Two types of ISE:
diseases (e.g thalassemia), rapid HbA1c A. Direct ISE (without sample dilution) – not
test (within 5 minutes) subject to pseudohyponatremia caused by
• Liquid Chromatography-Mass Spectroscopy (IL- hyperlipidemic or hyperproteinemic samples
MS) B. Indirect ISE (with sample dilution) – prone to
➢ For non-volatile substances in body pseudo hyponatremia
fluids 2. Coulometry Technique
➢ A complementary method GC-MS in - an electrochemical titration in
confirming positive screening tests for which the titrant is
illicit drugs. electrochemically generated and
VII. FLUOROMETRY/MOLECULAR LUMINESCENCE the end point is detected by
SPECTROPHOTOMETRY (very sensitive) amperometry
• Principle: measures light emitted by a molecule - measures amount of electricity (in
after excitation by electromagnetic radiation. Coulombs) at a fixed potential
• Measures amount of light intensity present over - uses: Chloride test (CSF, serum,
zero background sweat)
• It uses 2 monochromators (either filter, gratings, - interference: bromide, cyanide and
prisms) cysteine
• Light source: mercury arc or xenon lamp 3. Amperometry – measurement of the current
• Detector, PMT or phototube flow produced by an oxidation -reduction
VIII. CHEMILUMINESCENCE reaction
• Does not require light source - Used in pO2, glucose and
• Principle: the chemical reaction yields an peroxidase determination
electronically excited compound that emits light Polography
as it returns to its ground state, or that transfers
its energy to another compound, which then - measures differences in current at a constant
produces emission voltage
• Use: immunoassays - Amount of increase in current (i.e. the wave
• Detector: PMT (luminometer) height) is proportional to the concentration of
• More sensitive than fluorescence analyte
- Follow llkovic equation
4. Voltammetry – the measurement of current
after a potential is applied to an electrochemical
cell
Example:
Anodic stripping voltammetry
- For Lead and Iron testing
CLINICAL CHEMISTRY 1 adipose tissue to be stored as glycogen and
fat; inhibits the release of glucose from liver.
LESSON 4
2. Somatostatin – synthesized by delta cells of
CARBOHYDRATES the pancreatic islets of Langerhans; inhibits
secretion of insulin, glucagon, and growth
 Hydrates of aldehyde or ketone derivatives hormone, resulting in an increase in plasma
based on the location of the carboxyl functional glucose level.
group 3. Growth hormone and Adrenocorticotropic
 Carbohydrates: monosaccharides, hormone (ACTH) – hormones secreted by the
disaccharides, oligosaccharides, anterior pituitary that raise blood glucose levels
polysaccharides by acting as insulin antagonists
 Glycol aldehyde is the SIMPLEST GH – decreases glucose entry to cells,
carbohydrate increases glycolysis
 Glucose is the ONLY carbohydrate to be ACTH – releases cortisol
directly used for energy or stored as glycogen 4. Cortisol – secreted by the adrenal glands;
with the help of INSULIN stimulates glycogenolysis, lipolysis, and
 glucose metabolism generates pyruvic acid, gluconeogenesis.
lactic acid, and acetyl coenzyme-a as 5. Epinephrine – secreted by the medulla of the
intermediate products and carbon dioxide, adrenal glands. It stimulates glycogenolysis and
water and ATP as end-products after complete lipolysis; it inhibits secretion of insulin.
oxidation Physical or emotional stress causes increased
 Reducing sugars: glucose, maltose, fructose, secretion of epinephrine and an immediate
lactose and galactose increase in blood glucose levels.
 Nonreducing: Sucrose (do not contain an active 6. Glucagon – secreted by the alpha cells of the
ketone or aldehyde group), and Trehalose pancreatic islets of Langerhans; increases blood
(found in mushroom) glucose by stimulating glycogenolysis and
gluconeogenesis
PANCREAS 7. Thyroxine – secreted by the thyroid gland;
 Function as both endocrine and exocrine organ stimulates glycogenolysis and
in the control of carbohydrate metabolism gluconeogenesis; increases glucose absorption
from the intestines
 As an exocrine gland: it produces and secretes
an amylase responsible for the breakdown of GLUCOSE METABOLIC PROCESS
ingested complex CHO
 As an endocrine gland: it secretes hormones  GLYCOLYSIS
such as insulin, glucagon, and somatostatin Metabolism of glucose to lactate or pyruvate for
from different cells residing in the islets of energy new glucose
Langerhans in pancreas.
Substrate : Glucose
Product : Pyruvate/lactate + ATP
 GLUCONEOGENESIS
Formation of glucose-6-phosphate from non-
carbohydrate source; conversion of fatty acids
and amino acids to glucose by the liver
Substrate : Amino acid. Lactate, Glycerol
Product: Glucose, Ketones (fats), Urea nitrogen
(proteins)
 GLYCOGENOLYSIS
Breakdown of glycogen to glucose for use as
energy
HORMONES AFFECTING BLOOD GLUCOSE Substrate: glycogen
LEVELS Product: glucose
1. Insulin – produced by the beta cells of the  GLYCOGENESIS
pancreatic islets of Langerhans; promotes the Conversion of glucose to glycogen for storage
entry of glucose into liver, muscle, and Substrate: Glucose
 Product: Glycogen  Diagnostic test: 5-hour glucose tolerance test
 LIPOGENESIS 65 mg/dL to 70 mg/dL = glucagon and other
Conversion of carbohydrates to fatty acids glycemic hormones released
Substrate: Glucose ≤ 60 mg/dL = strongly suggest hypoglycemia
Product: Fatty Acid 50 mg/dL to 55 mg/dL = observable symptoms
 LIPOLYSIS of hypoglycemia appear
Decomposition of fat/metabolism of fats
Substrate: Glycogen CAUSES OF ABNORMAL GLUCOSE LEVELS
Product: Glucose HYPERGLYCEMIA
 TRICARBOXYLIC ACID AND PERSISTENT TRANSIENT
ELECTRON TRANSPORT SYSTEM 1. Diabetes 1. Pheochromocytoma
Energy production (24 ATP) 12 per Acetyl-CoA mellitus 2. Severe liver disease
Substrate: Pyruvate to acetyl CoA 2. Adrenal cortical 3. Acute stress
Product: ATP hyperactivity reaction (physical
 HEXOSE MONOPHOSPHATE SHUNT (Cushing’s and emotional)
Energy source of many anabolic reactions and syndrome) 4. Shock
glycolysis in RBCs since they lack 3. Hyperthyroidism 5. Convulsions
mitochondria 4. Acromegaly
Substrate: glucose 5. Obesity
Product: NADPH (Nicotinamide Adenine
Dinucleotide Phosphate) HYPOGLYCEMIA
PERSISTENT TRANSIENT
HYPERGLYCEMIA
1. Insulinoma 1. Acute alcohol
 Is an increase in blood glucose concentration 2. Adrenal cortical ingestion
 Toxic to beta cell function and impairs insulin insufficiency 2. Drugs: salicylates,
secretion (Addison’s anti-TB
Disease) 3. Severe liver disease
 Causes: stress, severe infection, dehydration or 3. Hypopituitarism 4. Severe glycogen
pregnancy, pancreatectomy, hemochromatosis, 4. Galactosemia storage disease (Von
insulin deficiency or abnormal insulin receptor 5. Ectopic insulin Gierke)
 FBS level = ≥ 126 mg/dL production from 5. Functional
 Glucosuria occurs when plasma glucose level tumors hypoglycemia
exceeds 160 – 180 mg/dL (9.99 mmol/L) with 6. Hereditary fructose
normal renal function intolerance
 In the presence of normal renal function,
plasma glucose reaches a “period of plateau” DIABETES MELLITUS
around 300 mg/dL to 500 mg/dL
 Laboratory findings:  Group of metabolic disorders characterized by
Increased glucose in plasma and urine hyperglycemia resulting from defects in insulin
Increased urine specific gravity secretion, insulin receptors or both
Ketones in serum and urine  Fasting plasma glucose concentrations ≥
Decreased blood and urine pH 126mg/dL on more than one test is diagnostic
Electrolyte imbalance (decreased Na and of DM
HCO3, increased K)  In severe DM, ratio of B-hydroxybutyrate to
acetoacetate is 6:1
HYPOGLYCEMIA
TYPE 1 DM
 Results from an imbalance between glucose
utilization and production  Characterized by insulinopenia
 Diagnosis of hypoglycemia should not be made  Require treatment with insulin to sustain life
unless a patient meets the criteria of Whipple’s  Most individuals exhibit it as an autoimmune
triad: low blood glucose, typical symptoms disorder where beta cells of the islets of
(CNS related), and symptoms alleviated by Langerhans are destroyed by anti-glutamic
glucose administration acid decarboxylase (GAD65) and insulin
 Infants and children with a deficiency of autoantibodies (IAA)
cortisol and growth hormone are prone to  Genetic association with HLA DR3 and DR4
develop hypoglycemia
  Primary symptoms include polyuria,
polydipsia, polyphagia, rapid weight loss,
 Women 45 years and older are recommended to
hyperventilation, confusion
be screened for diabetes every 3 years.
 80 – 90 % volume reduction of B-cells is
 IDIOPATHIC TYPE 1 DM: no known
required to induce symptoms
etiology, strongly inherited, episodic, B-cell
 Ketosis-prone: Can produce excess ketones,
autoantibodies.
resulting in diabetic ketoacidosis
 Complications: microvascular disorders – GESTATIONAL DIABETES MELLITUS (GDM)
nephropathy, neuropathy, retinopathy
 GDM is the onset of diabetes mellitus during
TYPE 2 DM pregnancy due to metabolic or hormonal
changes risk of intrauterine death or neonatal
 Defect in insulin secretion and cellular
complications, (infants born to diabetic mothers
resistance to insulin
are at increased risk for macrosomia,
 Individuals are not dependent on treatment with
hypoglycemia, hypocalcemia, polycythemia,
insulin
hyperbilirubinemia)
 Generally respond to dietary intervention and
 After childbirth, the individual generally
oral hypoglycemic agents
returns to normal metabolism however, there is
 Associated with strong genetic predisposition an increased chance that type 2 diabetes
but not related to autoimmunity: Geneticist’s mellitus may develop later in life (5 – 10 years
nightmare post-natal in 30-40 % of cases)
 Symptoms milder than type 1 DM  Screening is performed between 24 and 28
 If untreated, it will result to nonketotic weeks of gestation using 2-hr OGTT with 75g
hyperosmolar coma due to overproduction of glucose load
glucose (>300 mg/dL) with severe dehydration,
electrolyte imbalance (low Na, high K) and SCREENING FOR GESTATIONAL DIABETES
increased BUN and creatinine (GDM)
 Non-ketosis prone Pregnant women with risk Test for undiagnosed
factors type 2 at first prenatal
TYPE 1 DM TYPE 2 DM visit using standard
AKA Insulin Non-insulin diagnostic criteria
dependent, dependent. Adult Pregnant women without Test for GDM at 24-28
Juvenile onset, onset, Non ketosis prior diabetes weeks
Ketosis prone prone (after giving birth) Women Screen for persistent
Pathogenesis B-cell Insulin resistance with GDM diabetes 6-12 weeks
destruction postpartum using
OGTT and standard
Incidence rate 5-10% (10- 95% diagnostic criteria
15%)
Women with a history of Lifelong screening for
Onset Any; most Any; over 40 GDM diabetes or prediabetes
common to years of age every 23 yrs
children/teens Women with a history of Lifestyle interventions
Risk factors Genetic, Race/ethnicity, GDM and prediabetes or metformin for
autoimmune Genetic, obesity, diabetes prevention
lifestyle, PCOS,  Women with diabetes in the first trimester
dyslipidemia, have type 2 diabetes
hypertension  GDM is diagnosed in the second or third
C-peptide levels
Decreased or Detectable trimester and not clearly associated with type
undetectable 1 or type 2 diabetes
Pre-diabetes Autoantibodies Autoantibodies (-) ONE STEP DIAGNOSIS STRATEGY
(+)
Symptomatology Abrupt Gradual (some  Perform 75g OGTT with plasma glucose
asymptomatic) measurement
Ketosis Common, Rare  Test in the morning after the patient has fasted
poorly for ≥ 8 hours
controlled  Repeat test at 1 and 2 hours after initial
medication Injectable Oral agent measurement
insulin
  Diagnosis is confirmed when PG levels meet or  CSF glucose should be approximately 60% of
exceed: the plasma glucose
o Fasting : 92 mg/dL (5.1 mmol/L)  Peritoneal fluid glucose is almost the same as
o 1 hr : 180 mg/dL (10.0 mmol/L) plasma glucose
o 2 hr : 153 mg/dL (8.5 mmol/L)
DO NOT USE SODIUM FLUORIDE AS
TWO STEP DIAGNOSIS STRATEGY ANTICOAGULANT BECAUSE IT INHIBITS
ENZYMES AND BUN DETERMINATION
Step 1:
LABORATORY METHODS OF MEASURING
 Perform a 50g non-fasting GLT
GLUCOSE
 If Plasma Glucose measured 1 hour after the
load is ≥ 140 mg/dL (7.8 mmol/L), proceed to 1. RBS – requested during insulin shock and
100g OGTT hyperglycemic ketonic coma, for emergency
purposes
Step 2: 2. FBS – fasting blood glucose (FBG) should be
 Perform 100g OGTT while patient is fasting obtained in the morning after an approximately
 Diagnosis is confirmed when two or more PG 8 to 10 hours fast (not longer than 16 hours)
levels meet or exceed: (RV: 70-100 mg/dL); give the best indication of
o Fasting: 95 mg/dL or 105 mg/dL overall hemostasis of glucose
o 1 hr: 180 mg/dL or 190 mg/dL 3. POCT – strips impregnated with glucose
o 2 hr: 155 mg/dL or 165 mg/dL oxidase and peroxidase
o 3 hr: 140 mg/dL or 145 mg/dL  Principle: color change read by Reflectance
Photometry
DIABETES INSIPIDUS (DI)  Hemocue: utilizes transmittance
photometry and single test cartridges based
 Is an uncommon problem that causes the fluids
on glucose dehydrogenase method.
in the body to become out of balance. That
4. 2 HOUR POST PRANDIAL
prompts the body to make a large amount of
 Glucose load of 75g (adults) or 1.75g / kg
urine.
(children) of glucose is administered and a
 Deficiency of antidiuretic hormone
specimen is drawn 2 hours later
(vasopressin) released by posterior pituitary
 Normal value: < 140 mg/dL
gland
 Doesn’t have hyperglycemia but only normal  Diabetic: > 180 mg/dL
glycemia 5. HbA1c (Glycosylated hemoglobin/ glycated
hemoglobin)
 Clinical picture of DI: normoglycemia,
 Is the largest subfraction of normal
polyuria, polydipsia, polyphagia
hemoglobin A in both diabetic/nondiabetic
LABORATORY METHODS OF MEASURING individuals
GLUCOSE  Reliable method/index for long term
plasma glucose control 2-3 months
SPECIMEN HANDLING:
indicating compliance and efficacy of DM
 Standard clinical specimen: venous plasma therapy
glucose  It reflects the ave. blood glucose level over
 Fasting glucose in whole blood is 11% lower 2-3 months; doesn’t _____________
than in serum or plasma  Weighted average of plasma glucose level
 Venous blood glucose is 5 mg/dL lower than  Formed by attachment of glucose to
capillary blood; capillary and arterial blood HbA1c fraction to form a ketoamine
glucose is the same
HbA1c (Glycosylated hemoglobin/ glycated
 Separate plasma/serum from cells within 1 hour
hemoglobin)
of collection to prevent losses of glucose
 In normal uncentrifuged coagulated blood,  Specimen: EDTA whole blood (non-
glycolysis decreases glucose by 7 mg/dL/hr at fasting)
RT (20-25°C) and about 2 mg/dL/hr at 4°C;  Should be performed every 3-6 months
even at -20°C, glucose decreases significantly (at least twice a year) in DM patients
and progressively
  Methods: Electrophoresis,  Urinary glucose detected at any point is
immunoassay, HPLC, affinity evidence for DM, but its absence does not
chromatography in any way rule out DM
 Reference method: Diabetes Control 8. FRUCTOSAMINE
And Complications Trial (DCCT)  AKA: GLYCOSYLATED/
Assay GLYCATED ALBUMIN/ PLASMA
 5.7 – 6.4 % pre-diabetes PROTEIN KETOAMINE
 ≥ 6.5 % on at least 2 occasions is  Reflection of short term glucose
indicative control (2-3 weeks)
 1% HbA1c = 35 mg/dL plasma glucose  Useful for monitoring diabetic
6. KETONE TEST – ketones are products of fat individuals with chronic hemolytic
decomposition produced by the liver anemias and Hb varients
 Test recommended when plasma glucose (such as HB S, HB C, or decreased
reaches 300 mg/dL RBC lifespan)
 3 ketone bodies:  It should not be measured in cases of
1. Acetone (2%) low plasma albumin (<30g/L)
2. Acetoacetic acid (20%)  Method: affinity chromatography,
3. 3-B-hydroxybutyric acid (78%) HPLC and Photometric
 Normal ratio of B-hydroxybutyrate to AAA is  Reference value: 205-285 umol/L
1:1 (0.5-1.0 mmol/L each) 9. GLYCOMARKBLOOD TEST
 Specimen: fresh urine or serum  Monitors glycemic control for 1-2 weeks
 Methods:  Measures 1,5-anhydro-D-glucitol
1. Gerhardt’s ferric chloride test – reacts only
10. LACTOSE TOLERANCE TEST
with ACETOACETIC ACID (AAA)
2. Nitroprusside test – 10x more sensitive to  Provides a presumptive diagnosis of lactase
AAA than to acetone deficiency
3. Acetest tablets – detects AAA and acetone  Following an overnight fast, a blood sample is
(lesser degree) drawn, and 50g of lactose dissolved in 400 mL
4. Ketostix – detects AAA better than acetone of water is orally administered.
5. Ketosite assay  A 100g lactose dose has been reported to yield
– detects B-hydroxybutyrate; not common more definitive results
 Calculation of the anion gap is commonly done  Blood samples are collected at 30, 60, and 120
to monitor recovery from diabetic ketoacidosis mins after
(DKA)  An optional 5-hour stool specimen can be
7. OGTT (Oral Glucose Tolerance Test) collected, and its appearance, consistency, and
NOTE: American Diabetes Association does not pH noted.
recommend the OGTT for routine clinical use. 11. C-Peptide Test
 Patient preparation: unrestricted  C – Peptide is formed during conversion of pro-
carbohydrate rich diet (150g) for 3 days insulin to insulin
before the test with physical activity,  Mainly evaluates hypoglycemia and continuous
restrict medication on the test day assessment of insulin secretion (B-cell
 10 hour fast required, no smoking function)
 The test should be performed in the  Specimen: fasting serum
morning because of the hormonal diurnal  Method: immunometric assay
effect on glucose
 Increased: insulinoma, Type 2 DM, Ingestion of
 Adult patient: 75 grams of glucose in 300-
hypoglycemic drugs
400 mL of water
 Decreased: type 1 DM
 Children: 1.75g/kg up to 75 g of glucose
 Reference value: 0.90-4.3 ng/mL (CF : 0.333)
 For assessment of GDM, 50g, 75g, or 100g
 Normal ratio C – peptide : insulin = 5:1 to 15:1
of glucose may be used
 Collect fasting sample 10 mins before
glucose load and every 30 mins for 2 hours
(Calbreath)
TEST NORMAL IMPAIRED DIAGNOSTIC Folin Wu
GLUCOSE DM
TOLERANCE Glucose/Cuprous ions
Random <140 mg/dL 140-199 mg ≥200 mg/dL +  Phosphomolybdic Acid or
Plasma Phosphomolybdate Phosphomolybdenum
Glucose
(RBS) Neocuproine Method (2,9-dimethyl-1,10-phenantroline HCI)
Fasting <100 mg/dL 100-125 mg/dL ≥126 mg/dL
Plasma Cuprous ions
Glucose +  Cuprous-neocuproine Complex
(FBS) Neocuproine (yellow/yellow orange)
2-hr <140 mg/dL 140–199 mg/dL ≥200 mg/dL
OGTT Benedict’s method (modification of Folin Wu)
HbA1c 4 – 5.7% 5.7 – 6.5 % ≥ 6.5%  Detection and quantitation of reducing
substances in body fluids like blood and urine
MICROALBUMINURIA  Use citrate or tartrate as stabilizing agents

