MEDT 13 CLINICAL CHEMISTRY I 1

UNIT 1: SCOPE OF CLINICAL CHEMISTRY

INTRODUCTION  For instance, an increase in concentration of urea
 Clinical chemistry is a science, a service, and an nitrogen in the blood may indicate kidney failure and
industry. As a science, clinical chemistry links the increased concentration of blood cholesterol may
knowledge of general chemistry, organic chemistry, indicate increased risk of cardiac disorders.
and biochemistry with an understanding of human  The clinical chemistry laboratory provides accurate,
physiology precise measurements of selected biochemical
 As a service, the clinical chemistry laboratory markers, accompanied by reference, or comparison,
produces objective evidence from which medical ranges of the concentration of these biochemical
decisions may be made. As an industry, clinical markers in healthy individuals.
chemistry laboratories are businesses, which operate  Biochemical marker analysis is one factor in the
under the regulations and practices governed by the assessment of the patient.
state.  Physicians also gather history and symptoms of the
complaint; examination findings, such as blood
CLINICAL CHEMISTRY pressure; and testing by other health-care team
 Also known as chemical pathology, clinical members in ancillary fields, such as radiology and
biochemistry, or medical biochemistry respiratory therapy.
 The systematic study of biochemical processes  The physician uses all data to assess the patient and
associated with health & disease and the implement a plan for treatment.
measurement of constituents in body fluids or tissues  Organic biochemical markers are used for assessment
to facilitate timely, relevant, accurate and precise and diagnosis of disease: carbohydrates, lipids,
diagnosis of disease proteins, and nucleic acids and the derivatives of
 Because chemistry is the foundation of the science these markers.
that underlies biochemistry and pathophysiology, it is  The assessment of inorganic chemicals, such as ions,
appropriate to use the term clinical chemistry minerals, and dissolved gases, provide a measure of
homeostasis in the body. In addition to the
AREAS OF INTEREST OF CLINICAL CHEMISTRY measurement of these endogenous substances, the
Analytical Chemistry clinical chemistry laboratory provides measurement
of chemicals that are exogenous to the body, both
Biochemistry beneficial chemicals, such as therapeutic drugs, and
CLINICAL CHEMISTRY

harmful substances, such as poisons.

Computer

Instrumentation  Biochemical Marker – any biochemical compound,

such as an antigen, antibody, abnormal enzyme, or
Immunology hormone, that is sufficiently altered in a disease to
serve as an aid in diagnosing or in predicting
Toxicology susceptibility to the disease
Pharmacology
 Homeostasis – the state of dynamic equilibrium of
the internal environment of the body that is
maintained by processes of feedback and regulation
THE BIOCHEMISTRY OF DISEASE in response to external or internal changes
 The clinical chemistry laboratory measures change in  Endogenous – originating inside the body
biochemical compounds as an indicator of health  Exogenous – originating outside an organ or part of
status or disease processes. the body
 Because the biochemical changes of compounds are
not uniform in tissue and organs in response to Carbohydrates
disease, the measurement of selected biochemical  Carbohydrates are chemical substances that contain
markers can be used to monitor diseases processes only carbon, hydrogen, and oxygen.
as they occur in specific living cell systems.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 2
 Often carbohydrate molecules consist of one H2O  Although the concentration of cholesterol in blood is
molecule per carbon, hence the nomenclature dependent on many factors such as genetics, age, sex,
“carbohydrate,” or hydrate of carbon. diet, and physical activity, total cholesterol
 The simplest carbohydrates are monosaccharides, measurement is used clinically to monitor disease.
which usually contain 3 to 6 carbons. Monosaccharides  In addition to its role as a risk factor for coronary
are the units that make up more complex artery disease, increased cholesterol concentration
carbohydrates. may be the result of hypothyroidism, liver disease,
 Glucose, fructose, ribose, and galactose are common renal disease, or diabetes.
monosaccharides of living organisms.  Decreased cholesterol concentration may be the result
of hyperthyroidism, digestive malabsorption, or
CARBOHYDRATES AS BIOCHEMICAL MARKERS OF DISEASE impaired liver function.
 The most common carbohydrate disorder in humans is  Factors that increase HDL-C include increased
diabetes mellitus. estrogen in women, increased exercise, and the
 This disease is caused by an inability to produce or to effects of certain blood pressure medicines.
respond to the hormone insulin.  Factors that decrease HDL-C include increased
 Laboratory tests of body fluids of individuals with this progesterone, obesity, smoking, and diabetes.
disease show increased concentrations of glucose. Increased triacylglycerol may be the result of
 The laboratory tests for ketones, acids, and pancreatitis, diabetes mellitus, acute alcohol
glycosylated proteins provide measures of disease consumption, or certain liver diseases.
severity.  In addition, triacylglycerol may be increased
artifactually in non-fasting blood samples.
 Clinical Correlation: The most common
carbohydrate disorder in humans is diabetes mellitus Proteins and Amino Acids
 The human body requires 20 amino acids as the
Lipids building blocks of proteins.
 Lipids are, by definition, organic compounds that are  Humans make some of these amino acids but must
poorly soluble in solutions such as water and soluble gain the rest, as essential nutrients, through the diet.
in organic solutions such as ether.  Plants and bacteria produce the essential amino acids
 As a group of molecules that is defined functionally by and many others.
solubility, lipids consist of diverse chemical  The production of proteins from amino acid building
structures. Chemically, lipids are either compounds blocks is directed by a template that is produced by
that yield fatty acids when hydrolyzed or complex the DNA of the cell.
alcohols that can combine with fatty acids to form  Therefore, the amino acid sequence of protein is a
esters. reflection of the genetic information of the cell.
 Only a limited number of lipids are clinically  Most enzymes are proteins that are able to catalyze
important. This group includes fatty acids, reactions.
triacylglycerols (or triglycerides), cholesterol, and  Through regulation of reactions via enzymes, the
phospholipids. genetic information directs the diverse metabolic
reactions of the cell.
LIPIDS AS BIOCHEMICAL MARKERS OF DISEASE  In addition to their function as an energy source and
 Clinical chemistry laboratories offer many tests for in catalysis, amino acids and proteins are used as
lipid disorders. One of the most common tests is the transport molecules, structural components of cells
lipid profile. and tissues, hormones, clotting agents, and immune
 This panel of tests includes measures of agents.
triacylglycerol and cholesterol in the form of
lipoprotein-cholesterol molecules, low density TOTAL SERUM/PLASMA PROTEINS AND PLASMA ALBUMIN
lipoprotein cholesterol (LDL-C) and high-density AS BIOCHEMICAL MARKERS OF DISEASE
lipoprotein cholesterol (HDL-C).  Plasma proteins have functions in many organ and
 The results of testing for these lipids provide tissue systems. They are carrier molecules, receptor
measures of risk for coronary artery disease. chemicals, immune response agents, and enzymes or
catalytic proteins.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 3
 Total plasma protein is a measure of nutrition, the due to metabolic or genetic states or proliferation of
status of many organs and tissues that are involved neoplasms.
in protein metabolism, and the process of breakdown  In the latter case, increased enzyme activity can be
and excretion of protein metabolites. used as a tumor marker.
 The measurement of plasma protein fractions
provides more specific evidence for diagnosis and ENZYMES AS BIOCHEMICAL MARKERS OF DISEASE
assessment of disorders.  Damage to tissue can release different types of
 Because of its importance in maintaining osmotic enzymes based on their location.
pressure, the measure of albumin concentration is a  For example, mild inflammation of the liver reversibly
reflection of this pressure. increases the permeability of the cell membrane and
 As a transport protein, the measurement of albumin releases cytoplasmic enzymes such as lactate
monitors the ability of the body to transport such dehydrogenase (LD), alkaline phosphatase (ALP), and
diverse substances as bilirubin, fatty acids, and aspartate transaminase (AST), while necrosis will
calcium through the blood. release mitochondrial sources of alanine
 Measurement of the transport proteins of a specific transaminase (ALT) as well as AST.
substance provides information about the metabolism  Distribution of these enzymes within specific types of
of that substance. hepatic tissues varies.
 For example, the measurement of transferrin helps  ALP and gamma-glutamyltransferase (GGT) are more
the physician understand the metabolic processes concentrated in the biliary tree or canalicular tissues,
involving iron. while AST, ALT, and LD are found in parenchymal
 Most amino acids are broken down in the liver and hepatic cells.
then excreted through the kidney.
 Therefore, blood and urine tests that measure the  Clinical Correlation: Damage to tissue can release
concentration of these metabolites monitor both liver different types of enzymes based on their location.
and urine function. The measurement of increases in specific enzyme
 The urea nitrogen test is an indicator of renal activity may be used to determine the location of
disorders and may be used to determine the source of tissue damage.
the disorder: prerenal, renal or postrenal.
 Other breakdown products, such as ammonia, are DUTIES OF MEDICAL TECHNOLOGIST IN CLINICAL
measured as toxic compounds of metabolism. CHEMISTRY SECTION
1. Conduct chemical analysis of body fluids, including
Enzymes blood, urine, or spinal fluid, to measure or quantitate
 An enzyme is a protein catalyst that accelerates the biochemical compounds.
speed of a chemical reaction by binding specifically to 2. Operate, calibrate, or maintain equipment used in
a substrate, forming a complex. quantitative or qualitative analysis.
 This complex lowers the activation energy in the 3. Establish or monitor quality assurance programs or
reaction without the enzyme becoming consumed or activities to ensure the accuracy of laboratory results.
without changing the equilibrium of the reaction. 4. Verify records and reports laboratory results on all
 Enzyme analysis is used to aid in diagnosis and performed tests.
treatment of disease. In particular, enzymes 5. Supervise, train, or direct lab assistants, medical and
synthesized intracellularly carry out their functions clinical laboratory technicians or technologists, or
within cells, and are released into body fluids when other medical laboratory workers engaged in
those cells become diseased. laboratory testing.
 Thus, an increase in enzyme activity when compared 6. Develop, standardize, evaluate, or modify procedures,
to the reference range can indicate pathological techniques, or tests used in the analysis of specimens
changes in certain cells and tissues. or in medical laboratory experiments.
 Enzyme activity levels in body fluids can reflect 7. Demonstrate the ability to communicate test results
leakage from cells due to cellular injury, or changes in effectively with physicians, pathologists and nursing
enzyme production rate, or actual enzyme induction staff as a member of an interdisciplinary team
focused on providing exemplary quality of care.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 4