 a condition where a small amount of protein Hagedorn Jensen

called albumin leaks into the urine from the Ferric Reduction Method (Inverse Colorimetry)
blood Glucose
 is an early sign of diabetic nephropathy/ kidney +  Ferrocyanide (colorless)
damage (need to catch it early) Ferricyanide (yellow)
 it also is a risk factor for cardiovascular disease
 persistent albumin excretion between 30 and Ortho-toluidine
300 mg/day (20 to 200 mcg/min) – diagnostic Condensation of carbohydrates with aromatic amines
microalbuminuria producing Schiff’s bases (green)
 the normal rate of albumin excretion is less than
30 mg/day (20 mcg/min) ENZYMATIC METHODS OF GLUCOSE
MEASUREMENT
NON-ENZYMATIC METHODS OF GLUCOSE
MEASUREMENT 1. GLUCOSE OXIDASE (Saifer
ALKALINE COPPER REDUCTION METHOD Gernstenfield)
 Coupled enzyme reaction (Trinder’s Reaction)
 reduction of cupric ions to cuprous ions – colorimetric
forming cuprous oxide in hot alkaline solution  Measures B-D-glucose
by glucose
 chemical method that utilizes the nonspecific B-D-glucose + O2 + H2O – glucose oxidase gluconic
reducing property of glucose acid acid + H2O2
 glucose is 5 – 15 mg/dl higher because of the H2O2
presence of other reducing substances such as +  oxidized chromogen-red dye
urea reduced chromogen + H2O
 automated chemistry analyzers measure by (o-Dianisidine) peroxidase
color change of series of coupled chemical
reactions or by consumption of oxygen on an  For urine and whole blood glucose rapid
oxygen sensing device reagent strip testing.
 calculate glucose by the rate of the reaction  Also for automated methods for plasma and
which is proportional to the concentration of serum interference by: uric acid, bilirubin,
glucose ascorbic acid
 Increased uric acid, ascorbic acid, bilirubin,
Nelson Somogyi tetracycline, hemoglobin and glutathione 
Copper reduction method that uses BaSO4 to remove falsely low glucose result
saccharoids
Glucose/Cuprous ions
+  Arsenomolybdenum Blue
Arsenomolybdic Acid
 2. GLUCOSE OXIDASE-O 2 CONSUMPTION GLYCOGEN STORAGE DISEASE
(Polarographic Glucose Oxidase Method) Ia-Von Gierke Glucose-6-phosphatase
Ib Glucose-6-phosphatase
B-D-glucose + O2 + H2O – glucose oxidase gluconic translocase
acid acid + H2O2 II- Pompe’s Lysosomal 1,4-
glucosidase
O2 consumption is measured by O2 electrode IIIa – Cori Forbes Debranching enzyme
 Less common than hexokinase method (liver and muscle)
 Commonly used for glucose meter testing IIIb Debranching enzyme
 Accurate and precise method, virtually no (liver)
interferences IV – Andersen’s Branching enzyme
Disease/Amylopectinosis
3. HEXOKINASE METHOD V – McArdle Muscle phosphorylase
 Most specific and REFERENCE METHOD VI – Hers Disease Glycogen phosphorylase
(liver)
 Specific glucose method which employs
VII – tarui Phosphofructokinase
G6PD as a second coupling step requiring
XI – Fanconi-Bickel Glucose transporter 2
magnesium Syndrome (GLUT)

Glucose + ATP – Hexokinase  Glucose-PO4 + ADP LESSON 5 - PROTEINS

 Proteins are macromolecules made of amino
Glucose-6-PO4 acids, with each amino acid being linked to
+  NADPH + H+ + 6-phosphogiuconate another via a peptide bond
NADP+ - G6PD a. Peptide bond is formed when the carboxyl
(-COOH) group of one amino acid links to
 Based on formation of NADH followed by the amino (-NH”) group of another amino
increase in absorbance at 340nm (directly acid with the loss of a water molecule
proportional to glucose concentration) b. N-terminal: end of protein structure with a
 Falsely low: gross hemolysis (inhibits free amino group
peroxidase), extremely ↑ bilirubin c. C-terminal: end of protein structure with a
 Not affected by ascorbic acid or uric acid free carboxyl group
 May be performed on serum or plasma d. Nitrogen Content: proteins consist of 16%
collected using heparin, EDTA, fluoride, nitrogen, which differentiates proteins
oxalate, or citrate from carbohydrates and lipids
 Can also be used for urine, cerebrospinal fluid,  Nitrogen balance: a balance between
and serous fluids anabolism and catabolism
PROTEIN STRUCTURE
INBORN ERRORS OF CARBOHYDRATE A. Primary structure
METABOLISM B. Secondary structure
C. Tertiary structure
GALACTOSEMIA D. Quaternary structure
 Congenital deficiency of one of three enzymes
involved in galactose metabolism: galactose-1-
phosphate uridyl transferase (GPUT; most
common), galactokinase (GALK) and uridine
diphosphate galactose-4-epimerase (GALE)
 GPUT converts galactose-1-phosphate to
glucose
 Laboratory features: elevated blood and urine
galactose
 Diagnostic test: Erythrocyte galactose-1-
phosphate uridyl transferase activity
(Erythrocyte GPUT)
 CLASSIFICATION OF PROTEINS  Decreased: liver disorders, GIT associated
1. Simple proteins – polypeptides composed of malabsorption, muscle wasting disease,
only amino acids severe burns, renal disease (nephrotic
2. Conjugated proteins: composed of protein syndrome), glomerulonephritis, starvation
(apoprotein) and nonprotein (prosthetic group) and malnutrition
components; prosthetic groups are commonly ALPHA 1 GLOBULINS
metal, liquid, and carbohydrate in nature 1. a1-antitrypsin (AAT)
a. Metalloproteins: protein with a metal prosthetic  Primary Acute Phase Reactant
group (e.g., Cerulosplasmin)  Protease inhibitor
b. Lipoproteins: Protein with a lipid prosthetic  90% of a1-globulin band
group (e.g., Cholesterol, triglyceride)  Inactivates trypsin and other proteolytic
c. Glycoproteins: Protein with 10-40% enzymes
carbohydrates attached (e.g., Haptoglobin)  RV: 140-270 mg/dL
d. Mucoproteins: Protein with >40%  Increased: inflammatory response.
carbohydrates attached (e.g., Mucin)
Estrogen use, pregnancy
e. Nucleoproteins: Protein with DNA or RNA
 Decreased: Hereditary defect (AAT
nucleic acids attached (e.g., Chromatin)
deficiency), Emphysematous Pulmonary
Disease, Juvenile hepatic Cirrhosis
PROTEIN FUNCTIONS
2. a1-fetoprotein (AFP)
1. Energy production
 (+) Acute Phase Reactant
2. Water distribution
 Principal fetal protein
3. Buffer: the ionizable R groups of the individual
amino acids of a protein provide buffering  Detectable in maternal blood up to 7th to 8th
capacity by binding or releasing H+ ions as month of gestation
needed.  Screening done between 15 and 20 weeks
4. Transporter gestation
5. Antibodies  RV: 5 ng/mL
6. Cellular proteins: function as receptors for  Increased: Neural Tube Defects, Spina
hormones so that the hormonal message can Bifida, Fetal Distress, Twins, Hepatoma
activate cellular components; some hormones (>1000 ng/mL) or Gonodal Cancer, HDN,
are protein in nature tyrosinosis
7. Structural proteins  Decreased: Trisomy 21 (Down’s
8. Enzymes Syndrome), Trisomy 18 (Edward’s
syndrome)
PLASMA PROTEIN 3. a1-acid GLYCOPROTEIN
1. PREALBUMIN/ TRANSTHYRETIN  (+) Acute Phase Reactant
 Indicator of malnutrition  AKA: Orosomucoid
 Landmark to confirm specimen is CSF  Useful diagnostic tool in neonatal bacterial
 Transports thyroxine and retinol by binding infection
Retinol Binding Protein  Increased: pregnancy, Cancer, Pneumonia,
 RV: 18-45 mg/dL Rheumatoid Arthritis, Cell Proliferation
 Increased: steroid therapy, chronic renal failure,  Decreased: Nephrotic syndrome
alcoholism 4. a1-lipoprotein
 Decreased: liver disorders, inflammation,  (+) Acute Phase Reactant
malignancy and poor nutrition  Transports lipid (HDL)
2. ALBUMIN  Decreased: Positive risk factor for
 Negative APR atherosclerosis
 Binds bilirubin, steroids, fatty acids 5. a1-antichymotrypsin
 Major contributor to Oncotic Pressure  (+) Serine protease inhibitor
 (1/2 plasma protein mass)  Binds and inactivates PSA (Prostate
 Indicator of nutritional status Specific Antigen)
 Reservoir of amino acids  Vital component of amyloid deposits in the
 RV: 3.5 – 5.0 g/dL brain
 Increased: Dehydration  Increased: infection, malignancy, Liver
disease, burn, AMI Alzheimer’s disease
  Decreased: Liver disease  Decreased: Hemolytic Anemia,
6. Inter-a-trypsin inhibitor Intravascular hemolysis
 Serine protease inhibitor
7. Gc-globulin/Group specific component (Gc) 5. B2-macroglobulin
 Transport vitamin D and binds action  Single polypeptide chain that is a component
ALPHA 2 GLOBULINS of the light chain of HLA class I
1. HAPTOGLOBIN  Freely filtered by the glomerulus and
 (+) Acute Phase Reactant completely reabsorbed
 binds free hemoglobin to prevent urinary  Increased: inflammatory disease (SLE, RA),
loss Renal failure, multiple myeloma, HIV
 increased: stressful conditions, 6. Complement
myoglobinuria, inflammation  C1r, C2, C4, and C5
 decreased: burns and trauma, intravascular  Increased: Immune Response
hemolysis, liver disease, low RBC  Decreased: Hypocomplementemia, DIC,
production, hemoglobinuria hemolytic anemia, malnutrition
2. CERULOPLASMIN 7. Fibrinogen
 (+) Acute Phase Reactant  Precursor of fibrin clot, APR
 Copper-carrying protein with enzymatic  Increased: Inflammatory disorders,
activities pregnancy, contraceptives
 Marker for Wilson’s disease (0.1 g/L)  Decreased: extensive coagulation
 Increased: inflammatory response, cancer, 8. C-reactive protein
pregnancy  Primary APR, promotes phagocytosis
 Decreased: malnutrition, Wilson’s disease,  Cardiac marker and inflammatory marker
Menke’s syndrome, Nephritic disease  RV: < 1.0 mg/dL
3. a2-macroglobulin  Increased: Acute rheumatic fever, MI, RA,
 protease inhibitors gout, infection
 largest non-Ig plasma protein (800,000 D)  Decreased: Liver disease
 complexes with PSA GAMMA GLOBULINS
 increased: nephrotic Syndrome (10x 1. Immunoglobulins
increase), Diabetes, Liver Disease  Glycoproteins produced by plasma cells
BETA GLOBULINS  IgG, IgA, Ig M, IgD, IgE
1. Pre-B-lipoprotein  Increased: Humoral Immune Response,
 Transports lipids (VLDL) / transport Auto-immune Disorder, Allergic Reaction
endogenous TAG from liver to muscle, fat  Decreased: Hypogammaglobulinemia,
depots, peripheral tissues Immune deficiency
 Increased: hyperlipoproteinemia,
atherosclerosis
 Decreased: starvation
2. B-lipoprotein
 Transports lipids (LDL)
 Increased: hyperlipoproteinemia,
atherosclerosis, cardiovascular disease
3. Transferrin (Siderophilin)
 Binds free ferric iron (Fe3+), Negative
APR
 Increased: Hemochromatosis, Iron
deficiency anemia, estrogen use
 Decreased: Infections, liver disease,
malnutrition, nephrotic syndrome, anemia
of chronic disease
4. Hemopexin
 Binds free Heme; APR
 Increased: Acute Inflammation
CC 1 ↑ alpha 1 and a2,
Lesson 5 – Protein (Con nua on) chronic ↑ a1, a2,
gamma)
PLASMA PROTEIN ↓ ↓ N Inadequate diet,
 MYOGLOBIN nephro c
A red protein containing heme, which carries and syndrome
stores oxygen in muscle cells (Oxygen carrier in ↓ N ↓ Immunodeficiency
muscles). syndrome
- Cardiac marker; earliest to elevate but ↑ ↑ ↑ Dehydra on
nonspecific ↑ N ↑ Mul ple
↑1-4 hours of onset, peaks at 6-9 hrs myeloma,
 TROPONIN (cTn) monoclonal and
Cardiac marker (most specific) for Acute polyclonal
Myocardial Infarc on (AMI) or Acute Coronary gammopathies
Syndrome
RV: < 0.1 ng/mL GENERAL CLINICAL SIGNIFICANCE
↑4-10 hours of onset, peaks at 12-48 hours, CAUSES OF CAUSES OF
elevated 4-10 days INCREASED DECREASED
 FETAL FIBRONECTIN (fFN) VALUES VALUES
Placental adherence to the uterus TOTAL Dehydra on, Gastrointes nal
↑ preterm labor and delivery PROTEIN severe exercise, cancers, liver
 FIBRONECTIN infec ons, damage,
Mediates a wide variety of cellular interac ons cancer malnutri on, low
with extracellular matrix (ECM) and plays thiamine,
important roles in cell adhesion, migra on, growth glomerulonephri s
and differen a on.
- Also a marker for nutri on.
ALBUMIN Dehydra on, Pregnancy,
 CROSS-LINKED C-TELOPEPTIDES
sunstroke, malnutri on,
- Proteoly c fragment of collagen type I
exercise, malabsorp on, liver
- Marker of bone resorp on
mul ple disease, kidney
 B-TRACE PROTEIN
sclerosis, disease, burns
AKA: Prostaglandin D Synthase or Lipocalin
hypothyroidism
Prostaglandin D2 Synthase is an important
cons tuent of cerebral spinal fluid and is found in
much lower concentra on in blood
- Marker for CSF leakage
Normal Condi on
 CYSTATIN C
Cysteine proteinase inhibitor Albumin
Marker for kidney func on (GFR)
 ADIPONECTIN
- Recent studies have shown an inverse
correla on between body mass index and Alpha1 Beta
adiponec n values Alpha2 Gamma
- Lower levels of adiponec n correlate with an
increased risk of heart disease, type 2 diabetes,
metabolic syndrome, and obesity.
TOTAL PROTEIN ABNORMALITIES
Serum protein electrophoresis showing pa erns of
TP ALBUMIN GLOBULIN DISEASE
norm Ciesla B. Hematology in Prac ce, 2nd ed.
CORRELATION
Philadelphia FA Davis
N/↓ ↓ ↑
Condi on Pa ern
Hepa c damage Acute inflamma on ↑ alpha-1 & alpha-2
(Cirrhosis beta- Chronic infec on ↑ alpha-1, alpha-2, &
gamma bridging, gamma
Hepa s) Cirrhosis Polyclonal ↑ (all frac ons) in
Burns, trauma, gamma with beta-
infec ons (acute gamma bridging
 Monoclonal gammopathy sharp ↑ in 1  Albumin has a higher concentra on than
immunoglobulin globulin.
(“M Spike”). ↓ in  Certain disease states may affect rela ve
other frac ons amount of albumin and globulin.
Polyclonal gammopathy Diffuse ↑ in gamma  The A/G ra o provides a clue as to the cause of
Hypogammaglobulinemia ↓gamma the change in protein levels.
Nephro c syndrome ↓ albumin, ↑ alpha-2
Alpha-1-an trypsin ↓ alpha-1 LOW A/G Ra o HIGH A/G Ra o
deficiency Can reflect Suggests underproduc on
Hemolyzed specimen ↑ beta or unusual overproduc on of of globulins
band between alpha-2 globulins  Immunodeficiency
& beta  Mul ple syndromes
Plasma extra band myeloma  Alpha-1 an trypsin
(fibrinogen) between  autoimmune (AAT) deficiency
beta & gamma diseases
Or there can be
underproduc on of
albumin, or selec ve
loss of albumin
 Nephro c
syndrome
 Kwashiorkor,
malnutri on
 Cirrhosis
 Ascites
 Enteropathy