UNIT 2: LABORATORY MATHEMATICS

INTRODUCTION
 In the clinical laboratory, laboratory mathematics are Basic and Derived Properties of the SI System
frequently used by the laboratory specialist to
calculate the amount of a specific chemical needed to PROPERTY SYMBOL UNIT UNIT SYMBOL
prepare a reagent or to determine the concentration Basic Properties
of a chemical constituent in a clinical sample. Mass M, m Kilogram kg
Length L, l Meter m
 To enable the laboratory specialist to perform
Time T, t Second s
calculations with greater efficiency, the use of Current Ampere A
laboratory mathematics must be mastered. Temperature T Kelvin K
Luminous
I, J Candela Cd
UNIT OF MEASUREMENT Intensity
 Measurement is expressed as a numerical description Amount N, n Mole Mol
of properties in units, or units of measure. Derived Properties
Volume V cubic meter, liter m3 , L
 A unit of measure defines the dimension or magnitude
Area A square meter m2
of something. kilogram per
 In blood analysis, a red blood cell can be described in
Density Kg/L
liter
terms of its diameter, or a blood sample may be Rate unit per second unit/s
described in terms of the number of red blood cells
per unit volume. SI Prefixes Used with the Metric System
 There are seven basic dimensions: amount (number of
units), mass, length, time, temperature, electric
current, and luminosity (luminous intensity).
 A group of units measured together is a system of
measure.
 The commonly used systems of measure are the
metric system and the Systeme Internationale (SI).
 The SI system consists of seven basic properties and
derived properties.
 Basic properties, such as mass and length, are not
calculated from other properties; they are directly
measured.
 In contrast, the derived properties, such as density
and concentration, are multiples or ratios of basic
properties.

ROUNDING-OFF RULES
RULE EXAMPLE
In rounding off numbers, the last figure kept should be unchanged if the first figure If only one decimal place is to be kept, then 6.422
dropped is less than 5. becomes 6.4.
In rounding off numbers, the last figure kept should be increased by 1, if the first figure If only two decimal places are to be kept, then
dropped is greater than 5. 6.4872 becomes 6.49. Similarly, 6.997 becomes 7.00.
If only one decimal place is to be kept, then 6.6501
In rounding off numbers, if the first figure dropped is 5 and there are any figures
becomes 6.6
following the five that are not zero, then the last figure kept should be increased by 1.
If only two decimal places are to be kept then
(if no number or zero after 5, the last figure kept is unchanged)
7.4852007 becomes 7.49.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 5
significant digits to the same number as present in
the figure with the fewest significant digits.
Practice  Example:
Round off the following numbers to the number of decimal places 0.0211 × 25.63 × 1.058810.0211
indicated in the column on the right. has fewest significant figures
DECIMAL 0.0211 × 25.63 × 1.05881 = 0.5725970363 = 0.573
PROBLEM NUMBER ANSWER
PLACE
A 2.1988 1 2.2
B 5.7322 2 5.73 Practice
C 0.3552 2 0.36 1. 0.272 ÷ 30 = 9.066666667 x 10-3 = 0.009
D 4.09997 4 4.1000 2. 0.0013 x 2.71 = 3.523 x 10-3 = 0.0035
E 9.999517 3 10.000 3. 5.0637 + 10.823 + 1.29583 = 17.18253 = 17.183
F 6.8652 2 6.87
G 19.4745 3 19.474
H 2.7500 1 2.8
SCIENTIFIC NOTATION
 It is a way of writing numbers that accommodates

SIGNIFICANT FIGURES values too large or small to be conveniently written in

standard decimal notation.
 All numbers can be expressed as a number between 1
Determination of Significant Figures
1. All non-zero digit are significant
to 10 with an exponential term (base 10) multiplied by
2. Significance of Zero
the number to account for the position of the decimal
a. Trailing zero (any zero that follows the last nonzero
point.
Significand — 3.1 × 104 — Exponential term
digit) – significant only if there is a decimal point in significand = must be between 1 and 10
the number.
b. Leading zero (any zero that precedes the first
nonzero digit) – never significant Practice
c. Embedded zero (A zero that occurs anywhere 1. 4,300 = 4.3 x 103
2. 0.0211 = 2.11 x 10-2
between two nonzero digits) – lways significant 3. 4,500,000 = 4.5 x 106
4. 0.00000000007002 = 7.002 x 10-11
5. 4321.768 = 4.321768 x 103
Practice
1. 47 = 2 5. 340 = 2 9. 1.018 = 4
2. 45.6 = 3 6. 34.00 = 4 10. 47.8006 = 6
3. 31.999 = 5 7. 0.00083 = 2
PREPARATION AND STANDARDIZATION OF
4. 34.50 = 4 8. 404 = 3 SOLUTIONS AND DILUTIONS

Components of a solution
Calculations Using Significant Figures 1. Solute
 Addition and Subtraction  Dispersed phase on the dissolved substance (ex.
 When adding and subtracting figures with differing salt, sugar)
number of digits, adjust the number of significant  Not all solute can be dissolved by solvent (ex. sand)
digits appearing to the right of the decimal point so  If the solute is water-loving, it is soluble
that all figures have the same number of significant  If the solute is water-fearing, it is insoluble
digits appearing to the right of the decimal point.  Things that can help dissolve solute: mixing,
 Example: quantity of solvent, stirring, time
Addition 2. Solvent
0.0213 + 29.64 + 1.056931 = 30.718213 = 30.72  Dispersion medium or the substance in which the
Subtraction solute is dissolved (water, alcohol)
668.94 − 72 = 596.94 = 597
 Multiplication and Division Types of Solution
 When multiplying and dividing figures with
1. Dilute solution – contains a relatively small
differing significant digits, reduce the number of proportion of solute