LABORATORY METHODS OF MEASURING

PROTEINS
Specimen: SERUM (avoid hemolysis. Lipemia and
Normal serum protein electrophoresis
hemoconcentra on)
Reference value:
Total protein:
Ambulatory: 6.5 – 8.5 g/dL
Recumbent: 6.0 – 7.8 g/dL
Albumin: 3.5 – 5.0 g/dl
PROTEIN ELECTROPHORESIS
Monoclonal gammopathy Rate of migra on: Depends on size, shape, &
charge of molecule
Support medium: Cellulose acetate & agarose
Buffer: Barbital buffer, pH 8.6
Stains: Ponceau S, Amido blue, Bromphenol blue,
Coomassie brilliant blue
Charge: at pH 8.6, proteins are nega vely charged
& move toward anode
ALBUMIN: GLOBULIN RATIO Largest frac on: albumin
 Total Protein = Albumin + Globulin
 Normal A/G ra o 2:1 (0.8 to 2.0)
Electroendosmosis: Buffer flow toward cathode. METHODS OF MEASURING ALBUMIN
Causes gamma a region to be cathodic to point of SALT Globulins are Reagents:
applica on PRECIPITATION precipitated in sodium sulfate,
high salt sodium sulfite,
Urine: must be concentrated first because of low
concentra on. ammonium
protein concentra on. Bence jones proteins
Albumin in sulfate and
migrate to gamma region
supernatant is methanol
CSF: Must be concentrated first because of low quan tated by
protein concentra on. CSF has a prealbumin band. biuret reac on
DYE BINDING
METHYL Non-specific
ORANGE dye for
albumin
HABA [2,(4’- Specific for Interferences:
hydroxyazoben albumin but Hemolysis-
zene)-benzoic subject to increase
acid] interferences. Heparin-
Absorbs increase
strongly below Bilirubin-
500 nm decrease
Salicylates-
slight decrease
BCG Sensi ve, most Interferences:
LABORATORY METHODS OF MEASURING (bromcresol commonly Hemolysis –
PROTEINS green) used. Absorbs slight increase
strongly below Salicylate- no
METHOD OF DESCRIPTION 500nm with effect
MEASURING TOTAL addi onal Bilirubin –
PROTEIN maximum decrease
KJELDAHL REFERENCE METHOD absorbance at Heparin-
Total protein calculated 630nm (less decrease
based on assumed 16% affected by (remedy: add
nitrogen content of bilirubin and detergent)
proteins Hb)
Reagents: CH2SO4 + CuSO4 Overes mates
REFRACTOMETRY Measurement of refrac ve low albumin
index due to solute in levels
serum. Protein is highly
refrac ve BCP Specific and
BIURET Forma on of violet-colored (bromcresol precise
chelate between Cuprous purple)
ions and pep de bonds in
an alkaline medium. AMINOACIDOPATHIES
Absorbance at 540 nm is  Inherited errors of metabolism due to enzyme
propor onal to total defects of deficiencies
protein concentra on.
Requires at least two DISEASE: PHENYLKETONURIA
pep de bonds. CAUSE: Deficiency of enzyme phenylalanine
Recommended for hydroxylase that converts phenylalanine to
measuring total protein. tyrosine. Phenylpyruvic acid in blood & urine.
DYE BINDING Protein binds to dye and EFFECT: Mental retarda on. Urine has “mousy”
causes a spectral shi in the odor.
absorbance of maximum DIAGNOSIS: Guthrie bacterial inhibi on assay (B-
dye at 560-630 nm. 2-thienylalanine), HPLC, tandem mass
spectrometry (MS/MS), fluorometric & enzyma c
methods. All newborns are screened
DISEASE: TYROSINEMIA 2. Citrullinemia
CAUSE: Enzyme deficiency  Type I: lack of arginosuccinic acid
Type I: fumarylacetoacetate hydrolase synthetase (ASAS)
Type II: Tyrosine aminotransferase  Type II: muta on of gene that codes for
Type III: 4-hydroxyphenylpyruvate citrin
dioxygenase 3. Agrinosuccinic aciduria – lack of arginosuccinic
EFFECT: Liver and kidney disease, death (Type I). acid lyase (ASL)
o Cabbage odor urine
DIAGNOSIS: MS/MS LIPIDS