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 6
2. Concentrated solution – contains a relatively Removal of undesirable substances (protein in PFF
2.
large proportion of solute (hand or dish soap, syrup) preparation).
3. Saturated solution 3. Preparation of working standards from the stock
 Solution in which the dissolved and the solutions.
undissolved portions of the solute are in  Usually expressed as a ratio, e.g., 1:10. This means
equilibrium with each other that 1 unit of the original solution was diluted to a
 There is only a fixed amount of solute that can be final volume of 10 units and will give a concentration
dissolved by the solvent of 1/10 of the original solution.
 Intermediate color; balanced  The total volume increases, the concentration
4. Supersaturated solution decreases, but the amount of solute remains
 Solution in which there is more solute in the same.
solution than is present in a saturated solution of  Expression of concentrations, not volume and can be
the same substance at the same temperature and written in many ways, such as:
pressure; darker color  1 to 5 dilution
 Unsaturated solution – less solute; lighter  1:5 dilution
color  1/5 dilution
5. Standard solution  1 part of solution A to 4 parts of solution B
 A solution whose precise concentration is  1 part of solution A to 4 parts of solution B
known  1 part in 5 parts dilution
 The process of determining of adjusting the
concentration of the standard solution is known as  Diluent – is a liquid added to a solution to make it
standardization. less concentrated
 Dilution – the act of making a dilute solution; the
Three Methods of Standardization degree to which a solution is made less concentrated
1. Direct preparation of the standard solution by  Proportion – an equation with a ratio on each side.
dissolving a weighed amount of a pure, dry chemical It is a statement that two ratios are equal. 2:6 = 1:3
and diluting the solution to an exactly known volume.
2. Titration of a solution of a weighed portion of pure,
dry chemical by the solution to be standardized. Practice
3. Titration against a primary standard such as Complete the example of 1:10 dilutions.
1. 100 µl of serum and 900 µl of saline
hydrochloric acid that has been made up from a 100 × 10 = 1000 − 100 = 900
constant boiling hydrochloric acid. The solution 2. 20 µl of NaOH and 180 µl of saline
20 × 10 = 200 − 20 = 180
standardized is known as secondary standard. 3. 1 ml of Bleach and 9 ml of water
1 × 10 = 10 − 1 = 9

Ratio 4. 2 ml of serum and 18 ml of saline

2 × 10 = 20 − 2 = 18
 Relationship in number or degree between two
quantities. It is expressed as the amount of one
substance:amount of second substance. Using Proportion to Know the Dilution
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑜𝑛𝑒 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
 A simple formula for calculating dilution is:
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑒𝑐𝑜𝑛𝑑 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
(𝐴)
= (𝐶)
(𝐴) + (𝐵)
 Example: 5 mL of serum is added with 20 mL of saline. where: A = part or volume of substance being diluted
What is the ratio? 1:4 (5:20) B = part or volume of diluents added, and
C = the dilution expressed as a fraction
Dilution (A and B must be in the same units of volume)
 Example
 Involves the preparation of a weaker solution from a
stronger one. This is done by adding a diluent such as 1. 5 ml of serum is added to a total volume of 25 ml
water which contains none of the solute, to a solution. with saline. What is the dilution? 1:5 dilution
 Reasons for Dilution:
wherein 5 is the dilution factor.
5 5
1. Concentration of the material in solution is too high = = 5: 25 = 1: 5
5 + 20 25
to be accurately measure.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 7
2. 3 ml of NaOH is added to 6 ml of saline. What is the 3 3
= = 3: 9 = 1
3+6 9
dilution? 3/3+6=3/9 or 3:9 or 1:3 wherein 3 is the
dilution factor.

PREPARING A SOLUTION USING PROPORTION

A buffer is made by adding 2 parts of “solution A” to 5 parts “solution B”. How much of solution A and solution B would be required to
Problem
make 70 ml of the buffer?
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 (𝐶) = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑜𝑛𝑒 𝑝𝑎𝑟𝑡
Formula "𝑃𝑎𝑟𝑡𝑠 𝑜𝑓 𝐴" + "𝑃𝑎𝑟𝑡𝑠 𝑜𝑓 𝐵"
70 𝑚𝑙 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑
= 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑜𝑛𝑒 𝑝𝑎𝑟𝑡
2 "𝑃𝑎𝑟𝑡𝑠 𝑜𝑓 𝐴" + 5 "𝑃𝑎𝑟𝑡𝑠 𝑜𝑓 𝐵"

70
Solution = 10 𝑚𝑙 (𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑜𝑛𝑒 𝑝𝑎𝑟𝑡)
7

2 𝑝𝑎𝑟𝑡𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 "A" = 2 × 10 = 20 𝑚𝑙

5 𝑝𝑎𝑟𝑡𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 "B" = 2 × 10 = 20 𝑚𝑙
Answer The buffer would be made by mixing 20 ml of solution A with 50 ml of solution B to give a total volume of 70 ml.

Calculating the Concentration of Diluted albumin concentration of the original sample is found
Solutions to be 40 g/L.
 The general rule for calculating the concentration 4 𝑔/𝐿 × 10 = 40 𝑔/𝐿
of a diluted solution is to divide the
concentration of the original solution by the Using a Concentrated Solution to Make a Dilute
dilution factor, which is the reciprocal of the Solution
dilution.  Sometimes it is necessary to prepare a dilute action

 For instance, if a 2.0 M solution is diluted 1:5, the from a concentrated solution. The general formula is:
𝐶1𝑉1 = 𝐶2𝑉2
dilution factor is 5, and the concentration of the where: C1 = concentration of the solution of a greater
dilution is 0.4 M. concentration
𝟐. 𝟎 𝑴 = 0.4 𝑀 V1 = volume required of the solution of greater
5 concentration
 This rule can also be used to find
the original C2 = concentration of final (dilute) solution
concentration of a diluted solution. V2 = volume of final (dilute) solution
 For example, the albumin concentration of a 1:10  Example: Using the formula 𝐶1𝑉1 = 𝐶2𝑉2 to prepare a
serum dilution was measured 4g/L. By multiplying solution
the concentration by the dilution factor, the

Problem Prepare 100 mL of 0.1 M HCl using 1.0 HCl

Formula 𝐶1𝑉1 = 𝐶2𝑉2
Solution (1.0 𝑀)(𝑉1) = (100 𝑚𝐿)(0.1 𝑀)
100 𝑚𝑙 × 0.1 𝑀
𝑉1 =
1.0 𝑀
𝑉1 = 10 𝑚𝑙
Answer 10 ml of 1.0 M HCl is added to 90 mL of H20 to make 100 ml of 0.1 M HCl solution.

Multiple or Serial Dilution

 This refers to multiple progressive dilutions ranging
from more concentrated solutions to less concentrated
ones.
 This is extremely useful when the volume of
concentrate or diluents is in short supply and needs to
be minimized or a number of dilutions are required,
such as determining a titer.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 8

𝐷𝑡𝑢𝑏𝑒 = 1 𝑚𝑙/(1 𝑚𝑙 + 19 𝑚; ) = 1/20 𝑜𝑟 1: 20

Practice
𝐷𝑠𝑎𝑚𝑝𝑙𝑒 = (1/20)3 = 1/8000 𝑜𝑟 1: 8000
1. Prepare a 10% (v/v) solution of ethyl alcohol in water.
Type equation here.

CONCENTRATION OF SOLUTIONS 2. If you measured 10 ml of ethanol and diluted to 50 ml of water,

what is the concentration of the solution in %v/v?
𝑣 10 𝑚𝑙
% = × 100 = 20%
Percent Solutions 𝑣 50 𝑚𝑙

 Refers to parts of solute per 100 total weigh units or

volume units of solution; percent refers to parts per
100. 3.weight/weight (%w/w)
1. weight/volume (%w/v)  Percentage of weight of solute in the total weight of
 Percent of weight solution in the total volume of the solution
solution  Number of grams of solute per 100 grams of final
 Number of grams of solute in 100 ml of solution solution
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
%𝑤/𝑣 = × 100 %𝑤/𝑤 = × 100
𝑣𝑜𝑙 𝑜𝑓 𝑠𝑜𝑙𝑛 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑛
 Example:  Example:
 0.85% (w/v) physiological saline solution  5 g Na2SO4 dissolved in 95 g of water (approx. 95
contains 0.85 grams of sodium chloride (NaCl) in ml) would result in concentration of 5% w/w.
the 100 mL of solution.  10% w/w solution of aqueous NaCl is prepared by
 5 g of Na2SO4 dissolved in water and diluted to a weighing 10 grams of NaCl and dissolving it in 90
final volume of 100 ml solution can be grams of water.
designated as 5% (w/v) solution of Na2SO4.
Practice
Practice 1. What is the percent concentration of 5 grams of chemical added
1. What is the percent concentration of 4 g of NaCl in 100 ml of to about 90 ml of water in a flask placed on a balance wherein
solution? water is added until the total solution weight is 100 grams?
Type equation here.
4 𝑔 𝑁𝑎𝐶𝑙
% 𝑁𝑎𝐶𝑙 =
100 𝑚𝑙
× 100 = 4% 𝑁𝑎𝐶𝑙 2. Prepare 10%(w/w) NaCl solution.
2. Prepare 10% (w/v) solution of aqueous NaCl.
Type equation here.
3. Prepare 500 ml of 0.85% saline.
0.85% =
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
× 100 Molecular Weight (MW)
500 𝑚𝑙
0.85
× 100 = 0.17𝑔 𝑁𝑎𝐶𝑙
 Weight in atomic mass units of all the atoms in a
500
given formula
 Tells us how many grams are in one mole of a

2. volume/volume (%v/v) substance

 Example: What is the MW of glucose (C 6H12O6)
 Percent of volume of solute in the total volume of
𝐶 = 12.01 × 6 = 72.06
solution 𝐻 = 1.008 × 12 = 12.096
 Number of milliliters of solute in 100 ml of solution 𝑂 = 16.00 × 6 = 96.00
𝑣𝑜𝑙 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 72.06 + 12.096 + 96.00 = 180.156 𝑔/𝑚𝑜𝑙
%𝑣/𝑣 = × 100
𝑣𝑜𝑙 𝑜𝑓 𝑠𝑜𝑙𝑛
 Example: Equivalent Weight (EW)
 100 ml of a 10% solution of bleach can be  Like moles, equivalent are related to a specific weight
prepared by adding 10 mL of household bleach to (called “combining weight”) of material
90 ml of water.  It is computed by the following formula:
 5 ml of glacial acetic acid diluted with water to a 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡
𝐸𝑊 =
total volume of 100 ml can be described as 5% 𝑛
where n is: 1. # of replaceable H+ in an acid
(v/v) glacial acetic acid. 2. # of replaceable OH+ in an base
3. charge on a cation or anion
4. # of electrons lost (or gained) in an
oxidation-reduction reaction

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 9
 Example: Calculate the equivalent weight of H2SO4. Relationship between M and N
(H2SO4 is an acid with 2 replaceable hydrogens)  Normality is always equal to or greater than molarity
98 𝑔/𝑚𝑜𝑙  Molarity is always equal to or less than normality
𝐻2 𝑆𝑂4 𝑀𝑊 = = 49 𝑔/𝐸𝑞
2
 Valence, or the number of replaceable H+, may be

Molarity (M) used to calculate a solution’s normality.

 Concentration of chemical solutions which is
𝑴 = 𝑵/𝒗𝒂𝒍𝒆𝒏𝒄𝒆 𝑵 = 𝑴 × 𝒗𝒂𝒍𝒆𝒏𝒄𝒆
expressed as moles per liter (mol/L) of solution, 2𝑁 𝐻𝐶𝑙 = 2/1 = 2𝑀 2𝑀 𝐻𝐶𝑙 = 2 × 1 = 2𝑁
replacing the older term molarity. 2𝑁 𝐻2 𝑆𝑂4 = 2/2 = 1𝑀 2𝑀 𝐻2 𝑆𝑂4 = 2 × 2 = 4𝑁
 A mole of a pure compound is the formula weight 2𝑁 𝐻3 𝑃𝑂4 = 2/3 = 0.67𝑀 2𝑀 𝐻3 𝑃𝑂4 = 2 × 3 = 6𝑁

(F.W.) or molecular weight (M.W.) of that compound

expressed in grams. Conversion of units: mg/dL to nmol/L and SI
 Number of moles per 1 Liter of solution
units
 Concentrations of solutions may be expressed in
 Formula: 𝑀𝑜𝑙𝑒𝑠/𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 =
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒/𝑀𝑊
𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
many ways, and a lab scientist must be able to
convert units of concentration from one form to
Normality another.
 The equivalent of solute per liter (Eq/L) of solution
 Example: A 350mg/dL chloride is equal to how many
 Formula:
(𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒/𝑀𝑊) mmol/L? (MW Cl = 35)
𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡/𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 =
𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

350 𝑚𝑔 10 𝑑𝐿 1𝑔 1 𝑚𝑜𝑙 𝐶𝑙 1000 𝑚𝑚𝑜𝑙

× × × × = 100 𝑚𝑚𝑜𝑙/𝐿
𝑑𝐿 1𝐿 1000 𝑚𝑔 35 𝑔 𝐶𝑙 1 𝑚𝑜𝑙
or use this formula: [10 × 𝑚𝑔/𝑑𝐿]/𝑀𝑊 = 𝑚𝑚𝑜𝑙/𝐿