 Primary source of fuel, provide stability to

DISEASE: ALKAPTONURIA cell membranes and allow for
CAUSE: Deficiency of enzyme homogen sate transmembrane transport
oxidase needed in metabolism of tyrosine &  Insoluble in water but soluble in organic
phenylalanine. Buildup of homogen sic acid. solvents (chloroform, ether) – non polar
EFFECT: Diapers stain black due to homogen sic  Major lipids: phospholipids, cholesterol,
acid in urine. Later in life, darkening of ssues, hip
triglycerides, fa y acid, fat soluble vitamins
& back pain. 1. PHOSPHOLIPID (Conjugated Lipid)
DIAGNOSIS: GC-MS
 MOST ABUNDANT LIPID from phospha dic
acid; originate in liver and intes ne.
DISEASE: MAPLE SYRUP URINE DISEASE (MSUD)
 Par cipates in cellular metabolism and
CAUSE: Branched chain a-ketoacid decarboxylase
blood coagula on
enzyme deficiency leading to buildup of leucine,
 Formed from conjuga on of two fa y
isoleucine, valine, and ketoacids.
acids and phosphorylated glycerol =
EFFECT: Burnt-sugar odor to urine, breath, skin.
amphipathic lipid
Failure to thrive, mental retarda on, acidosis,
seizures, coma, death.  Sphingomyelin is an essen al component
of cell membranes of RBCs and nerve
DIAGNOSIS: Modified Guthrie test (azaleucine),
sheath
MS/MS
 RV: 150-380 mg/dL (serum)
DISEASE: HOMOCYSTINURIA o Forms of phospholipids
CAUSE: Deficiency in enzyme cystathionine-B-  Lecithin/phospha dyl
synthetase needed for metabolism of methionine. choline 70%
Methionine & homocysteine build up in plasma &  Sphingomyelin 20%
urine.  Cephalin 10%
EFFECT: Osteoporosis, dislocated lenses in eye,  Phospha dyl
mental retarda on, thromboembolic events. ethanolamine
DIAGNOSIS: Guthrie test (L-methionine  Phospha dyl
sulfoximime), MS/MS, LC-MS/MS serine
 Lysolecithin +
DISEASE: CYSTINURIA inositol
CAUSE: Increased excre on of cys ne due to defect phospha de
in renal reabsorp on. 2. CHOLESTEROL (3-hydroxy-5,6-cholestene)
EFFECT: Recurring kidney stones. Greasy  Unsaturated steroid alcohol containing
resembling old soap four rings with a single C-H side chain tail
DIAGNOSIS: Test urine with cyanide nitroprusside. similar to fa y acid
Posi ve = red-purple color  Found on the surface of lipid layers;
regulates fluidity of lipid bilayers
 Synthesized in the liver; almost exclusively
OTHERS: synthesized by animals
1. Isovaleric Acidemia  Not catabolized by most cells – not a
 Deficient isovaleryl-CoA dehydrogenase source of energy
(increased isovaleric acid, metabolite of  Not lyse during exercise
leucine, in blood)  Should be measured in all adults 20 years
 Sweaty feet odor of urine and skin and older at least once every 5 years
 Tandem MS/MS
  Important in the assembly of cell Color Developer:
membranes, bile acids and steroid
a. GLACIAL ACETIC ACID
hormones
b. ACETIC ANHYDRIDE
 Transport and excre on is promoted by
c. CONCENTRATED SULFURIC AICD
estrogen
 Evaluates risk for atherosclerosis, GENERAL METHODS
myocardial infarc on and coronary arterial
occlusions ONE STEP METHOD
 TWO (2) FORMS:  Pearson, Stern, and Mac Gavack
o Cholesterol esters (CE)-70%  Colorimetry
 Cholesterol bound to fa y acids
and is found in plasma and TWO STEP METHOD
serum  Bloors
 Neutral lipid: found in the center  Extrac on + Colorimetry
of lipid drops and lipoproteins  Cholesterol is extracted using an alcohol ether
(along with TG) mixture
 Esterified by LCAT  Measured using L-B
 Excess cholesterol is re-
esterified by the microsomal THREE STEP METHOD
enzyme acyl cholesterol acyl
transferase (ACAT) and is stored  Abell-Kendall
un l needed.  Saponifica on + E + Colorimetry
 Lecithin-Cholesterol Acyl  Cholesterol saponified/hydrolyzed with
Transferase (LCAT) – ac vated alcoholic KOH
 Extracted with petroleum jelly
by Apo A-1 and enable to HDL to
accumulate cholesterol esters by  Measured with L-B
promo ng transfer of fa y acids FOUR STEP METHOD
from lecithin to cholesterol =
lysolecithin and cholesterol  Shoenheimer Sperry, Parekh and Jung
ester.  S+E+C+Precipita on
o Free cholesterol (FC) - 30 % Precau ons:
 Polar nonesterified alcohol
found in plasma, serum, and 1. Avoid hemolyzed blood – falsely increased
rbcs 2. Avoid icteric specimens – 5-6 mg/dL increase in
 Produced by lysosomal cholesterol per mg of bilirubin above normal
hydrolysis and becomes 3. Avoid water contamina on
available for membrane, 4. Observe precise and accurate ming for color
hormone and bile acid synthesis development
 Laboratory methods:
ENZYMATIC METHOD
Specimen: Fas ng (12-14 hrs) or non-fas ng
plasma or serum  Most common method of quan fying the
 Use tube with gel separator to avoid cholesterol oxidase reac on is to measure the
exchange of cholesterol with RBC amount of hydrogen peroxide produced
membranes if not refrigerated at 4°C  If the cholesteryl ester hydrolase step is
 2 weeks prior to tes ng: pa ent should omi ed, it can be used to measured
be in their usual diet and neither losing unesterified cholesterol
nor gaining weight
Cholesterol ester + H2O-cholesterol-esterasecholesterol + fa y acid
CHEMICAL METHODS Cholesterol + O2-cholesterol-oxidase  cholest-4-en-3-one + H2O2
Principle: Dehydra on and oxida on of cholesterol to H2O2 + phenol + 4-aminoan pyrine-peroxidasequinoneimine dye
form a colored compound (COLORIMETRY)
1. Salkowski Reac on – End Product:
Cholestadienyl Disulfonic Acid (RED)
2. Liebermann-Burchardt – End-product
Cholestadienyl Monosu onic Acid (GREEN)
 REFERENCE METHOD CHEMICAL METHODS
1. COLORIMETRIC (Van Handel & Zilversmith)
CDC REFERENCE METHOD: Abell, Levy and Brodie
Method Triglycerides – alcohol KOH  Glycerol + fa y acids (FA)
 Hydrolysis/saponifica on with alcohol KOH + Glycerol oxidized by Periodic acid  Formaldehyde (HCHO)
Hexane extrac on Colorimetry (L-B reagent)
HCHO + Chromotropic Acid  (+) BLUE colored compound
CURRENT REFERENCE METHOD
2. FLUOROMETRIC (Hantzsch Condensa on)
 GC-MS
Triglycerides – alcohol KOH  Glycerol + fa y acids (FA)
NIST GOLD STANDARD/ Defini ve method: ISOTOPE
Glycerol oxidized by Periodic acid  Formaldehyde (HCHO)
DILUTION/ MASS SPECTROMETRY (IDMS)
HCHO + diacetyl Acetone + NH3  Diacetyl Lu dine compound
 Used for research se ng (yellow)
NCEP GUIDELINE RECOMMENDATION FOR ADULTS IN ENZYMATIC METHOD
TERMS OF RISK FOR CHD:
 Specific, rapid and easy to use
DESIRABLE <200 mg/dL (<5.8 mmol/L)  Major interference: glycerol (corrected by
BORDERLINE HIGH 200-239 mg/dL (5.18-6.19 using blank assay – without addi on of lipase)
mmol/L) 1. GLYCEROL KINASE METHOD
HIGH CHOLESTEROL > 240 mg/dL (>6.72 mmol/L)  Involves hydrolysis of TG to FA and
AGE MODERATE HIGH RISK glycerol followed by phosphoryla on
RISK of glycerol to glycerophosphate
2 – 19 > 170 mg/dL >185 mg/dL  Disappearance of NADH is measured
20 – 29 >200 mg/dL >220 mg/dL at 340nm
30 – 39 >220 mg/dL >240 mg/dL
Enzymes: Lipase, Glycerol kinase, LDH,
40 - 49 >240 mg/dL >260 mg/dL
3. TRIGLYCERIDE/ TRIACYLGLYCEROL (neutral fat) Pyruvate kinase, Glycerol phosphate
 Main storage lipid in man dehydrogenase, Diaphorase
 Resynthesized in the intes nal epithelial cells REFERENCE METHOD
a er absorp on then combines with cholesterol
and Apo-B48 to from chylomicrons 1. MODIFIED VAN HANDEL & ZILVERSMITH
 Contains 3 molecules of fa y acids and one (COLORIMETRIC)
molecule of glycerol  Time consuming manual method
 The breakdown is facilitated by lipoprotein  Involves alkaline hydrolysis
lipase (LPL) epinephrine and cor sol (saponifica on) with alc. KOH, solvent
 Fas ng TAG ≥200 mg/dL are at risk for coronary extrac on with chloroform and
artery disease because of atherogenic VLDL treatment with silicic acid
remnants
(chromatography) to remove
 TAG and cholesterol are the most important
phospholipids and isolate TG
lipids in the management of CAD
 Laboratory measurement: based on hydrolysis Glycerol + sodium periodate  formaldehyde (HCHO) + formic acid
of fa y acids to produce glycerol
HCHO + Chromotropic Acid  (+) PINK colored compound
 Specimen: fas ng for 12-14 hours plasma or
serum CDC TRIGLYCERIDE LEVELS
 Interference: ascorbic acid, bilirubin and
hemolysis NORMAL < 150 mg/dL
< 200 mg/dL TG = clear serum BORDERLINE HIGH 150-199 mg/dL
300 mg/dL TG = turbid serum HIGH 200-499 mg/dL
600 mg/dL = opaque or milky serum VERY HIGH ≥ 500 mg/dL
Elevated triglyceride levels may be seen in Fredrickson
Type I, IIb, IV, AND V Hyperlipoproteinemia,
pancrea s, alcoholism, obesity, hypothyroidism,
nephro c syndrome, and storage diseases (Gaucher,
Niemann-Pick)
LIPOPROTEINS 3. Reverse Cholesterol Transport Pathway
o HDL par cles mobilize Ch from ssues
 Spherical lipid and protein complex, liquid core
and reintroduces it into the circula on
(TG and CE) and an outer shell of
for exchange with VLDL for further
phospholipids, protein and free cholesterol
metabolism or back to the liver for
 Transport lipids throughout the body excre on into the bile
 A ached with apolipoproteins
 3 main func ons of apolipoproteins: MAJOR LIPOPROTEINS
o Ac vate enzymes to aid in lipid
 CHYLOMICRONS
metabolism
 Largest and least dense produced in the
o Maintain structural integrity of
intes ne
lipoprotein molecule
 Delivers dietary lipids to hepa c and
o Enhance cellular uptake of lipoproteins
peripheral cells
 Used in assessment of atherosclerosis and
 Triglycerides are the predominant lipid
Coronary Artery disease
component
 Lipoprotein metabolism/lipoly c enzymes:
 Major apoliprotein: main: Apo B-48;
1. Lipoprotein Lipase – hydrolyzed TAG to
others: Apo A1, Apo C, Apo E
glycerol, monoglycerol and fa y acids
 Cleared 6-9 hours post prandial; NON-
which are rapidly removed by the liver
ATHEROGENIC
2. Hepa c lipase – hydrolyzes TAG and
 Density: < 0.95 kg/L
phospholipids form HDL; and lipids in VLDL
 VLDL/ VERY LOW-DENSITY LIPOPROTEIN
and IDL
 AKA: Pre-beta-lipoprotein
3. Lecithin cholesterol acyltransferase (LCAT)
– converts free cholesterol to CE  Secreted in the liver
4. Endothelial lipase – hydrolyzes HDL for the  Major apolipoprotein: Apo B-100, Apo C
release of TG and phospholipids and Apo E
5. ATP-binding casse e protein A1 (ABCA1) –  Transports endogenous TAG from the
for efflux of cholesterol from peripheral liver to peripheral ssues
ssues into HDL  ATHEROGENIC
 Lipid metabolism:  Density: 0.95 – 1.006 kg/L
1. Dietary or exogenous pathway of lipid  LDL/ LOW-DENSITY LIPOPROTEIN
transport:  AKA: Beta-lipoprotein
o Absorp on of cholesterol and  Major catabolic end-product of VLDL
triglycerides through the intes ne with  Cons tutes about 50% of the total
the forma on of chylomicrons (CM) lipoproteins in plasma
into the lymph (chyle) and into the  Primary target of cholesterol lowering
blood by way of the thoracic duct therapy and primary marker for CHD risk
o LPL liberates fa y acids from TAG,  Major apolipoprotein: Apo B-100 and
reducing the size of CM to become CM Apo E
remnants which are taken up by the  Transports cholesterol to peripheral
liver ssues: MOST ATHEROGENIC
o Free fa y acids are taken up by the  Density: 1.019 – 1.063 kg/L
muscles and adipose ssue  Research method: Beta Quan fica on
2. Endogenous pathway  HDL/ HIGH-DENSITY LIPOPROTEIN
o Produc on of TAG from free fa y acids  AKA: Alpha-lipoprotein
by the liver with the synthesis of VLDL  Smallest (5-12nm) and most dense
o VLDL par cles are converted to IDL  Produced in the liver and the intes ne
that can either be removed by the liver  Major apolipoprotein: Apo A-1
by apo-E or converted to cholesterol-  Transports excess cholesterol from ssue
rich LDL back to liver
o LDL can be taken up by the liver or into  HDL2 transports more effec vely and is
other ssues for steroid or cell more CARDIOPROTECTIVE
membrane synthesis  Density: 1.063 – 1.21 kg/L
  CDC Reference method: 
Increased levels: premature CHD and
Ultracentrifuga on precipitated with stroke
heparin- MnCl2 and Abel-Kendall assay  Electrophore c mobility: like VLDL
(some mes between LDL and albumin)
The phospholipid content of HDL is more important
 Density: like LDL (1.045 – 1.080 kg/L)
than the cholesterol or protein content in reverse
 Isola on in the LDL-HDL density range by
cholesterol transport abnormali es in the
ultracentrifuga on and measured by
phospholipid composi on have greater effect on HDL
immunoassay
func on.
 Lipoprotein X (LpX)
OTHER LIPOPROTEINS  Found in obstruc ve jaundice and LCAT
deficiency
 IDL (INTERMEDIATE DENSITY LIPOPROTEINS)
 Specific and sensi ve indicator of
 Product of VLDL catabolism/ “VLDL
cholestasis
remnant”
 Lipid content is mostly phospholipid and
 Converted to LDL: “Subclass of LDL”
free cholesterol (90%)
 Migrates either in the pre-B or Beta
 Contains albumin and Apo C
region
 B-VLDL
 Defec ve clearance of IDL: Type 3
 AKA: “Floa ng B-Lipoprotein”
hyperlipoproteinemia due to deficiency
 Abnormally migra ng B-VLDL,
of Apo E-III
cholesterol-rich VLDL
 Major apolipoprotein: Apo B-100
 Electrophore c mobility: Like LDL
 Density: 1.006 – 1.019 kg/L
 Density: like VLDL (< 1.006 kg/L)
 Lp(a) / Lipoprotein (a)
 Due to accumula on of IDL because of
 AKA: “sinking pre-B Lipoprotein”
failure to fully convert VLDL to IDL
 LDL variant that has a molecule of Apo (a)
 Found in Type 3 hyperlipoproteinemia/
linked to Apo B-100 by a disulfide bond
Dysbetalipoproteinemia
 Independent risk factor for
atherosclerosis

Summary of Major Lipoprotein Comparison

LIPOPROTEIN DENSITY MAJOR LIPIDS ELECTROPHORETIC PROTEIN MAJOR

(DIAMETIER) (g/mL) MOBILITY CONTENT PROTEIN
Chylomicrons < 0.95 Dietary/Exogenous TG Origin 1 – 2% Apo B-48
(> 70 nm) (90%)
VLDL (26-70 0.95 – 1.006 Endogenous TG (65%) Pre-beta 6 – 10% Apo B-100
nm) Cholesterol (15%)
LDL (19-23 nm) 1.019 – 1.063 Cholesterol (50%) Beta 18 -20% Apo B-100
HDL (4-10 nm) 1.063 – 1.21 Cholesterol (20%) Alpha 45 – 55% Apo A-1
Phospholipid (25%)
Comparison of Major Lipoproteins Based on Content

LIPOPROTEIN TAG CHOLESTEROL FREE PHOSPHOLIPID PROTEIN

ESTER CHOLESTEROL
CM 80 – 95% 2 – 4% 1 – 3% 3 – 6% 1 – 2%
VLDL 45 – 65 % 16 – 22% 4 – 8% 15 – 20% 6 – 10%
LDL 4 – 8% 45 – 50% 6 – 8% 18 – 24% 18 – 20%
HDL 2 – 7% 15 – 20% 3 – 5% 26 – 32% 45 – 55%

ApoLIPOPROTEIN PRIMARY SOURCE LIPOPROTEIN FUNCTION

ASSOCIATION
ApoA-I Intes ne, liver HDL, Chylomicrons Structural protein for HDL
Ac vates LCAT
 ApoA-II Liver HDL, Chylomicrons Structural protein for HDL
ApoA-IV Intes ne HDL, Chylomicrons Unknown
ApoA-V Liver VLDL, Chylomicrons Promotes LPL-mediated
triglyceride lipolysis
Apo(a) Liver Lp(a) Unknown
ApoB-48 Intes ne Chylomicrons Structural protein for
chylomicrons
ApoB-100 Liver VLDL, IDL, LDL, Lp(a) Structural protein for
VLDL, LDL, IDL, Lp(a)
Ligand for binding to LDL
receptor
ApoC-I Liver Chylomicrons, VLDL, HDL Unknown
ApoC-II Liver Chylomicrons, VLDL, HDL Cofactor for LPL
ApoC-III Liver Chylomicrons, VLDL, HDL Inhibits lipoprotein
binding to receptors
ApoE Liver Chylomicron remnants, Ligand for binding to LDL
IDL, HDL receptor
ApoH Liver Chylomicrons, VLDL, LDL, B2 glycoprotein I
HDL
ApoJ Liver HDL Unknown
ApoL Unknown HDL Unknown
ApoM Liver HDL Unknown
Abbrevia ons: HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LCAT, lecithin-cholesterol
acyltransferase; LDL, low-density lipoprotein; Lp(a), lipoprotein A; LPL, lipoprotein lipase; VLDL, very low-density
lipoprotein.

 Apo A-1 is the major protein found in HDL. It  Based on the protein and TG content of
ac vates lecithin-cholesterol acyltransferase lipoproteins
(LCAT) and removes free cholesterol from
Order from most to least dense: HDL, LDL, VLDL,
extrahepa c ssues. Thus, it is considered
CM
an atherogenic.
 Apo B-100 is the major protein found in LDL. It 2. ELECTROPHORESIS
is associated with increased risk of coronary  Separa on based on size and charge
artery disease. Lp(a) is an independent risk  Lipid stains: Oil red O, Sudan black B and Sudan
factor associated with impaired plasminogen III
ac va on and thus decreased fibrinolysis. A
high level suggests increased risk for coronary Order from fastest/most anodic: HDL, VLDL, LDL, CM
heart disease and stroke. LABORATORY METHODS FOR LIPOPROTEINS:
TEST METHODOLOGY STANDING PLASMA TEST
Apo-A, Apo-B, and Lp(a) are measured by  An aliquot of plasma (2 mL) is placed into a
immunochemical methods such as 10 x 75-mm test tube and allowed to stand in
immunoturbidimetric and immunonephelometric. the refrigerator at 4°C undisturbed overnight.
LABORATORY METHODS FOR LIPOPROTEINS:  Chylomicrons accumulate as a floa ng “cream”
layer and can be detected visually.
 Specimen: EDTA PLASMA, 12 -14 hrs fas ng, but  The presence of chylomicrons in fas ng plasma
preferred now is serum collected in a serum is considered to be abnormal.
separator tube
 A plasma sample that remains turbid a er
1. ULTRACENTRIFUGATION
standing overnight contains excessive amounts
 Sample is adjusted to density of 1.063 with of VLDL; if floa ng “cream” layer also forms,
Potassium Bromide and centrifuged at high chylomicrons are present as well.
speed for 24 hours
 Sample separates based on density HDL MEASUREMENT – to measure, combine
 REFERENCE METHOD ultracentrifuga on with polyanion precipita on
 1. Polyanion precipita on (3-step chemical  Remaining solu on is measured for
precipita on) cholesterol
 Apo B containing lipoproteins (CM, VLDL, LDL,  LDL is precipitated out and the solu on is
IDL) are precipitated out using a polyanion again measure for cholesterol
(heparin sulfate, dextran sulfate, or  The difference between the 2
phosphotungstate) and divalent ca on (Mg, measurements is the concentra on of LDL
Ca, or Mn) solu ons Reagent: dextran sulfate-
magnesium chloride or heparin sulfate- 4. HOMOGENOUS DIRECT LDL-c METHOD
manganese chloride  Useful when TG is elevated (> 600 mg/dL)
 HDL is then quan tated in the supernatant by  Use a combina on of two reagents.
cholesterol oxidase and cholesterol esterase o First reagent: selec vely removes
 Cannot be automated non-LDL and/or inhibits it from
2. Homogenous assay reac ng with enzymes
 Uses an an body to Apo B-100 to bind LDL and o Second reagent: releases
VLDL so that they will not react cholesterol from LDL to be
 HDL is then measured enzyma cally measured enzyma cally
 Principle used in automated measurements 5. GEL CHROMATOGRAPHY OR AFFINITY
CHROMATOGRAPHY
LDL MEASUREMENT
6. IMMUNOCHEMICAL METHODS
1. FRIEDEWALD EQUATION: 7. IMMUNOASSAY OR IMMUNONEPHELOMETRY
LDLc = Total cholesterol – (HDLC + VLDL) DISEASES ASSOCIATED WITH LIPIDS AND
VLDL = TG ÷ 5 (mg/dL) LIPOPROTEINS

VLDL = TG ÷ 2.175 (mmol/L) NCEP – NATIONAL CHOLESTEROL EDUCATION

PROGRAM (USA)
Not reliable when TG > 400 mg/dL or for pa ents
with B-VLDL  Provides evidence-based guidelines for
cholesterol tes ng and management, and
2. DELONG EQUATION provides detailed informa on on other topics,
More accurate than Friedewald when TG >400 including the classifica on of lipids and
lipoprotein par cles, CHD risk assessment,
mg/dL
lifestyle interven on, drug treatment, specific
LDLC = TC – (HDLc + VLDL) dyslipidemias, and treatment adherence
VLDL = TG ÷ 6.5 (mg/dL) issues.