UNIT 3: LABORATORY INFORMATICS

INTRODUCTION timeliness, and converts data into information for
 Laboratory informatics is defined as the specialized health-related decision-making.
application of information technology to optimize and
extend laboratory operations.  Health Informatics – the application of both
 Rising with the tide of informatics in general, technology and systems in a healthcare setting
laboratory informatics is one of the fastest growing  Health Information Systems – refer to any
areas of laboratory-related technology. system that “captures, stores, manages or transmits
information related to the health of individuals or the
HEALTH INFORMATION SYSTEM activities of organizations that work within the health
 Sound and reliable information is the foundation of sector.”
decision-making across all health system building
blocks and is essential for health system policy Sources of Information about the Country
development and implementation, governance and Health Information System
regulation, health research, human resources  Information about the functioning of the health
development, health education and training, service information system can be obtained from the different
delivery and financing. sectors and agencies that have responsibilities for the
 The health information system provides the generation, synthesis, analysis and use of data at
underpinnings for decision-making and has four key country, regional and global levels.
functions: data generation, compilation, analysis and  At country level, Ministries of Health record the
synthesis, and communication and use. timeliness and quality of data reported through health
 The health information system collects data from the services and disease surveillance systems.
health sector and other relevant sectors, analyses the  National Statistics Offices maintain of information
data and ensures their overall quality, relevance and about the availability and quality of data generated
through major data collection undertaking such as the
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 10
decennial census, large scale household surveys, and  Electronic medical records (EMRs) and electronic
the civil registration system. health records (EHRs) are some programs where it
is deployed and extensively used.
HOSPITAL INFORMATION SYSTEM  Most system offer support 24/7 to facilitate easier
 Health care plays a vital role in a society and people usage of the system by healthcare professionals.
expect efficiency from health care providers and 3. Radiology Information System (RIS)
health institutions which face the challenge of  RIS are capable of providing billing services and
handling the numerous patients that seek their appointment scheduling aside from reporting and
services. database storage in the radiology department.
 Proper management of clinical and operational  Technological advances have made the practice of
records is therefore necessary. radiology more complicated such that more
 Presently, most hospitals have shifted from tedious hospitals turn to RIS to address the commercial
manual recording to the use of a hospital information concerns of their radiology departments.
system (HIS) to assist them in maintaining the 4. Pharmacy Information System
different records of the institution.  It helps monitor the utilization of medicines in
 Hospital information system is an element of health health institutions.
informatics that focuses mainly on the  The system also handles information on
administrational needs of hospitals. medication-related complications and drug allergies
of patients.
 A hospital information system is fundamentally a 5. Laboratory Information System (LIS)
computer system that could manage all the  It is a software that allows for effective
information (medical, administrative, financial, and management of sample and associated data in the
legal) to permit health care providers to do their jobs laboratory.
efficiently.  It is designed to help process information to
 HIS was introduced in the 1960s and has evolved improve the efficiency of the department’s services
since then to cope with the changes and demands of & laboratory operations by reducing manual tasks
the modern times. and procedures which saves time and reduces
 Back then the features of HIS were used mainly for typographical error.
billing and inventory.
 However, all of these have changed through time, and LABORATORY INFORMATICS (LABORATORY
now it is also integrated with other financial, INFORMATION SYSTEM)
scientific, and administrative programs.  Laboratory informatics is the specialized application
of information through a platform of instruments,
Hospital Information System for Different software, and data management tools that allow
Departments scientific data to be captured, migrated, processed,
1. Nursing Information Systems (NIS) and interpreted for immediate use, as well as stored,
 NIS are developed to enhance patient care by managed, and shared to support future research,
providing nurses with accurate information to development, and lab testing efforts while
assist them in performing their duties more maximizing the efficiency of laboratory operations.
efficiently.  In a busy laboratory for example, one must learn to
 Carries out numerous functions including the work fast but still remain efficient while staying on
handling of personnel schedule, accurate patient top of patient requests, analyzing specimens, and
charting and better clinical data integration. releasing lab test reports and results.
 This is also useful in designing the patients’ care  The sheer number of patients and the amount of work
plan since the medical information integration that needs to be done can get overwhelming, but
function allows nurses to collect and examine thanks to modern day technology, the Laboratory
retrieved medical records. informatics can help automate workflows, integrate
2. Physician Information Systems instruments, and manage samples and associated
 It is designed to improve the practiced of information.
physicians.  Each machine or instrument in the lab can be hooked
up into the main system and can be controlled in a
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 11
computer, from what samples to run, to the  When does the sample need to be removed or
transmission of results, once the machines have discarded?
already run the tests. Information can thus be – With LIS, it will be easier to check that all the
communicated and managed very easily. test ordered on the sample has been properly
performed and no further verification of test
FEATURES OF LABORATORY INFORMATION results is needed, helping you make sure that
the sample can now be prepared for disposal.
Sample Management
 It is a process of ensuring accurate and detailed Workflow Management
records to avoid mixed-up or loss of samples once it  Workflow Management by definition is the execution
enters or is endorsed to the laboratory. and automation of a set of procedural rules used to
 We avoid mixed up of samples by using radio- coordinate tasks between people and systems, while
frequency identification (RFID) or barcodes from the ensuring that all steps and requirements of the
very moment the sample is received up to the process are correctly followed.
reporting of its results.  With this feature in LIS, workflow of laboratory
 Information that a LIS can store includes the operations can be easily managed by the system
following: ensuring that all steps from sample login to reporting
 From whom was the sample taken from? of results is done orderly while being regulated
– This includes personal information of the patient continuously with other workflow features like Data
whom the sample was collected from. security.
– Example of this is the name, age, gender and  LIS can also manage workflow in the laboratory by
birthday of the patient. the following ways:
 What is the sample taken?  Saving time by automating records and workflows
– These are the information that will detail the which eliminates the need for manual repetitive
type of sample that is collected. entry of data
– Is the sample collected blood, urine, sputum, or a  Taking part in the decision process by using
body fluid? existing coding methods, making the software able
 Who is working with it? to delegate decision when a certain scenario is met
– These reports the medical technician or medical while performing analysis. For example, you may
technologist currently carrying out the task set coded procedure in the system to make the
concerning the sample. analyzer perform the test twice to any sample
– Let us say you are tracking a sample; you may which present initial test results that appears
check whose lab section or personnel is currently abnormally high or low.
working on it via the LIS.  Using preset rules, it can suggest the instrument
 Who handled the sample? needed for the procedure, allocating it based on the
– These are record of individuals or personnel who test requested on the sample.
have handled or processed the sample.  Automatically assign the med techs or specialists to
– This information is very helpful especially on complete the test ordered on the sample.
cases where you are investigating incident  In a nutshell, the workflow management feature
reports. coordinates tasks that make up the work an
 Where does it go next? organization does.
– With the help of LIS, all the diagnostic tests to be
performed on the sample may be easily accessed Reporting
for you to where the sample needs to go next if  Every laboratory should have a system that allows
it has other additional ordered test. both analyzing and reporting of results in a
 How to store sample? standardized and customizable format.
– These information enables the personnel to  Standardization is important so that there is a
review on how to properly store and preserve template to follow that allows data to be universally
different samples for future testing. understandable to both humans and machines.
– This may include special instructions for samples
which may be tested for specific analytes.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 12
 Whereas customization is also important so as to be  Enter the mobile LIS, also known as the pocket
able to place the laboratory’ name, logo, address, laboratory.
contact details and other pertinent information.  By the name of this feature, one is able to access LIS
 Information generated by this reports can also include on a smartphone or a tablet.
data that is useful to the daily operations of the lab  Mobility is about having technology where it’s
such as which instrument is the most frequently used, needed, whether it’s at the lab bench, at the
the average handling time of samples and list of instrument side, or in the field.
backlogs which are accumulated or uncompleted  This software is able to collect data from the lab, track
tasks. inventory, manage users and view information, all in
 We call this as clinical and technical data which can a handy device which can be brought anywhere and
help the laboratory improve more on its day to day accessed anytime.
operation.  Although the companies that offer mobile LIS may still
 Furthermore, these reports generated are useful in be limited to certain countries at the moment, it could
data analysis and formulation of recommendation for definitely be more common in the years ahead.
future policy making as well.
 In other words, the laboratory can not only generate Enterprise Resource Planning (ERP)
patient results but also provide information to policy  ERP is one of the most important business technology
makers about morbidity, mortality, prevalence, up to date and has a number of powerful tools usually
incidence rates and other data that affects public used by top management.
health.  ERP and LIMS may be distinct from one another, but
when used in tandem, can provide great benefits for
Electronic Medical/Health Records the laboratory.
 Another integral part that any LIS may have is the  Having the ERP combined with LIMS allows easy
ability to view and handle patient records and billing viewing of current supplies, calculating storage
information. capacity and managing location among other
 This is done through integration of patient’s Electronic functionalities.
Health Record.  With the ERP installed you get the functionality and
 EHR is its own type of software but some LIS have this scope of ERP while still maintaining the robustness of
as a built-in functionality. the LIMS without compromising its capability.
 This feature helps the medical technologists manage  That means that the ERP can perform the daily
and interpret clinical laboratory procedures and micromanaging of other laboratory essential tasks
results more accurately. such as inventory management while allowing the
 Say for example, you find a spike in the leukocytes of LIMS to just concentrate and do what it does best: and
a patient while running his/her CBC request, you that is creating results based upon sample requests.
would then check the smear and find abnormal cells.
 Using the LIS that is hooked up with EHRs, you can LABORATORY INFORMATICS: PATIENT CARE &
easily correlate your results with the doctor’s initial PUBLIC HEALTH GOALS
or presumptive diagnosis, therefore confidently  In the past few years, technology has significantly
releasing a lab diagnosis for the said patient. improved the healthcare system in context to quality
 In this way, which just a click of a mouse button and a of care, patient safety, and operational efficiency.
simple search, the medical technologist can make  Technology is one of the key driving forces in
sense of the results through knowing important healthcare that is set to enhance the overall provider-
demographic and medical histories of their patients. patient experience.
 The future of healthcare is aimed at improving patient
Mobile LIS satisfaction, quality of care, care process, and practice
 Today, the laboratory industry is seeing challenges methods.
that did not exists back in the day.  The adoption of technology would improve 3 key
 To manage operations in such day and age, a robust, areas of patient-centered care: monitoring,
agile, and modern LIS is needed. consultation, and treatment.
 But what is the answer to those challenges.  Efficient treatment and management of patients
across healthcare sectors, specifically during a
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 13
medical emergency has become possible with the help collect, process, store, and manage such processes is
of advanced health information technology. required.
 In the past decade, one such technological  The LIS has helped in improving laboratory operations
development has been witnessed which has emerged and overall patient care.
to improve overall diagnostic and assessment  LIMS or LIS has helped several healthcare facilities to
processes in the healthcare system, i.e. laboratory combat high influx of patients efficiently and
information systems (LIS). effectively.
 The modern-day or 21st century laboratory requires  The use of LIMS/LIS replaces conventional paper-
an efficient system that would handle and process based collection storage, and handling of patient
large amounts of information daily. reports or information.
 The LIS has emerged as a live-saver for medium-to-  Data collected and archived is in digital/electronic
large scale laboratories in context to improving format which has lesser risk of loss or theft when
pathology workflow processes, management of compared to paper-based collection and storage.
reports, and allied monitoring operations.  Most of the LIS/LIMS can develop and produce reports
 The LIS is a fundamental component of the diagnostic in real-time to patients which increases overall
or pathology department. credibility and reliability of the laboratory.
 In the era, where laboratory reports have become  LIS can also be designed for use in Public Health such
complex and high-dimensional, the need to develop as reporting and analysis of a variety of significant
and implement a cost-effective platform or tool to laboratory testing component which are of public
health importance like outbreak-related information.

UNIT 4: QUALITY MANAGEMENT

INTRODUCTION acceptably; method evaluation is used to verify the
 In order to achieve the highest level of accuracy and acceptability of new methods prior to reporting
reliability, it is essential to perform all processes and patient results.
procedures in the laboratory in the best possible way.  Once a method has been implemented, it is essential
 The laboratory is a complex system, involving many that the laboratory ensures it remains valid over
steps of activity and many people. time; this is achieved by a process known as quality
 The complexity of the system requires that many control (QC) and quality assurance (QA).
processes and procedures be performed properly.  All of these concepts fall under the umbrella of quality
 Therefore, quality management is very important for management (QM), where the entire testing process is
achieving good laboratory performance. directed with the overall goal improving the accuracy
of laboratory results.
QUALITY MANAGEMENT IN CLINICAL
LABORATORY  Quality Management – approach focusing on
 It is widely accepted that the majority of medical process improvement as a means to meet a set
decisions are made using laboratory data. standard.
 It is therefore critical that results generated by the  Quality Assurance – measurement of the broader
laboratory be accurate. dimensions of quality, from the perspective of the
 Determining and maintaining accuracy requires end-users. In the laboratory it might involve the
considerable effort and cost, entailing the use of a monitoring of specimen acquisition, turnaround times,
series of approaches depending on the complexity of or proficiency testing of materials to determine
the test. To begin, one must appreciate what quality is analytic performance.
and how quality is measured.  Quality Control – the main purpose of QC is to
 It is vital to understand basic statistical concepts that detect and repair performance, and sporadic spikes to
enable the laboratorian to measure quality. the prior chronic level, either by prompt action to
 Before implementing a new test, it is important to restore the status quo or, preferably, by preventing
determine if the test is capable of performing the damage from occurring in the first place.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 14
QUALITY CONTROL  It involves proficiency testing programs that
 A system of ensuring precision and accuracy in the periodically provide samples unknown concentration
laboratory using quality control reagents in every of analytes to participating laboratories.
series of measurements.  It is also used to determine estimates of the state-of-
 It is a system of techniques to ensure with a specified the-art interlaboratory performance.
degree of confidence that the result obtained from
each series of analysis is true and correct. Goals of Quality Control
 A good quality control program monitors test  Initial goal: to develop methods with high analytical
performances, helps identify problems with a specific accuracy and precision
assay system, and helps health care staff assess the  Second goal: to ensure that the methods will be
reliability of results. performed continuously in an accurate and precise
manner
Types of Quality Control System
1. Intralab QC (Internal Quality Control) Purposes of Quality Control
 Quality control performed in a certain laboratory to 1. To check the stability of the machine
see that day to day performance is not deviating 2. To check the quality of reagents
from an established standard. We are interested in 3. To check for technical error if the operator committed
precision and accuracy. any
 Involves the analyses of control samples together 4. To make results obtained in various laboratories
with the patient specimens. comparable to each other.
2. Interlab QC (External Quality Control) 5. To enhance the role of the laboratories in the
 Quality control in several laboratories which infers prevention, diagnosis and treatment of disease.
that a determination performed in several 6. To improve the performance of clinical laboratory.
laboratories will yield the same values.

Characteristics of a Good Quality Control

1. Sensitivity
 Ability of a method to detect and measure even the smallest amount of the particular substance tested for. It is also
the degree by which significant deviation can be detected.
 Diagnostic Sensitivity – ability of a test to detect the proportion of individuals with disease who test (+) with the test
𝑛𝑜. 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ 𝑎 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝐷𝑆 (%) = × 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟𝑒 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑡𝑒𝑠𝑡𝑒𝑑
𝑇𝑃
= × 100
[𝑇𝑃 + 𝐹𝑁]
2. Specificity
 Ability of a method to detect a particular substance w/o the interference of other substances present in the sample.
 Diagnostic Specificity – ability of a test to detect the proportion of individuals w/o the disease who test ( with the test
𝑛𝑜. 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 𝑤𝑖𝑡ℎ 𝑎 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝐷𝑆 (%) = × 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟𝑒 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑡𝑒𝑠𝑡𝑒𝑑 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒
𝑇𝑃
= × 100
[𝑇𝑃 + 𝐹𝑁]
Predictive Values – predictive ability of a test to identify disease or its absence in individuals
 Positive predictive value – the probability that subjects with a positive screening test truly have the disease
𝑛𝑜. 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ 𝑎 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝑃𝑃𝑉 = × 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑤𝑖𝑡ℎ 𝑎 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝑇𝑃
= × 100
[𝑇𝑃 + 𝐹𝑁]

 Negative predictive value – the probability that subjects with a negative screening test truly don't have the
disease
𝑛𝑜. 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 𝑤𝑖𝑡ℎ 𝑎 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝑁𝑃𝑉 = × 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑤𝑖𝑡ℎ 𝑎 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡
𝑇𝑃
= × 100
[𝑇𝑃 + 𝐹𝑁]

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 15
3. Practicability QUALITY ASSURANCE
 Ability of a method to be easily repeated  Concerned with the total process, not simply assay
4. Reliability validation.
 Ability of an analytical method to maintain accuracy  Involved with maintaining integrity of the entire
and precision over an extended period of time process from patient identification to accurate
during which equipment, reagents and personnel reporting and logging of the laboratory data.
may changed
5. Accuracy Phases of Quality Assurance
 Refers to how close the measured value is to the 1. Pre-analytical Phase
true value  Test ordering
 This parameter is not defined by statistics, but by  Patient preparation
comparison of the measured value to the “known  Patient identification
value” obtained from a reference material  Specimen collection
6. Precision  Specimen transport
 Refers to the reproducibility of a measurement  Specimen processing
 It indicates how close the single values are to one 2. Analytical Phase
another  Test analysis
 It is expressed by the Standard Deviation, the  Quality control
smaller the SD, the more precise the values are  Reagents
 Calibration
 Preventive maintenance
3. Post-analytical Phase
 Verification of calculations and reference ranges
 Review of results
 Procedures for notification of critical values
 Reporting/releasing of result

Relevant Terminologies  Test interpretation by physician

 Variations – errors in quality control; the fundamental Quality Assurance Cycle

basis of any statistical analysis
1. Random error – a non-recurring error due to
factors such as dirty glassware, use of wrong
pipette, voltage fluctuations or sampling error
– Clerical Error – error on the part of the
personnel; example is wrong release of result
due to bad penmanship or wrong patient ID.
2. Systematic error – recurring error that affects all
results. A faulty machine and contamination of
standard or reagents would cause systematic error
– Aging phenomena – variations due to reagents Laboratory Testing Processes and its Potential
or failing instruments Errors
– Personal Bias – variation caused by operator
 Delta Check PROCESS POTENTIAL ERRORS
 Most commonly used patient based-QC technique
 Inappropriate test
 Handwriting not legible
where the most recent result of a patient is Test ordering
 Wrong patient identification
compared to the previous value  Special instructions not given to patient
 Could detect errors otherwise overlooked but at the  Incorrect tube or container
cost of investigating many false positives  Incorrect patient identification
Specimen  Inadequate volume (QNS)
acquisition  Invalid specimen (e.g. hemolyzed)
 Collected at wrong time
 Improper transport condition

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 16
 Instrument not calibrated correlated 𝑀𝑒𝑎𝑛 = 110 𝑚𝑔/𝑑𝑙
 Specimen mixed-up 𝑆𝐷 = 9 𝑚𝑔/𝑑𝑙
Analytical
 Incorrect volume of specimen
measurements
 Interfering substance present
 Instrument precision problem
 Wrong patient identification
 Report not legible
Test reporting
 Report delayed
 Transcription error

QUALITY CONTROL CHARTS

Measures of Dispersion or Variability

1. Mean or Average
2. Standard Deviation
3. Coefficient of Variation
4. Variance

MEAN OR AVERAGE
 The mathematical result when the summation of data  The reference range is usually set at mean +/- 2SD
is divided by the total number of data. range – also known as the 95% limits or 95%
𝑀𝑒𝑎𝑛 (x̅) =
𝑠𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 𝑡ℎ𝑒 𝑣𝑎𝑙𝑢𝑒𝑠 confidence limit. Therefore, in our example, the
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑣𝑎𝑙𝑢𝑒𝑠 reference range would be 92-128mg/dl. 5% chance
 A measure of central tendency (peak of the curve)
that not really healthy.
 Measure accuracy
 Associated with symmetrical or normal distribution
COEFFICIENT OF VARIATION
𝑆𝐷
𝐶𝑉 = × 100
𝑋̅
 The percentile expression of the mean which is a
measure of the relative magnitude of variability.
 Assess the consistency and precision of an assay.
 It simplifies comparison of standard deviations of test
results expressed in different units and
concentrations.
STANDARD DEVIATION  Ideally should be less than 5%.
 Measure of the distribution (range) of values around
the mean VARIATION
 Indicates the amount of difference among the 𝑉 = (𝑆𝐷)2
individual data  Statement of variability and measures the significant

∑(𝑥 − 𝑥)2
differences between groups of data
𝑆𝐷 = √
𝑛−1
Types of Quality Control Charts
1. Gaussian Curve
Practical Meaning of Standard Deviation
2. Cummulative Sum Graph
 68.2% of all the values – lies between mean ±1SD
3. Youden Plot/Twin Plot
 95.5% of all the values – lies between mean ±2SD
4. Levey-Jennings Chart
 99.7% of all the values – lies between mean ±3SD

Application to Reference Range

 We analyze FBS (fasting blood sugar) from 1000
presumably healthy normal adults and the data
gives a Gaussian distribution (normal values) when
plotted. Let us assume that we have this data
obtained:
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 17
GAUSSIAN CURVE

 Obtained by plotting the values from multiple

LEVEY-JENNINGS CHART
 Most commonly used histogram in Quality Control
analyses of a sample
 A chart is needed for each method & control w/control
 Population probability distribution that’s symmetric
about the mean results being plotted over a specified period of time
 The value obtained for the control material is plotted

CUMMULATIVE on the Y-axis vs the date of analysis on the X-axis.