VLDL = TG ÷ 2.825 (mmol/L) ATP (Adult treatment panel) III recognizes addi onal
posi ve risk factors for CHD, including eleva ons in
Example:
Lp(a), remnant lipoproteins, small LDL par cles,
TOTAL CHOLE – 145 mg/dL fibrinogen, homocysteine, high-sensi vity C-reac ve
protein (hs-CRP), impaired fas ng plasma glucose (110-
HDL – 36.5
125 mg/dL), and preexis ng subclinical atherosclerosis
TAG – 79.2 (as evidenced by myocardial ischemia on exercise
tes ng, caro d in mal-medial thickening, and/or
LDLc = TC – (HDLC + VLDL) coronary artery calcium deposi on).
= 145 – (36.5 + 15.84) DYSLIPIDEMIAS
= 145 – (52.34) Can be subdivided into two major categories:
= 92.66 mg/dL hyperlipoproteinemia’s, which are diseases associated
with elevated lipoprotein levels, and
3. BETA QUANTIFICATION hypolipoproteinemia’s, which are associated with
 Uses ultracentrifuga on (at least 18 hours decreased lipoprotein levels.
at 105 K x g) to separate VLDL and CM
HYPERLIPOPROTEINEMIAS 200-499 high
≥500 very high
Have been classified using the Frederickson-Levy
VLDL (Calculated)
classifica on system, which is not commonly used
≤ 30 mg/dL Desirable
today.

The hyperlipoproteinemia’s can be subdivided into Major Risk Factors That Modify LDL Goals
hypercholesterolemia, hypertriglyceridemia, and
1. Cigare e smoking
combined hyperlipidemia, with eleva ons of both
2. Hypertension (BP ≥140/190 or on
cholesterol and triglycerides.
an hypertensive medica on)
NCEP GUILDLINE RECOMMENDATIONS FOR ADULTS 3. Low HDL cholesterol (<40 mg/dL)
IN TERMS OF RISK FOR CHD 4. Family history of premature CHD (CHD in a
LDL CHOLESTEROL male 1st degree rela ve <55 years; CHD in a
< 100 Op mal (nega ve risk) female 1st-degree rela ve <65 years)
100-129 near op mal/ above op mal 5. Age (men ≥ 45; women ≥ 55)
130-159 borderline high 6. Diabetes mellitus
160-189 high 7. Preexis ng CHD
≥190 very high
NCEP Therapeu cs goals:
TOTAL CHOLESTEROL
LDL:
<200 Desirable
≤ 100 mg/dL if CHD is present
200-239 borderline high
< 129 mg/dL if no CHD with 2 or more risk factors
≥240 high
< 159 mg/dL in no CHD
HDL CHOLESTEROL
<40 POSITIVE RISK FACTOR
TC < 200 mg/dL
≥60 NEGATIVE RISK FACTOR
TAG <150 mg/dL
TRIGLYCERIDES
HDL >60 mg/dL
<150 Normal
150-199 borderline high

FREDRICKSON CLASSIFICATION OF HYPERLIPIDEMIAS

TYPE SYNONYM DEFECT SERUM CLINICAL FEATURES TREATMENT SERUM
ABNORMALITY APPREARANCE
Type I Familial Low LDL Chylomicron ↑ Pancrea s, lipemia Diet Creamy top
hyperchylomicronemia Altered re nalis, skin layer
ApoC2 erup ons,
Xanthoma,
Hepatosplenomegaly
Type Familial ↓LDL LDL ↑ Xanthelasma, Arcus Cholestyramine Clear
IIa hypercholestrolemia receptor senilis, Tendon or choles pol,
xanthomas Sta ns, Niacin
Type Familial combined ↓LDL LDL & VLDL ↑ Sta ns, Niacin, Clear
IIb hypercholestrolemia receptor & Fibrate
↑Apo B
Type Familial Apo E2 IDL ↑ Tubo-erup ve Fibrate, Sta ns Turbid
III dysbetalipoproteinemia synthesis xanthomas, palmar
defect xanthoma
Type Familial hyperlipemia ↑VLDL VLDL ↑ Sta ns, Niacin, Turbid
IV produc on, Fibrate
↓
elimina on
Type Endogenous ↑VLDL VLDL & Niacin, Fibrate Creamy top
V hypertriglyceridemia produc on, Chylomicron ↑ layer & turbid
↓LPL bo om
TYPE I HYPERLIPOPROTEINEMIA: ELEVATED Hyperapobetalipoproteinemia is associated with VLDL
CHYLOMICRONS and Apo B-100 overproduc on in the liver. It is
1) Serum appearance: Creamy layer of characterized by normal or moderate eleva on of LDL
chylomicrons over clear serum cholesterol with an elevated Apo B-100. Total
2) Total cholesterol: Normal to moderately cholesterol and triglyceride are generally elevated but
elevated may be normal. HDL cholesterol and apo a-I levels are
3) Triglyceride: Extremely elevated decreased.
4) Apo B-48 increased, Apo A-IV increased
Secondary lipoproteinemia: Many condi ons cause
TYPE IIA HYPERLIPOPROTEINEMIA: INCREASED LDL lipoproteins to be abnormally metabolized. Some of
those condi ons include diabetes mellitus,
1) Serum appearance: Clear
hypothyroidism, obesity, pregnancy, nephro c
2) Total cholesterol: Generally elevated
syndrome, pancrea s, alcoholism, and myxedema.
3) Triglyceride: Normal
4) Apo-B 100: increased HYPOLIPOPROTEINEMIAS

TYPE IIB HYPERLIPOPROTEINEMIA: INCREASED LDL a. Abetalipoproteinemia: (also known

AND VLDL as Bassen- Kornzweig syndrome)
Total cholesterol level very low,
1) Serum appearance: Clear or slightly turbid
triglyceride level nearly undetectable,
2) Total cholesterol: Elevated
LDL and Apo B-100 absent
3) Triglyceride: Elevated
b. Hypobetalipoproteinemia: unable to
4) Apo B-100 increased
synthesize apo B-100 and apo B-48,
TYPE III HYPERLIPOPROTEINEMIA: INCREASED IDL low cholesterol level and normal to
low triglyceride level
1) Serum appearance: Creamy layer some mes c. Hypoalphalipoproteinemia: severely
present over a turbid layer elevated triglyceride level and low HDL
2) Total cholesterol: Elevated level
3) Triglyceride: Elevated d. Tangier Disease: HDL absent, Apo A-I
4) Apo E-II increased, Apo E-II decreased, and Apo and Apo A-II very low levels, LDL low;
E-IV decreased total cholesterol level low, triglyceride
TYPE IV HYPERLIPOPROTEINEMIA: INCREASED VLDL level normal to slightly increased. Due
to a muta on in the ABCA 1 gene on
1) Serum appearance: turbid chromosome 9.
2) Total cholesterol: Normal to slightly elevated
3) Triglyceride: Moderately to severely elevated Lipoprotein Lipase (LPL) Deficiency
4) Apo C-II either increased or decreased, Apo B-
 Results to inability to clear chylomicron
100 increased par cles crea ng the classic type I
TYPE V HYPERLIPOPROTEINEMIA: INCREASED VLDL chylomicronemia syndrome which has TAG =
WITH INCREASED CHYLOMICRONS 10,000 mg/dL or 113.0 mmol/L
 Pa ents with this condi on do not develop
1) Serum appearance: Turbid with creamy layer premature coronary disease, implying that
2) Total cholesterol: Sightly to moderately chylomicron themselves are not atherogenic
elevated  Characterized by abdominal pain and
3) Triglyceride: Severely elevated pancrea s
4) Apo C-II increased or decreased, Apo B-48
increased, and Apo B-100 increased LCAT/Lecithin Cholesterol Acyltransferase Deficiency

The most common familial form is familial combined  AKA: Fish Eye disease
hyperlipidemia (FCHL). FCHL is characterized by  Due to muta on in the LCAT gene
increased plasma levels of total and LDL cholesterol  HDLc levels are typically < 10 mg/dL; TC is
(Type IIa), or triglyceride (Type IV), or a combina on of normal or high
both (Type IIb). Also, Apo B-100 is increased. The level
of HDL cholesterol may be decreased.
Niemann-Pick dx (lipid storage disease) Bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
 An inherited disorder of lipid metabolism;
bbbbbbbbbbbbbbb
accumula ons of sphingomyelin in bone
marrow, spleen, and lymph nodes
 Involves in deficiency of enzymes responsible
Bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
for removing phosphorylcholine from
bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
sphingomyelin
B
Arteriosclerosis – hardening and narrowing of arteries

Atherosclerosis – narrowing of arterial due to plaque

build up on arterial walls due to deposi on of B
cholesterol and TG

 Coronary artery disease  heart

B
 Peripheral vascular disease  arms/legs
 Cerebrovascular disease  brain (stroke) B

Sitosterolemia – is an extremely rare autosomal B

recessive disorder wherein phytosterols (plant sterols)
B
are absorbed and accumulate in plasma and peripheral
ssues B
CETP deficiency – is an autosomal recessive disorder in
which the transfer of cholesterol esters is inhibited. As
a result, HDL par cles are large and laden with
cholesterol ester, and apo a-l is increased, as is HDL-C
(typically > 100 mg/dl).

Chylomicron reten on disease (Anderson’s Disease) –

presents in childhood with fat malabsorp on and low
levels of plasma lipids.

METABOLIC SYNDROME:

Group of risk factors that seem to promote

development of atherosclero c cardiovascular disease
& type 2 diabetes mellitus.

Risk Factors: ↓ HDL-C, ↑ LDL-C, ↑ triglycerides, ↑

blood glucose, ↑ blood pressure

Chylomicrons – transporters of exogenous lipids/TAG

VLDL – transporters of endogenous lipids/TAG

LDL – transporter of chole to peripheral ssues; “BAD

CHOLESTEROL”