 Allows laboratorians to apply multiple rules w/o
 Taken by calculating the actual difference between the
individual values and the expected mean value, then computer.
 Can detect both random and systematic error.
sum those differences to determine the cumulative
effect for all control observations collected.
 Gives earliest indication of a trend (only for
systematic error)
 Identifies consistent bias problem

Interpretation of Quality Control Results

1. In-Control – when the values of the control fall within
the confidence limit; indicates that result is reliable
2. Out-of-Control – when the values of the control fall
outside the confidence limit; indicates that result is
YOUDEN PLOT/TWIN PLOT unreliable
 Used to compare results obtained on a high and low
control by several different laboratories Type of Out-of-Control Charts
 Particularly useful for interlaboratory quality control 1. Trend

programs 2. Shift
3. Outliers
4. Westgard Rules

TREND
 Formed by the control values that continue either to
increase (upward) or decrease (downward) for a
period of six (6) consecutive days by passing the
mean.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 18
 Conditions when dealing with outlier:
 One outlier in 20 days (in control)
 Two outliers in 20 days (out-of-control)

 A trend indicates a gradual loss of reliability in the

test system. Trends are usually subtle.
 Causes of trending may include:
 Deterioration of the instrument WESTGARD RULES
 In 1981, Dr. James Westgard of the University of
 Gradual accumulation of debris in sample/reagent
tubing Wisconsin published an article on laboratory quality
 Gradual accumulation of debris on electrode control that set the basis for evaluating analytical run
surfaces quality for medical laboratories.
 The elements of the Westgard system are based on
 Aging of reagents
 Gradual deterioration of control materials principles of statistical process control used in
 Gradual deterioration of calibration materials industry nationwide since the 1950s.
 There are six basic rules in the Westgard scheme: 12s,

SHIFT 22s, 13s, R4s, 41s, 10x

 These rules are used individually or in combination to
 Formed by the control values that distribute
themselves on one side of the mean for a period of evaluate the quality of analytical runs (Intralab).
 Westgard devised a shorthand notation for expressing
six (6) consecutive days.
quality control rules.
 Most of the quality control rules can be expressed as
NL where N represents the number of control
observations to be evaluated and L represents the
statistical limit for evaluating the control
observations.
 Thus, 13s represents a control rule that is violated
when one control observation exceeds the ±3s
control limits.
 There are two applications to this rule:
 Within-run – affects all control results obtained for
 Shifts in QC data represent a sudden and dramatic the current analytical run
positive or negative change in test system
performance. DATE QC LEVELS
 Shifts may be caused by: Day 1 Level 1 Level 2
Day 2 Level 1 Level 2
 Sudden failure or change in the light source
Day 3 Level 1 Level 2
 Change in reagent formulation
 Major instrument maintenance
 Across-run – affects all control results obtained for
 Change in room temperature or humidity
the current control material
 Failure in the sampling system
 Failure in reagent dispense system
DATE QC LEVELS
 Inaccurate calibration/recalibration Day 1 Level 1 Level 2
OUTLIERS Day 2 Level 1 Level 2
 Values which are far from the main set of values due Day 3 Level 1 Level 2
to wild errors
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 19
 When rule is violated: – Do not report patient results until problem is
 Warning rule – use other rules to inspect control solved and controls indicate proper performance
points  Allows for detection of random analytical errors
 Rejection rule – “out-of-control” versus systematic analytical error
– Stop testing  Rules 12s, 13s and R4s – random analytical error
– Identify and correct problem  Rules 22s, 41s and 10x – systematic analytical error
– Repeat testing on patient samples and controls

RULE DESCRIPTION

One control observation is exceeding the mean ±2SD

control limits
Used only as “warning” rule that initiates testing of the
control data by the other control rules

Application: Across-run

Two consecutive control values for fall outside of ±2SD in

the same direction

Application: Within-run & Across-run

One control observation exceeding the mean ±3SD control

limits

Application: Across-run

A range of 4s difference between control values within a

single run

Application: Within-run

For example, assume both Level I and Level II have been

assayed within the current run. Level I is +2s above the
mean and Level II is -2s below the mean. The total
difference between the two control levels is ≥4s. (e.g.
[+2s – (-2s)] = 4s).

Four consecutive control observations are on the same

side of the mean and exceeding the ±1SD control limit

Application: Within-run & Across-run

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 20

Ten consecutive control observations falling on one side of

the mean (above or below, with no other requirement on
size of deviation)

Application: Within-run & Across-run

UNIT 5: ANALYTICAL TECHNIQUES, AUTOMATION AND ROBOTICS

INTRODUCTION  The relationship between wavelength and energy
 Instrumentation is the mainstay of clinical chemistry. (E) is described by Plank’s formula:
 Of all departments in the clinical laboratory, clinical 𝐸 = ℎ𝑣
chemistry is the most automated and dependent upon Where h = a constant (6.62 x 10-27 erg sec) known as
Plank’s constant
large-volume automated instrumentation. v = frequency
 Automated instrumentation employs the same  Light energy is directly related to frequency, that
chemistry reactions used in manual biochemistry and is, light of greater frequency possesses more
general chemistry laboratories. energy than light of lesser frequency.
 Knowledge of laboratory equipment and function of  The greater the frequency, the shorter the
components within instruments will be discussed wavelength and vice versa.
further in this chapter through the eyes of a student.  Light energy and wavelength are, therefore,
 This chapter describes the student’s first experience inversely related.
in the clinical chemistry section of the laboratory. 3. Amplitude
 Height of the crest of the wave from the midline
ANALYTICAL TECHNIQUES, AUTOMATION AND  The higher amplitude, the more intense the light
ROBOTICS and the more light energy is produced at that
wavelength.
ELECTROMAGENTIC ENERGY (LIGHT)
 Radiant energy behaving as though it possessed an
electrical field and a magnetic field
 Photons of energy travelling in a wavelike manner
 Other non-visible forms of electromagnetic radiation:
gamma, x-ray, ultraviolet, infrared, microwave, and
radio waves.

Properties of Light
1. Wavelength
Range of Electromagnetic Spectrum
 Distance between 2 successive wave crests
ENERGY AND APPROXIMATE
 The factor that determines the color in the visible TYPE OF
FREQUENCY RANGE OF
spectrum RADIATION
RELATIONSHIP WAVELENGTH
 Wavelength visible to the naked eye = 400 – 700 More energy Gamma
nm Greater frequency x-rays
-10 Short wavelength
 Expressed in angstrom (Å, 10 m), millimicrons Ultraviolet 200-400 nm
(mu) and nanometer (nm) Visible 400-700 nm
2. Frequency
Less energy Infrared 700-2000 nm
Lesser frequency Microwaves
 Number of wave cycles that are completed in one
Long wavelength Radiowaves
second (Hertz/Hz)

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 21
 It also states that the “source of light should be
INCREASING WAVELENGTH monochromatic, and the distance travelled by the light
through the cuvette should be fixed”.
𝐴=𝑎𝑏𝑐
where: A = Absorbance being sought for
a = absorptivity of substance
b = length of the light path
c = concentration of substance sought for
(unknown)
 Light transmitted through a solution can be measured.

2 Kinds of Light
INCREASING FREQUENCY

INCREASING ENERGY
INCIDENT LIGHT TRANSMITTED LIGHT

1. Incident Light (Io) – light entering cuvette

The Visible Light 2. Transmitted light (I) – light leaving the cuvette
WAVELENGTH COLOR COLOR TRANSMITTED
(nm) ABSORBED (OBSERVED)
% Transmittance is the ratio of the transmitted light
400-435 Violet Yellow-green over incident light times 100 or;
𝐼
435-480 Blue Yellow %𝑇= × 100
𝐼0
480-490 Green-blue Orange
490-500 Blue-green Red
500-560 Green Purple
Absorbance (A) = popular measurement
560-580 Yellow-green Violet  the amount of light absorbed
580-595 Yellow Blue  directly proportional to concentration
595-605 Orange Green-blue  has no unit and can’t be measured directly by a
605-700 Red Green spectrophotometer, but rather is mathematically
derived from %T as follows:
 The visible color of a solution, that is, the color that %𝑇=
𝐼
× 100
you actually see, is determined by the wavelengths of 𝐼0

light that are transmitted, not observed. where: I = transmitted light thru the sample
 In photometry and spectrophotometry, the color that
I0 = intensity of light striking the sample
is absorbed is measured.
 The color absorbed is the complementary color of that
Absorbance is defined as:
transmitted. For example, a solution that appears A = -log (I/I0)
purple to the eye (color transmitted) is absorbed A = log (100%) – log %T
principally in the green portion of the spectrum. A = 2 – log %T
 The wavelength chosen to measure the absorbance of
 all photometers/spectrophotometers measure
the purple solution would be in the green portion of
the spectrum. %T or the light transmitted
 absorbance is derived from the transmittance