HDL – transporter of excess cholesterols from ssues

back to liver
CC1  About 180 liters of water is filtered daily; 150
Lesson 8 – NON-PROTEIN NITROGENOUS liters is reabsorbed in the proximal tubule and
SUBSTANCES about 5 liters in the descending limb of Henle
 The kidneys are paired, bean-shaped organs of cor cal nephrons.
located retroperitoneally on either side of the 1. Clearance
spinal column.  is the removal of the substance from plasma
 There are 2 regions of the kidneys – an outer into urine over a fixed me.
region called the cortex and an inner region  It represents the volume of plasma that would
called the medulla. contribute all the solute excreted.
 Nephrons, the func onal unit of each kidney,  It is expressed in milliliter/minute,
are composed of 5 basic parts namely, represen ng the volume of plasma that would
glomerulus, proximal convoluted loop of be totally cleared of the solute in one minute.
Henle, distal convoluted tubule, and collec ng  Plasma concentra on and clearance is
duct. inversely propor onal – as clearance of a
 The proximal convoluted tubule is responsible substance declines, its concentra on in plasma
for the reabsorp on of sodium, chloride, increases.
bicarbonate, and other ions; glucose; amino a. Inulin Clearance
acid and proteins; urea and uric acid. About 25  It is not rou nely done because of the
liters of dilute urine is delivered to the necessity for con nuous IV infusion –
ascending limb of Henle. Because the tubules requires an intravenous infusion and med
from this point to the beginning of the cor cal urine collec ons over many hours
collec ng duct are water impermeable, the  It has higher values in male due to larger
volume remains unchanged at 25 liters, but renal mass.
osmolality decreases progressively to about  Priming dose: 25 mL of 10% inulin solu on
60-80 mOsm/L.  Con nuous infusion: 500 mL of 1.5% inulin
 Collec ng duct is the final site for either solu on
concentra ng or dilu ng urine.  Reference values: male = 127 mL/min
 Plasma contains 20-35 mg/dL of NPN which Female = 118 mL/min
comprises urea 45%, amino acid 25%, uric acid Alterna ves to Inulin (Other Exogenous Substances);
10%, crea nine 5%, crea ne 1-2% and 1. Radioac ve markers 125I – iothalamate and
ammonia 0.2%. 99mTc-DTPA (metastable techne um99-
 Renal func on panel: glucose, BUN, crea nine, labeled diethylene triamine pentaace c acid)
sodium, potassium, chloride, phosphorus, 2. Iohexol and Chromium51-labeled
calcium, albumin, CO2. ethylenediaminetetraace c acid (51Cr-EDTA)
Func ons of the Kidneys:  The advantage of these substances is
1. Elimina on of waste products. that it does not require urine
2. Maintenance of blood volume. collec on.
3. Maintenance of electrolyte balance. 3. Nonradiolabeled iothalamate
4. Maintenance of acid-base balance.
5. Endocrine func on (erythropoie n secre on) b. Crea nine Clearance
TESTS FOR THE GLOMERULAR FILTRATION RATE  It provides an es mate of the amount
Glomerular Filtra on Rate of plasma that must flowed through
 is a measure of the clearance of normal the kidney glomeruli/minute.
molecules that are not bound to protein and  It is an excellent measure of renal
are freely filtered by the glomeruli neither func on – crea nine is freely filtered
reabsorbed nor secreted by the tubules. by the glomeruli but reabsorbed.
 It is considered the best overall indicator of the  It is a measure of the completeness of
level of kidney func on. 150 liters of a 24-hour urine collec on.
glomerular filtrate is produced daily. GFR  Produc on and excre on of crea nine
decreases by 1.0 mL/minute/year a er age 20 is related directly to muscle mass –
to 30 years. when renal func on is normal and
stable, crea nine excre on is almost
 equal to its produc on, which depends  It is freely filtered at the glomerulus, not
primarily on muscle mass. secreted by the renal tubules but
 The amount of crea nine generated reabsorbed.
from crea ne turnover tends to  It is completely reabsorbed and
remain constant for 24 hours. catabolized by the proximal convoluted
 Excre on of crea nine is not rou nely tubule, hence its presence in urine denotes
affected by diet = 1.2-1.5 g crea nine damage of that tubule – serum level is an
excreted/day indirect es mate of GFR
 Major limita on: accurate urine  It is not affected by muscle mass, age, diet
collec on and gender. It increases more rapidly than
 Reference values: crea nine in the early stages of GFR
Male = 85 – 125 mL/min impairment.
Female = 75 – 112 mL/min  Plasma level increases when glomerular
Increased Crea nine Clearance filtra on decreases, however, renal
1. High cardiac output clearance of this substance cannot be
2. Pregnancy measured because it is completely
3. Burns reabsorbed.
4. Carbon monoxide poisoning  Advantage: to assess GFR among pediatric
Decreased Crea nine Clearance and elderly pa ents, and renal transplant
1. Impaired kidney func on pa ents.
2. Shock, dehydra on  Specimen: serum or plasma (fas ng is not
3. Hemorrhage required)
4. Conges ve heart failure  Increased: acute and chronic renal failure,
c. Urea Clearance diabe c nephropathy
 It can demonstrate progression of renal disease or  Reference value:
response to therapy. 5 – 1.0 mg/dL (adults)
 It does not give reliable es mates of the GFR since 0.9 – 3.4 mg/dL (> 65 years old)
urea is freely filtered by the glomeruli but variably Modified Cysta n C Equa on:
reabsorbed by the tubules – in the presence of high GFR (m/min) = 84.69 x cysta n C (mg/L) x 1.384
urine osmolality and high urea concentra on, the +
amount reabsorbed in the inner medullary 3. Beta Trace Protein
collec ng duct is increased.  Is a low molecular weight glycoprotein.
 It is about 50% of crea nine clearance (50% GFR), in  It belongs to the lipocalin protein family
the presence of normal renal func on without and func ons as prostaglandin D synthase.
volume deple on, but in the presence of severe  Is isolated primarily from CSF – plasma BTP
volume deple on, urea clearance could be as li le originates from the brain and is freely
as 10% of crea nine clearance. filtered at the glomerulus, then is
 In advanced renal failure, urea clearance reabsorbed completely and catabolized by
approaches unity with GFR, and is a be er predictor the proximal tubule.
of GFR than crea nine clearance – as renal func on  Increased: renal disease (because of
declines, the frac on of urea reabsorbed declines reduced filtra on in the presence of
progressively, whereas the tubular secre on of constant produc on)
crea nine increases progressively. II. TEST FOR RENAL BLOOD FLOW
 Volume deple on decreases crea nine clearance A. Blood Urea Nitrogen (BUN)
only by reduced filtra on, and urea clearance by B. Crea nine
both reduced filtra on and increased reabsorp on. C. Blood Uric Acid
The faster the rate of urine flow, the less urea is Compound Approximate Approximate
reabsorbed and vice versa. plasma urine
2. Cysta n C concentra on concentra on
 Is a low molecular weight protease (% of total (% of excreted
inhibitor and produced at a constant rate NPN) nitrogen)
by all nucleated cells. Urea 45 - 50 86.0
Amino Acid 25 -
 Uric Acid 10 1.7 Indicator dye:
Crea nine 5 4.5 1. Nessler’s Reac on (Hgl2/mercuric (II)
Crea ne 1-2 - iodide, or Kl and gum gha ) – yellow end
Ammonia 0.2 2.8 product
UREA 2. Berthelot’s Reac on (alk. Hypochlorite and
 Major waste/end product of dietary and amino Na nitroprusside) – blue end product
acid catabolism b. Coupled Urease-GLD enzyme reac on – UV
 It is approximately 80% of the nitrogen enzyma c method
excreted - Glutamate dehydrogenase
 Synthesized in the liver from CO2, and the - Specific, more expensive
ammonia from the deamina on of amino acids - Measures disappearance of NADH at 340 nm
and is excreted by the kidneys Urea – urease > ammonia + CO2
 Free filtered at glomerulus but is reabsorbed Ammonium + 2-oxoglutarate + NADH – GLD 
substan ally in the proximal convoluted tubule glutamate + NAD + H2O
and inner medullary collec ng duct 3. CONDUCTIMETRIC (ISE)
 First metabolite to increase in kidney disease - Conversion of urea to ammonium and CO2
 Good indicator of nitrogen status and state of results in increased conduc vity
hydra on 4. ISOTOPE DILUTION MASS SPECTROMETRY
 Easily removed by dialysis (IDMS)
 The concentra on of urea is expressed only by - Reference method
nitrogen content of urea Increased BUN: Decreased BUN:
 Level of urea in the blood is affected by 1. Chronic renal 1. Poor nutri on
o Amount of dietary protein disease 2. Hepa c dx
o Kidney’s ability to excrete urea 2. Burns, stress 3. Impaired absorp on
(directly propor onal to GFR) 3. High protein diet (celiac dx)
o Protein metabolism 4. Dehydra on 4. Pregnancy
o Degree of hydra on
 INCREASED LEVELS IN THE BLOOD: UREMIA  Clinically, BUN rises in response to renal
 Its concentra on is expressed only as the dysfunc on.
nitrogen content of urea – blood urea nitrogen  Low BUN are not generally considered
(BUN) abnormal renal func on.
 To obtain the concentra on of urea from BUN:  Serum urea levels drop in severe hepa c
2.14 x BUN = Urea mg% or Urea x 0.467 = BUN disease because of a decline in the capacity of
 RV: 5 – 38 mg/dL (serum/plasma urea) the liver to generate urea from ammonia.
LABORATORY METHODS: CREATININE
 Specimen: fas ng plasma or serum (non-  Waste product of muscle metabolism derived
fas ng) may be acceptable) from crea ne, propor onal to skeletal muscle
 Avoid fluoride or citrate (inac vates/inhibit mass.
urease)  Amount generated is fairly constant
 Refrigerate samples if delay (bacteria  Not affected by protein diet (unlike UREA) and
u lize urea) not easily removed by dialysis
1. Chemical Method (Direct)  It is also produced by three amino acids such as
- Fearon Reac on or Diacetyl Monoxime methionine, arginine, and lysine
Method – simple, non-specific  It is par ally secreted by the proximal tubules
Urea + DAM  YELLOW Diazine deriva ve via the organic ca on transport pathway.
2. Enzyma c Method (INDIRECT)  Index of overall renal func on
- More specific, most common  It is a measures the completeness of 24 hour
a. Hydrolysis of Urea by Urease: urine collec on (< 0.8g in 24 hrs indicates
Urea-urease  ammonium + CO2 incomplete collec on)
Ammonium + indicator dye  color change  Used to evaluate fetal kidney maturity – as
gesta on progresses, more crea nine is
excreted by the fetus into the amnio c fluid
(2mg/dL).
  RV: Male = 0.9 – 1.3 mg/dL (80-115 umol/L) Kine c Principle: COLORIMETRY-
Female = 0.6 – 1.1 mg/dL (53-97 umol/L) Jaffe SPECTROPHOTOMETRY; Serum is mixed with
CREATININE CLEARANCE – provides an es mate of Method Jaffe reagent and the rate of change in
amount of plasma that flowed through the kidney absorbance is measured between 2 points
glomeruli per minute - Popular, inexpensive, rapid and easy to
o Crea nine is freely filtered by the perform – more rapid
glomeruli but not reabsorbed  Enzyma c methods
- more specific than Jaffe methods
o 1.2 – 1.5 g Crea excreted/day
- interference from bilirubin and catecholamines
o Specimen: Plasma sample, 24 hour
Crea nine - requires a large volume of pre-
urine
Aminohydrolase- incubated sample;
o RV : Male = 85 - 125 mL/min CK Method - not widely used
o Female = 75 – 112 mL/min - enzymes: Crea nine
( ) .
Formula: Clearance (mL/min) = x x aminohydrolase, CK, PK, and LDH
Where: - measures rate of disappearance
U – concentra on of the analyte in urine of NADH
P – concentra on of the analyte in plasma Crea nase- - more specific than Jaffe
Volume – volume of urine in milliliter in 24 hours Hydrogen method and may poten ally
Minutes – me required to collect urine (1440 minutes) Peroxide Method replace it
1.73 – constant value; average body surface of an adult - without interference from
individual acetoacetate and
A – body surface of the pa ent (obtained from a cephalosporins
nomogram; height and weight are taken) - crea nase = Crea nine
Aminohydrolase
Predic on equa on for pa ents with CKD: - Adapted for dry slide method
- End product: RED
( ) benzoquinoneimine dye
Ccr(mL/min) = x (0.85 if female)
 Reference method
Es mated glomerular filtra on rate (eGFR) uses only a
ISOTOPE DILUTION MASS SPECTROMETRY (IDMS)
blood crea nine and the MDRD (Modifica on Of Diet
- Detec on of characteris c fragments
In Renal Disease)
following ioniza on
 Correc on for gender and race required - Quan fica on using isotopically labeled
 Results only reported as a number if < 60 compound
mL/min/1.73 m2 Increased Serum Crea nine:
MEASUREMENT OF CREATININE: 1. Impaired renal func on
 CHEMICAL METHODS 2. Chronic nephri s
COLORIMETRIC, KINETIC: rapid, sensi ve and specific 3. Conges ve heart failure
COLORIMETRIC END-POINT: simple; less specific 4. Dehydra on
Direct Principle: COLORIMETRY; JAFFE REACTION: a Decreased Serum Crea nine:
Jaffe red-orange tautomer of crea nine picrate is 1. Decreased muscle mass
Method formed when crea nine is mixed with Jaffe 2. Advance and severe liver dx
reagent (Alkaline Picrate: saturated picric acid 3. Inadequate dietary protein
and 10% NaOH) 4. Pregnancy
a. Folin Wu method – sensi ve but
nonspecific (subject to interference by  Elevated plasma crea nine concentra on is
crea-like substances like bilirubin and associated with abnormal renal func on,
hemoglobin = falsely decreased) especially as it relates to glomerular func on.
b. Lloyd’s or Fuller’s Earth method –  Plasma crea nine is a rela vely insensi ve
sensi ve and specific by absorbing marker and may not be measurably increased
interferences; me consuming and not un l renal func on has deteriorated more than
readily automated 50%.
a. Lloyd’s reagent: Sodium aluminum  In muscle disease such as muscular dystrophy,
silicate poliomyeli s, hyperthyroidism, and trauma,
b. Fullers earth reagent: aluminum plasma crea ne and urinary crea nine are
magnesium silicate elevated.
  In the presence of normal renal func on,  There is striking BUN level, slowly rising
plasma crea nine concentra on is usually plasma crea nine, anemia and electrolyte
within reference limit or normal in muscular imbalance.
diseases.  Complica ons: coma and neuropsychiatric
 However, in severe muscle was ng, produc on changes
of crea nine could be reduced to less than 25%  Causes: acute/chronic renal disease,
of the amount predicted from the body weight. glomerulonephri s, nephrosclerosis,
BUN:CREATININE RATIO tubular necrosis, malignant hypertension,
 Be er indicator of the source of eleva on of nephrotoxic drugs or metals, renal cor cal
either substance necrosis, diabe c nephropathy,
 RV = 10:1 to 20:1 arteriosclerosis, collagen-vascular disease
BUN:CREA RATIO c. Post-renal Azotemia
LOW RATIO <10:1 Acute tubular necrosis, low protein  Is usually the result of urinary tract
intake, starva on, severe liver disease obstruc on (↓ GFR)
or vomi ng, repeated dialysis  Urea level is higher than crea nine due to
HIGH RATIO >20:1 Tissue breakdown, prerenal azotemia, back-diffusion of urea into the circula on;
with normal high protein intake, dehydra on, increased urea and crea nine in blood.
crea nine muscle was ng, dialysis increased  Causes: renal calculi (nephrolithiasis),
protein breakdown, treatment with cancer or tumors of genitourinary tract,
cor sol, decreased kidney perfusion, tumors of the bladder or prostate,
GI hemorrhage prosta s, inflamma on, surgical
HIGH RATIO >20:1 Post renal obstruc on misadventure
with high (nephrolithiasis), prerenal azotemia 2. UREMIA
crea nine with renal disease, prosta s, severe  Is a clinical syndrome comprised of a
infec on, acute renal failure marked eleva on in plasma urea and other
NORMAL RATIO End-stage renal disease, chronic renal nitrogenous waste products, accompanied
with elevated failure by acidemia and electrolyte imbalance (K
BUN and Crea eleva on).
Disease Correla on:  It is characterized by anemia (normocy c
1. Azotemia – is elevated con. of nitrogenous normochromic), uremic frost (dirty skin),
substances like urea and crea nine in blood generalized edema, foul breath and sweat
Types of Azotemia: is urine-like.
a. Pre-renal Azotemia  The kidneys fail to eliminate waste
 Is diminished glomerular filtra on with products of metabolism.
normal renal func on (↓GFR).  This condi on is responsible for changes in
 It is characterized by decreased/reduced red cell shape, with burr cells (echinocytes)
renal blood flow – GFR decreases and and ellipsoidal cells commonly present on
tubular reabsorp on increases leading to peripheral blood films – presence of burr
slower filtrate flow due to poor kidney cells during the course of illness may signal
perfusion the development of renal dysfunc on.
 Dehydra on should be considered when
BUN is elevated but the plasma crea nine
is normal
 Cause: dehydra on, shock, conges ve
heart failure, hemorrhage, increased
protein catabolism, high protein diet,
shock, blood loss, crush injuries, burns,
fever, hemolysis.
b. Renal azotemia
 is damaged within kidneys (↓GFR).
 Damage to filtering structures of the
kidney (glomerulus, tubules) Low Ra o (BUN:Crea) <10:1
 Lab result: BUN = > 100 mg/dL 1. Low protein diet
Crea nine = 20 mg/dL 2. Acute tubular necrosis
BUA = 12 mg/dL 3. Repeated dialysis
4. Hepa c disease
 High Ra o (BUN:Crea) >20:1 with normal crea nine 2. Increased nuclear metabolism
1. Pre-renal azotemia  Seen in leukemia, lymphoma, MM or
2. Dehydra on polycythemia, hemoly c and
3. Catabolic states megaloblas c anemias
4. GI hemorrhage  Monitoring is important
5. High protein diet  Treatment: allopurinol
High Ra o (BUN:Crea) >20:1 with increased crea nine 3. Chronic Renal Disease
1. Postrenal azotemia  Due to decreased GFR and tubular
2. Prerenal azotemia with renal disease secre on
3. Renal failure  BUA > 10mg/dL (can cause calculi
KIDNEY FUNCTION TESTS forma on)
Glomerular filtra on Cysta n C, Crea nine clearance, 4. Lesch-Nyhan Syndrome (Orange Sand Diaper
Inulin clearance, radioisotopes Syndrome)
Concentra on Mosenthal test, Fishberg test,  Inborn error of purine metabolism
Specific gravity, osmolality  Deficiency of hypoxanthine-guanine
Renal Blood flow p-aminohippuric acid (PAH) test phosphoribosyl transferase (HGPRT)
B2-microglobulin Test for tubular func on of the other cause of hyperuricemia: secondary to glycogen
kidney storage disease, toxemia of pregnancy, lac c acidosis,
Proteinuria Sensi ve but not specific for increased dietary intake, ethanol consump on
kidney Hypouricemia
Crea nine Single indicator of renal disease 1. Fanconi’s syndrome – renal type aminoaciduria
URIC ACID - Total loss of tubular reabsorp on
 Major product of purine (adenine and guanine) 2. Wilson’s disease
catabolism 3. Hodgkin’s disease
 Final breakdown of nucleic acids catabolism in 4. Overtreatment with allopurinol
humans 5. Chemotherapy with azathioprine or 6-
 Formed from xanthine by the ac on of mercaptopurine
xanthine oxidase in the liver and intes ne 6. Liver disease
 It is freely filtered, par ally reabsorbed and TREATMENT
secreted in the renal tubules 1. Allopurinol
 > 95 % exists as monosodium urates in plasma - Inhibits xanthine oxidase ac vity
 > 6.8 mg/dL urate crystals may precipitate in - Lowering serum urate levels without
ssues increasing excretory load in kidneys
 Organ meats, legumes and yeast = high purine 2. Uricosuric drugs
food - Ex: Probenecid and Sulfinpyrazone
 Assess inherited disorders of purine - Lowers serum urates by increasing uric acid
metabolism content of urine thereby posing a risk for
 Confirms and monitors gout stone forma on if the urine is not
maintained at alkaline pH
 Monitors for uric acid nephropathy during
3. Colchicine
chemotherapy
- Neither affects the produc on or excre on
 RV: (Uricase Method)
- It alters the phagocy c response of
Male = 3.5-7.2 mg/dL
leukocytes to urate crystals in ssues
Female = 2.6-6.0 mg/dL
LABORATORY METHODS:
Urine = 250-750 mg/day
 Specimen: heparinized plasma, serum or urine
Hyperuricemia
(fas ng preferred)
1. Gout
- Stable in room temp. for 3 days
 Found primarily in males between 3rd and
- Do not use potassium oxalate
5th decade of life
an coagulant
 Presence of birefringent crystals in the
- Interference: lipemia, bilirubin, hemolysis
synovial fluid causing pain and acute
(false decrease), salicylates and thiazides
inflammatory arthri s.
(falsely increase)
 Pa ents are highly suscep ble to 1. CHEMICAL METHOD
nephrolithiasis
 Caraway Method or Phosphotungs c
Method (PTA)
  Principle: Reduc on-Oxida on (Redox) B. Concentra on Test
reac on  It reflects the func on of the collec ng
Uric acid + PTA + O2-NaCN/NaCO3 Tungsten blue + tubules and the loops of Henle.
allantoin + CO2  It is used to assess the quality of solutes
2. ENZYMATIC METHOD (Blauch and Koch) present in urine, which reflects the ability
 Principle: UV spectrophotometry; of the kidneys to produce a concentrated
Decrease in absorbance measured at 293 urine
nm  It can detect renal damage that is not yet
Uric acid + O2 -uricase  allantoin + CO2 + H2O severe enough to cause elevated plasma
AMMONIA urea and crea nine levels.
 By product of amino acid deamina on  Monitoring the concentra on of chloride
 Converted to urea by the liver for final and sodium in urine reveals the ability of
excre on by the kidney the kidney to concentrate the ultrafiltrate
 Clinical applica ons: in tubules
o Assess and diagnose hepa c failure  3 most prevalent solutes excreted: urea,
and hepa c coma (ammonia increases chloride and sodium
CNS pH and inhibits GABA  Specimen: first morning urine
neurotransmi er) 1. Specific Gravity (SG)
o Diagnose Reye’s syndrome  Is the simplest test of renal concentra ng
o Inherited deficiencies of urea cycle ability.
 RV: 19-60 ug/dL (11-35 mmol/L)  It compares the weight of a fluid with that
 Increased levels: cirrhosis, hepa s, Reye’s of dis lled water at a reference
Syndrome, chronic renal disease, temperature.
acetaminophen poisoning  Measurement is affected by solute number
LABORATORY METHODS: and mass (high molecular weight).
 Specimen:  Increases in large urinary solutes such as
- Chilled heparinized or EDTA arterial plasma glucose; urea and protein increase the
- Sample is centrifuged at 0-4 °C within 20 “true specific gravity.”
minutes of collec on and the plasma  “fixa ve” of SG at 1.010 indicates severe
removed loss of concentra ng ability of the kidneys.
- Avoid cigare e smoking for several hours  Specific gravity of 1.010 is equal to SG of
prior to test (false increase) ultrafiltrate in Bowman’s space (same as
 Methods: protein-free plasma)
1. Chemical method  High molecular weight substances: X-ray
- ISE – Poten ometry with gas sensing dye and mannitol yield high SG (> 1.050).
electrode  Reference value: 1.005 – 1.030
- Spectrophotometry: ammonia + 2. Osmolality
bromphenol blue  blue dye  Is an expression of concentra on in terms
- Berthelot reac on = (+) blue of the total number of solute par cles
- Nessleriza on reac on = using diode; (+) present/kg of solvent (moles/kg solvent).
yellow  It is affected only by the number of solutes
2. Enzyma c method – GLD method present, thus more accurate than specific
- Decrease in absorbance of NADPH at gravity in assessing renal tubular func on
340nm (concentra on ability).
TESTS MEASURING TUBULAR FUNCTION  Urine osmolality is due primarily to urea;
A. Excre on Tests serum osmolality is primarily due to
1. Para-Amino Hippurate Test (Diodrast Test) sodium and chloride.
- It measures renal plasma flow.  Measurements of serum osmolality is
- This method requires clearance of the dye. useful for assessing water deficit or excess.
- Reference value: 600-700 mL/minute  Values are not affected by high molecular
2. Phenolsulfonthalein Dye Test weight substances as opposed to specific
- It measures excre on of dye propor onal gravity
to renal tubular mass.  Proteins and lipids do not contribute to
- Dose: 6 mg of PSP is administered IV osmolality.
- Reference value: 1200 mL blood  Normal ra o of urine osmolality to serum
flow/minute osmolality is 1:1
-
 1. Direct Method: LESSON 9 – LIVER FUNCTION TEST
Freezing point osmometry (popular method) LIVER ANATOMY AND PATHOPHYSIOLOGY
Vapor pressure osmometry (seebeck effect)  Largest/complex organ responsible for many
 An increase in osmolality (solute) major metabolic func ons in the body; chief
decreases the freezing point and metabolic organ in the body
vapor pressure.  Reddish brown and located under the
2. Indirect Method: Formula for Compu ng diaphragm in the right upper quadrant of the
Serum Osmolality abdomen
 To use glucose or urea in osmolality,  It receives 15 mL of blood per minute
calcula ons must be converted from  It is composed of 2 types of cells, hepatocytes
milligram units to molar units. and Kupffer cells (phagocy c).
Serum osmolality = 1.86 x Na+ +
( / )
+
( / )  The liver has a unique capacity to regenerate
.
Interpreta on of Results: by cell division, and hypertrophy of the
 Concentrated urine: 1.025 SG and >800 remaining ssue in case of ssues injury due to
mOsm/kg biliary obstruc on or toxic exposure.
 Loss of renal concentra ng ability: 1.2:1;  Severe loss of hepa c func ons may result to
diabetes insipidus = < 1:1 diagnos c changes in synthe c capaci es and
 If the ra o of urine : serum osmolality is > 1:1, in the func ons of excre on, detoxifica on and
it is glomerular disease and presence of metabolic ac vity that are reflected in mul ple
increase solute in the urinary filtrate. standard and specialized tests.
Interpreta on of Results:  To abolish liver ssue func on, more than 80%
 If the serum osmolality is > 2.1-2.3x the value of the liver must be destroyed.
of serum sodium, it may be due to  Has abundant blood supply receiving
hyperglycemia uremia and anion gap acidosis. approximately 1500mL/min from two major
 In DM, 5000 mOsm/day of solutes requires vessels:
significant amount of water for elimina on 1. Hepa c Artery
(polyuria), thus requiring excessive water - Branch of aorta that provides most of the
replacement (polydipsia). oxygen requirement: contributes 20% of
 An increase in plasma osmolality causes a blood supply
decrease in urine flow due to the reabsorp on 2. Portal Vein
of more water by vasopressin from the - Drains the gastrointes nal tract and
glomerular filtrate as it passes through the spleen, this transports most of the recently
tubules, from the glomerular filtrate as it absorbed materials from the intes nes to
passes through the tubules, and as result the liver
lowers plasma osmolality. - Drains from the general circula on
 With renal loss of water, the urine osmolality is  The metabolic ac vity of the liver takes place
decreased or normal. within the parenchymal cells, which cons tute
Notes to Remember: 80% of the organ mass; the liver also contains
 Urea is the only ineffec ve osmol that has Kupffer (re culoendothelial) cells and stellate
substan al concentra on in the normal cells (the major cell type responsible for
plasma, 5 mOsm/L. fibrosis)
 Hyperphosphatemia and hypocalcemia, in the LIVER LOBULE – are the basic func onal units of the
face of elevated BUN and crea nine, indica ve liver. They are classically described (‘classic lobules’) as
of renal disease, strongly suggest tubular hexagonal structures made of six ver cally aligned
failure. portal canals with a central vein; six-sided func onal
 Osmolal gap is the difference between unit
measured and calculated plasma osmolality.
 An osmolal gap > 12 mOsm/kg is significant as Component of liver lobule:
seen in DKA, drug ovedose and renal failure. 1. Branches of the hepa c portal vein and hepa c
 Osmolal gap is also a sensi ve indicator of artery
alcohol or drug overdose, causing a large 2. Central vein
“ostno gap” in ethanol intoxica on. 3. Sinusoids
4. Hepatocytes and Kupffer cells
5. Bile canaliculi
6. Bile ducts
 2. Bile pigments – heme waste products (bilirubin
LIVER PHYSIOLOGY and porphyrin products)
 The liver performs several hundred known V. SYNTHESIS
func ons each day, including metabolic, 1. CARBOHYDRATES – glucose is converted to
secretory and excretory func ons glycogen that is stored in the liver and later
 Total loss of liver func ons leads to death due reconverted to glucose as necessary
to HYPOGLYCEMIA within 24 hours 2. FAT – endogenous triglycerides, phospholipids,
SUMMARY OF LIVER FUNCTIONS and cholesterol
I. SYNTHETIC FUNCTION 3. LIPOPROTEIN – high and low density
 Liver secretes plasma proteins, carbohydrates, lipoproteins
lipids, lipoproteins, clo ng factors, ketone 4. KETONE BODIES – by product of
bodies and enzymes. gluconeogenesis
 The normal liver produces about 12 grams of 5. PROTEINS – albumin (12g/day) and majority of
albumin daily. alpha and beta globulins. All blood clo ng
 It is also involved in the metabolism of factors except FVIII VWF
cholesterol into bile acids. 6. HORMONES - somatomedin (mediates the
II. CONJUGATION FUNCTION ac vity of GH), angiotensinogen, and
 Involves in bilirubin metabolism. thrombopoie n.
 200 to 300mg of bilirubin is produced daily in VI. STORAGE FUNCTION
the healthy adult.  Storage site for all fat-soluble and water
III. DETOXIFICATION AND DRUG METABOLISM soluble vitamins
 Liver serves to protect the body from  Liver is also the storage depot for glycogen,
poten ally injurious substances absorbed which are released when glucose is
from the intes nal tract and toxic by-products depleted.
of metabolism.
 Ammonia (toxic by-product) is converted to BILIRUBIN
urea in the liver.  is the end product of hemoglobin metabolism
 Detoxifica on is responsible for the and principle pigment in bile. It is formed from
produc on of urea from ammonia, uric acid, destruc on of heme-containing proteins such
and other molecules that are less toxic than as myoglobin, catalase and cytochrome
their parent compounds. oxidase.
IV. EXCRETORY AND SECRETORY FUNCTION  B1 forms a complex with albumin for transport
 Metabolism (conjuga on and excre on of to the liver.
bilirubin)  Bilirubin (B1) is taken up by the hepatocytes by
 Excre on of bile – bile acids or salts, pigments, ligandin
cholesterol.  Conjugated in the endoplasmic re culum with
 Bile acids (primary bile acids: cholic acid and 2 molecules of glucuronic acid to form bilirubin
chenodeoxycholic acid) are conjugated with diglucuronide (conjugated bilirubin or B2)
the amino acids glycine and taurine to form  The reac on is catalyzed by uridine
bile salts. diphosphate (UDP) glucuronyltransferase.
 Synthesis of bile salts and bile acids  Conjugated bilirubin is excreted into the bile for
(assignment) storage in the gallbladder, secreted into the
1. Bile Acids duodenum in response to gallbladder
BILE ACID FORMATION: s mula on, and reduced by anaerobic bacteria
Cholesterol  liver  primary bile acids (cholic and in the intes ne to urobilinogen.
chenodeoxycholic acid) conjugated with glycine and  Some intes nal urobilinogen is reabsorbed
taurine (for water solubility)  stored in gall bladder as  A por on returns to the liver (reconjugated)
bile salts  goes to the small intes ne and increases and some enters the circula on for excre on in
absorp on of dietary fats  intes nal bacteria  the urine
forma on of secondary bile acids (deoxycholic acid and  The remaining por on in the intes nes is
litholic acid); secondary bile acids are produced in the oxidized by anerobic bacteria for excre on in
colon by bacterial ac on; and reabsorbed in the liver the stool as urobilin/stercobilin (an orange-
brown pigment that gives stool its
Decreased levels: loss of func oning hepatocytes – characteris c color)
cirrhosis and hepa s  The remaining por on in the intes nes is
Increased levels: regurgita on from hepatocytes – oxidized by anaerobic bacteria for excre on in
biliary obstruc on and hepatocellular disease the stool as urobilin/stercobilin (an orange-
 brown pigment that gives stool its Very sensi ve and
characteris c color) specific for
UNCONJUGATED CONJUGATED hepatobiliary
BILIRUBIN (B1) BILIRUBIN (B2) disease
Structure Bilirubin Bilirubin
monoglucuronide,  5’ nucleo dase
bilirubin (5 NT)
diglucuronide, & Liver  Serum  Heme
delta bilirubin excretory bilirubin product from
Bound to Yes (albumin) No (except delta catalysis and
protein bilirubin) conjuga on
Type of Nonpolar Polar with
compound glucuronic
Soluble in No Yes func on acid
water?  Three
Present in No Yes frac ons:
urine? conjugated,
Reac on Indirect (only Direct (reacts w/o unconjugated
with reacts in accelerator) , and delta
diazo zed presence of bilirubin
sulfanilic accelerator) (albumin-
acid bound)
Affinity for High Low  Causes
brain ssue jaundice
when
LABORATORY ASSAYS TO ASSESS LIVER DISEASE concentra on
Category Laboratory Assay Side Note s exceed 1.5
Hepatocellul  Aspartate Found in mg/dL
ar damage aminotransfer numerous ssues
ase (AST) including liver, Performed
cardiac muscle, qualita vely using
skeletal muscle,  Urine bilirubin a urine dips ck
kidneys, brains,
and pancreas  Liver converts
ammonia to
Found primarily in urea
liver; considered  Blood  Significant
 Alanine best laboratory ammonia liver
aminotransfer test for liver injury dysfunc on
ase (ALT) results in
Cholestasis  Alkaline Usually increased elevated
phosphatase during periods of serum
(ALP) growth (e.g., ammonia
children,  Poor
teenagers) and correla on
during pregnancy between
ammonia
Very sensi ve to level and
small liver insults degree of liver
 Gammaglutam (e.g., alcohol disease
yl transferase consump on) Assays of  Total protein, Altered ra o of
(GGT) May be elevated in biosynthe c albumin, and albumin to
hepatocellular Func on globulins globulin in liver
disease disease

 Coagula on  All factors

factors produced in
 liver factors IL, scarring and destruc on of the normal liver
VII, IX, and X architecture
are vitamin K  Primary biliary cirrhosis – progressive
sensi ve autoimmune disease characterized by
(meaning they destruc on of intrahepa c bile ducts,
require presence of an mitochondrial
adequate an bodies in the plasma
quan es of 3. TUMOURS – both primary but, more
vitamin K for frequently, secondary; for example, metastases
produc on) from cancers of the large bowel, stomach and
 Prothrombin bronchus.
me (PT) is a
collec ve STAGES OF ALCOHOL LIVER DISEASE
measure of  Steatosis (1st stage) / Alc. Fa y liver
factors II, V, Fa y deposits in the liver due to the effect of ethanol
VII, and X in lipid metabolism thus decreasing fa y acid
 Elevated PT oxida on and increasing the forma on or deposits of
unresponsive triglycerides in liver
to vitamin K  Steatonecrosis (2nd stage)
supplementa Further fat accumula on takes place with
on suggests inflamma on and development of fibrosis or necrosis
poor liver  Alc. Cirrhosis
func onality Extensive development of fibrosis takes place, further
inflamma on and hepatocellular carcinoma.
CHANGES IN SERUM PARAMETERS IN LIVER DISEASE
Bilirubin Increased Hemolysis, Delta Bilirubin
hereditary  It is conjugated bilirubin ghtly bound to
Erythrocyte albumin.
Enzymopathies  It has a longer half-life than other forms of
ALP Increased Bone cancer, bilirubin.
pregnancy  It is formed due to prolonged eleva on of
ALT Increased Muscle conjugated bilirubin in biliary obstruc on.
damage,  It helps in monitoring the decline of serum
cancer, bilirubin following surgical removal of gallstones.
pancrea s,  It reacts with diazo reagent in the direct bilirubin
renal disease assay.
AST Increased Myocardial  Is computed by using this formula: TB – DB + B =
infarc on, Delta bilirubin. It is not calculated on neonatal
skeletal muscle pa ents (≤14 days).
damage  Reference value: < 0.2 mg/dL (< 3umol/L)
Lactate Increased Myocardial
dehydrogenase infarc on, JAUNDICE OR ICTERUS
skeletal muscle  Yellowish pigmenta on of the skin, mucous
damage, renal membrane and sclera of the eyes due to
disease, cancer hyperbilirubinemia
cholinesterase DECREASED Pes cide  Not apparent un l the bilirubin exceeds 1.5-2
poisoning mg/dL

LIVER DISORDERS CLASSES OF JAUNDICE

The most common disease processes affec ng the liver 1. PRE-HEPATIC JAUNDICE
are:  Also known as HEMOLYTIC or
1. HEPATITIS – which may be acute or chronic or a RETENTION JAUNDICE
combina on of both, in which there is damage  CAUSES:
to and destruc on of liver cells 1) Excessive or too much destruc on
2. CIRRHOSIS is a process in which death of liver of RBC, elevated indirect Bilirubin
cells with regenera on leads to fibrosis,
 Free bilirubin is elevated due to circula ng inhibitor of
HDN or Erythroblastosis fetalis and bilirubin conjuga on
malaria  IMPAIRMENT OF HEPATIC EXCRETION
2) HEPATIC UPTAKE DECREASED: (ACQUIRED DISORDERS)
prolonged fas ng and intake of a. Dubin-Johnson Syndrome (EXCRETION
drugs DEFICIT) – presence of delta bilirubin,
2. HEPATIC JAUNDICE OR HEPATOCELLULAR / dark stained granules (lipofuscin) in liver
INDECTIOUS JAUNDICE biopsy
 CAUSES: b. Rotor Syndrome – reduced/defec ve
 Severe damage to the hepatocytes ligandin (UPTAKE/TRANSPORT DEFICT)
due to microorganisms (viruses, possibly viral in origin
parasites) or alcohol 3. POST-HEPATIC JAUNDICE
 Starva on and certain medica ons  Also known as OBSTRUCTIVE or
 Hepa s and cirrhosis CHOLESTIC JAUNDICE
 Parasi sm (Fasciola hepa ca)  Failure of bile to flow to the
 BILIRUBIN CONJUGATION DECREASED: intes ne/impaired bilirubin excre on
a. Physiologic Neonatal Jaundice  Bilirubin Assay: Elevated Direct
b. Gilbert’s disease – UDPGT (uridine Bilirubin
diphosphate  CAUSES:
glucuronyltransferase) is only 30% - obstruc on of the biliary flow
func onal, and a defect in the - intra-hepa c cholestasis
transport protein ligandin causes a - obstruc on of the extra-
TRANSPORT DEFICIT from the hepa c biliary tree
sinusoidal membrane of - cholelithiasis or gallstones
hepatocytes - strictures, spasms, atresia and
- Is characterized by microorganisms
impaired cellular uptake of - Ascaris lumbricoides infec on
bilirubin COMPARISON OF THE CAUSES OF JAUNDICE
- Is diagnosed in young NORMAL PRE- HEPATIC POST-
adults (20-30 yrs old); VALUE HEPATIC HEPATIC
affected individuals may Total 0.2-1.0 Increased Increased Increased
have no symptoms but bilirubin mg/dL
may have mild icterus Indirect 0.2-0.8 Increased Increased Normal
c. Crigler-Najjar syndromes I and II (free) mg/dL
bilirubin
(UDPGT deficiency –
Direct 0-0.2 Normal Increased/ Increased
CONJUGATION DEFICIT)
Bilirubin mg/dL normal
- Infants are treated by
Urine 0.1-1 Nega ve Variable Posi ve
means of phototherapy Bilirubin Ehrlich’s
 Type I Crigler-Najjar Syndrome unit
- Deficiency of the enzyme Urine Increased Normal Normal
glucoronyl transferase urobilinogen
(UDPGT) Stool Brown Dark Pale/Brown Clay
- Total absence of B2 brown
produc on Bilirubinuria reflects an increase in the plasma
- (+) kernicterus; bile is concentra on of conjugated bilirubin, and is always
colorless pathological.
 Type II Crigler-Najjar Syndrome
- It is characterized by par al NEONATAL JAUNDICE
deficiency of UDPGT  Buildup of unconjugated bilirubin
- Small amount of B2 is  Noted 2-3 days of neonatal life, rarely rises
produced greater than 5 mg/d/day (peaks at 4-5 days)
d. Lucey-Driscoll Syndrome –  Most common cause is HDN
circula ng inhibitor of conjuga on  Poorly developed blood brain barrier allowing
- Is a familial form of B1 to cause damage to central nervous system
unconjugated – kernicterus (B1 20 mg/dL)
hyperbilirubinemia caused by  Treatment: PHOTOTHERAPY
SUMMARY OF LIVER DISORDERS
DISEASE ANALYTE COMMENTS ALP, GGT, Total Indicates
CONCENTRATION bilirubin: mild cholestasis
Acute Viral AST/ALT: 10-110 x Rise early eleva on
Hepa s A-E, ULN prior to Albumin, Due to loss of
Cytomegalovirus bilirubin cholesterol: synthe c
(CMV), Epstein- Total bilirubin (B1 Elevated at 2- decreased ability
Barr Virus (EBV) and B2): 5-20 8 weeks post PT: prolonged Due to loss of
mg/dL (icteric infec on synthe c
phase) ability
ALP: 2 x ULN 5 x ULN if Primary biliary Total bilirubin and Due to
intrahepa c Cirrhosis B1: elevated progressive
cholestasis is destruc on
present of
GGT: 5 x ULN 10 x ULN if intrahepa c
intrahepa c bile ducts
cholestasis is ALP: 2-10 x ULN
present AST/ALT:
Chronic AST/ALT: slight Usually moderately
Hepa s persistent eleva on associated Hepa c Tumors LD: 2-10 x ULN Liver func on
with GGT: up to 20 x ULN altered when
persistent ALP: elevated ssue is
hepa s B compressed
infec on by tumor
Total bilirubin: mass
slight persistent Ra o of
eleva on metasta c
Total protein: tumors to
Decreased primary liver
Alcoholic Liver GGT: 2-3 x ULN Returns tumors is
Disease associated 20:1
with Presence of
abs nence elevated AFP
unless liver is used a tumor
damaged marker
AST/ALT: mild AST may be Cholestasis GGT and ALP Maybe
eleva on more markedly elevated intrahepa c
elevated if ALT/AST: slightly extrahepa c
concurrent elevated
alcoholic Bilirubin: elevated
myopathy
Albumin: Due to REYE’S SYNDROME
decreased decreased  Hepa c destruc on following recovery from
nutri onal viral infec on and aspirin inges on in children,
status wherein the pa ent develops neurologic
Globulins: elevated Due to abnormali es due to accumula on of
decreased ammonia in the CNS.
nutri onal  Non-inflammatory hepa c encephalopathy
status and fa y liver degenera on
Lipids: elevated Due to The degenera on of the liver is characterized by:
decreased  Mild hyperbilirubinemia
nutri onal  Threefold increases in ammonia, ALT, and AST
status  Without treatment, rapid clinical deteriora on
Cirrhosis AST/ALT, LD: slight Due to liver leading to death may occur
eleva on cell injury BILIRUBIN DETERMINATION/ASSAY
Specimen: fas ng SERUM
  Protect from light (light decreases bilirubin by Bromsulfonthalein (BSP) Dye Excre on Test
30-50% per hour)  It is a test for hepatocellular func on and
 Avoid hemolysis = falsely decreased potency of bile duct; rarely used.
 Lipemia = falsely increased  Dose (BSP Dye) Administra on Methods:
1. Rosenthal White (Double Collec on Method)
COLORIMETRIC METHOD Dose = 2mg/kg body weight (BW) of the
Principle: Van den Berg reac on (is a diazo za on of pa ent
bilirubin to produce azobilirubin) Specimen collec on = a er 5 minutes and a er
a. Evelyn and Malloy Method 30 minutes
 Coupling Accelerator (speed): Methanol Normal value:
 Diazo Reagents: 0.1% Sulfanilic Acid + HCI; A er 5 minutes = 50% dye reten on
0.5% Sodium Nitrite A er 30 minutes = 0% dye reten on
 Diazo Blank: 1.5% HCI
 Final reac on: pink to purple azobilirubin 2. Mac Donald Method (Single Collec on
b. Jendrassik and Groff Method)
 It is the most commonly used method. Dose = 5mg/kg body weight (BW) of the
 Is a popular technique for the discreet pa ent
analyzers. Specimen collec on = a er 45 minutes
 Is more sensi ve than Evelyn and Malloy Normal value:
method. A er 45 minutes = +/- 5% dye reten on
 Is not affected by hemoglobin up to 750
mg/dL and pH changes. Urobilinogen
 Coupling Accelerator: Caffeine Sodium  Is a colorless end product of bilirubin
Benzoate metabolism that is oxidized by intes nal
 Buffer: Sodium Acetate bacteria to the brown pigment urobilin.
 Ascorbic acid – terminates the ini al  It is either excreted in urine and feces, or
reac on and destroys the excess diazo reabsorbed into the portal blood and returned
reagent to the liver.
 Final reac on: pink to blue azobilirubin  Absence of this substance in urine or stool
denotes complete biliary obstruc on.
Notes to remember:  In the collec on of the sample, avoid exposure
 Conjugated bilirubin (B2) produced a color in to direct light.
aqueous solu on. It reacts directly with Diazo  Specimen: 2-hour freshly collected urine or
reagent freshly collected stool
 Unconjugated bilirubin (B1) is the frac on that  Method: Ehrlich’s method (p-dimethyl
produced a color only a er the addi on of aminobenzaldehyde reagent)
alcohol reacts with Diazo reagent with an  Reference value:
accelerator Urine = 0.1-1.0 Ehrlich units/2 hour or 0.54
 Delta bilirubin is the conjugated bilirubin Ehrlichs units/day
bound to albumin and behaves like B2; Stool = 75-275 Ehrlich units/100g of feces
elevated in obstruc ve jaundice. TEST FOR DETOXIFICATION FUNCTION
- Involves enzyme and ammonia tests.
Increased B1: A. Enzyme Tests
1. Gilbert’s Syndrome  Is used to assess the extent of liver damage
2. Crigler-Najjar Syndrome and to differen ate hepatocellular
3. Hemoly c Anemia (func onal) from obstruc ve (mechanical)
4. Hepatocellular dx disease.
5. Lucey Driscoll Syndrome  Any injury to the liver that results in
6. G6PD deficiency cytolysis necrosis causes the libera on of
various enzymes.
Decreased B2:  Enzyme tests are o en the only indica on
1. Biliary obstruc on (gall stones) of cell injury in early or localized liver
2. Pancrea c (head) cancer disease.
3. Dubin Johnson Syndrome  Enzymes secreted by the liver: ALP,
4. Alcoholic and viral hepa s aminotransferases, 5’ nucleo dase, GGT,
5. Biliary atresia OCT (Ornithine Transcarbamylase), LAP
6. Hepatocellular dx and LD
 B. Ammonia 2) GAMMA GLOBULINS
 It arises from deamina on of amino acids, a) Autoimmune hepa s: marked increased of
which occurs mainly through the ac on of polyclonal IgG
diges ve and bacterial enzymes (bacterial b) Primary biliary cirrhosis: marked increase of
proteases, ureases and amine oxidases) on polyclonal IgM
proteins in the intes nal tract. c) A/G ra o in liver disease is <1.0
 Is also released from metabolic reac ons 3) ALBUMIN
thar occur in skeletal muscles during  Concentra on of this protein is inversely
exercise. propor onal to the severity of the liver
 The liver normally removes most of this disease
NPN via the portal vein circula on and  Plasma levels decline when severe
converts it to urea, then eliminated by the hepatocellular disease lasts more than 3
kidneys (urine). weeks.
 Reference value: 19-60 ug/dL (11-35  In hepa c circulatory disorder, albumin is
mmol/L) used because its concentra on reflects the
shi of protein and fluid into ascites and,
Diagnos c Significance: its important contribu on to the plasma
 For the diagnosis of hepa c failure (hepa c onco c pressure.
coma) and Reye’s syndrome  Decreased serum albumin concentra on
 In severe liver disorder, it accumulates and may be due to decrease synthesis
reaches the systemic circula on, which is then  Low total protein + low albumin = hepa c
converted to glutamine in the brain, thus cirrhosis and nephro c syndrome
compromising the Kreb’s cycle leading to coma  Dyes used for measurement:
due to lack of ATP for the brain – ammonia 1. Bromcresol green (BCG) – most
increases CNS pH. commonly used
 Elevated plasma levels of ammonia are 2. Methyl orange (MO)
neurotoxic and are o en associated with 3. Hydroxyazobenzene benzoic acid
encephalopathy – an important mechanism by (HABA)
which ammonia can cause toxicity to the CNS is 4. Bromcresol purple (BCP) – most
its ability to lower the concentra on of y- specific dye
aminobutyric acid (GABA), a cri cally  Albumin can be measured by
important neurotransmi er in the CNS, by direct methods based on its dye-
reac ng with glutamic acid to form glutamine binding property.
via reversal of the glutaminase-catalyzed  BCG and BCP are ca onic dyes, and
reac on. free from interference from
 Increase levels: cirrhosis, hepa s, Reye’s bilirubin
syndrome, chronic renal disease and  BCG and BCP are not significantly
acetaminophen poisoning affected by used of hemolyzed
OTHER TESTS FOR HEPATIC FUNCTION samples. BCG is used extensively in
1) PT (Prothrombin me) – VITAMIN K RESPONSE automa c analyzers for
TEST determining serum albumin in
 Sensi ve marker for impaired hepa c parallel with Biuret reagent for
synthe c func on total protein.
 It differen ates intrahepa c disorder  The presence of penicillin may
(prolonged pro me) from extrahepa c cause falsely low result of serum
obstruc ve liver disease (normal pro me). albumin (BCG method).
 Prolonged pro me despite vitamin K Hyperalbuminemia:
administra on indicates loss of hepa c 1. Severe dehydra on
capacity synthesize the proteins. 2. Prolonged tourniquet applica on – ar factual
 In acute viral or toxic hepa s, prolonged hyperalbuminemia
pro me signifies massive cellular damage. Hypoalbuminemia:
 Vitamin K is administered intramuscularly, 1. Reduced synthesis
10 mg daily for 1 to 3 days. a. Chronic liver disease
 To differen ate prolonged PT if due to b. Malabsorp on syndrome
cholestasis or impaired vitamin K c. Malnutri on and muscle was ng disease
absorp on – administer parenteral vit. K 2. Increased loss
 a. Nephro c Syndrome (20-30g/day) – CONVERSION OF TRADITIONAL UNITS TO SI UNITS FOR
albumin excre on is increased when the COMMON CHEMISTRY ANALYTES
glomerulus no longer func ons to restrict
the passage of proteins from the blood
b. Massive burns
c. Proteins-losing enteropathy
d. Orthosta c albuminuria
3. Increased Catabolism
a. Massive burns
b. Widespread malignancy
c. Thyrotoxicosis
Notes to remember:
 Analbuminemia – is hereditary absence of
albumin; inability to synthesize albumin.
 Bisalbuminemia – is the presence of two
albumin bands instead of a single band in
electrophoresis.
- It is associated with excess
amount of therapeu c drugs
in serum.
- It is the presence of albumin
with unusual molecular
characteris cs in the blood.
Albumin/Globulin Ra o
 Albumin has a higher concentra on than
globulin.
 It is determined to validate if globulin is higher
than albumin.
 If globulin is greater than albumin, it is known
as inverted A/G ra o seen in cirrhosis, mul ple
myeloma and Waldenström’s
macroglobulinemia
 Serum and even urine protein electrophoresis
may help define the clinical situa ons.