LAMBERT-BOUGER-BUNSEN-ROSCOE-BEER’S LAW reading by using formula A = 2 - log %T

 Beer’s Law states that “Absorbance is directly
proportional to the concentration of the substance All instruments involved in the measurement of light
absorbing light and inversely proportional to the intensity will use one of the following principles:
1. Absorption
logarithm of the transmitted light”.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 22
 The concentration of a sample in solution is 1. Light Source – provide a source of radiant energy
determined by measuring the amount of light a. Tungsten-Iodide Lamp – commonly used for
transmitted through the sample. measurements at wavelengths in the visible and
 As the concentration of the sample increases, the near-infrared portion of the spectrum
amount of transmitted light decreases b. Mercury Arc Lamp and Xenon Arc Lamps – can be
logarithmically. used in UV region to the visible region deuterium
2. Reflectance lamp (provides a continuous spectrum in the UV
 The concentrate of a sample in solutions region)
determined by measuring the amount of light c. Hydrogen Lamp – can be used in the UV region
reflected. d. Infrared Energy Source – used above 800 nm
 The concentrate is inversely proportional to the (infrared region)
amount of light reflected by the sample.  Merst glower
3. Emission  Globar
 The concentrate of a sample in solution is 2. Entrance Slit – minimizes unwanted or stray light and
determined by measuring the amount of light prevents the entrance of scattered light into the
emitted or given off by the sample. The concentrate monochromator
of the sample and the amount of light emitted are  Stray Light – light striking the detector at
directly proportional wavelengths other than that for which the
4. Scatter wavelength dial is set.
 The concentrate of a sample in solution is  Causes of Stray Light
determined by measuring the amount of light – Reflection of light from scratches on optical
scattered by the solution. surfaces
 Light scatter is proportional to sample concentrate. – Reflection of light from dust particles anywhere
in light path
COLORIMETRY: SPECTROPHOTOMETRY AND – Darkened lamp envelope
PHOTOMETRY – Presence of second order spectra
 Sharp Cut-Off Filter – monochromator that can
Kinds of Colorimetry isolate stray light
1. Visual Colorimetry 3. Monochromator – selects the band of light that
2. Photoelectric Colorimetry – measure the light passes to the cuvette
transmitted by a solution to determine the  Monochromatic Light – light radiation of a single
concentration of light-absorbing substance present in wavelength
the solution  Bandpass/Bandwidth – the width of the band of
a. Spectrophotometry – Spectrophotometric light that is allowed to pass to the sample; the
measurements range of wavelengths between the points at which
 Measurement of light intensity in a much transmittance is one half peak transmittance
narrower wavelength a. Photometers – uses filter in isolating desired
b. Filter Photometry – Photometric measurements wavelength
 Measurements of light intensity of multiple i. Glass filter/colored filter
wavelength  Absorb light of wavelengths other than the
desired wavelength
Basic Components  Made of glass that absorbs some portion of
the electromagnetic spectrum and transmit
others
 Not true monochromators since they transmit
light of more than one wavelength
 Filters are simple, least expensive, not precise
but useful
ii. Interference filter
 Provides light of greater spectral purity than
is possible with glass filter
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 23
 Utilizes the wave character of light to enhance  Requires no external voltage source to operate
the intensity of the desired wavelength by but utilize internal electron transfer for current
constructive interference and eliminates production – low internal resistance
others by destructive interference and  Used mainly in filter photometers with a wide
reflections bandpass
b. Spectrophotometer – uses prism or diffraction b. Photoemissive or phototube
grating  Consists of 2 electrodes [cathode and anode]
i. Prism sealed in an evacuated glass to prevent
 Wedge-shaped pieces of glass, quartz or scattering of photoelectrons by collision with gas
sodium chloride molecules
 Can be rotated, allowing only the desired  Has photosensitive material that gives off
wavelength to pass thru an exit slit electron when light energy strikes it
– Ordinary glass prism – used in visible  Requires an outside voltage for operation
region
– Quartz/fused silica – used in UV
ii. Diffraction grating
 Most commonly used;
has better resolution
than prism
 Made by cutting
grooves or slits into
an aluminized surface
of a flat piece of DIFFRACTION
c. Photomultiplier (PM) tube
crown glass – GRATING
 Most commonly used detector – measures
wavelengths are bent visible and UV regions
as they pass a sharp corner  200 times more sensitive than the phototube and
4. Exit Slit – controls the width of light beam (bandpass); are used in instruments designed to be
allows only a narrow fraction of the spectrum to reach extremely sensitive to very low light levels and
the sample cuvette light flashes of very short duration
5. Analytical Cell or Cuvettes – used to hold the solution  It should never be exposed to room light because
in the instrument whose concentration is to be it will burn out
measured
 Types According to Shade
– Square
– Round
– Disposable plastic
cuvettes
 Types According Plastic
Cuvettes
– Glass cuvettes – visible
spectrum SQUARE
– Quartz/fused silica – UV CUVETTE
and visible
6. Detector – detect and converts transmitted light
energy in proportion to the intensity of the light d. Phototransistors or Photodynodes
 a.k.a. Solid state detector (e.g. Photodynodes)
striking the sensing area
 Newest entry among the light detectors
a. Photocell or Barrier Layer Cell
 Small durable and capable of high amplification
 Simplest detector, least expensive and
 Constructed of 2 types of semiconductors joined
temperature sensitive
 Made up of light sensitive materials that will
together that resist current flow between them
release electrons when exposed to light energy
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 24
7. Read-Out Device – provides the readout of the NEPHELOMETRY
transmitted light, either in %T or in absorbance units
8. Power Source Principle
 Nephelometry determines the amount of scattered
DOUBLE-BEAEM SPECTROPHOTOMETER light by a particulate matter suspended in a turbid
 Operates like single-beam spectrophotometers except solution.
that it is designed to compensate for possible
variations in intensity of the light source Basic Components
 The compensation is done by splitting the picture
monochromatic light into two components: one beam 1. Light source
passes through the sample, and the other through a a. Laser sources – provide monochromatic light
reference solution. Thus, any change in light intensity b. Tungsten-halogen sources – provide a broad
affects both cuvets simultaneously and thus is spectral range
canceled out 2. Monochromator
 The ratio of the sample light intensity to the reference 3. Cuvet
light intensity is measured 4. Detector – a photomultiplier tube is located at an
angle to the light beam
2 Types of Double-Beam Spectrophotometer 5. Read-out
1. Double-beam in space – uses 2 photodetectors (for
the sample beam and reference beam) Uses
Double-beam spectrophotometer with 2 detector  Used to quantitate the rate of insoluble
2. Double-beam in time – uses one photodetector and antigen:antibody complex formation during the assay
alternately passes the monochromatic light through of specific serum proteins
the sample cuvet and then reference cuvet using a  Ideal for measuring individual proteins:
rotating disc immunoglobulins, csf albumin, fibrinogen,
Double-beam spectrophotometer with only 1 detector haptoglobin, crp, complement components,
 Use: Ideally suited for making spectral scan rheumatoid factor, alpha-1 -antitrypsin, transferrin,
because the instrument automatically corrects for ceruloplasmin, alpha-2 – macroglobulin
the change in light transmission through the  Also for measuring drug concentrations
reference cuvet as the wavelength is changed
FLUOROMETRY AND PHOSPHOMETRY
TURBIDIMETRY
Principle
Principle  Fluorometry works by the principle that concentration
 It determines the amount of light blocked (reduction of a solution is directly proportional to its
of light) by a particulate matter in a turbid solution. fluorescence intensity.
 Some molecules fluoresce or emit light after being
Disadvantages exposed to light at a certain wavelength. Light is
 The decrease in light transmittance is related to the emitted within a brief period of time (10-9 to 10-6
number and size of the particles in the solution. seconds) and is of lower energy (longer wavelength)
 Particles may settle out upon standing or the turbidity than the light absorbed.
may change in time.  In order to fluoresce, a molecule in the ground state is
excited by light absorption and jumps to a higher
Uses energy level. Collisions and heat loss then cause the
 Measures total proteins in CSF and urine. molecule to drop to a lower, but still excited energy
 Determination of the activity of the enzymes, amylase level, but no lights is emitted. However, once it begins
and lipase dropping back quickly to the ground state, light is
 Usually reserved for the measurement of abundant finally emitted, and fluorescence occurs. This process,
large particles, such as bacteria in solution when completed within 10-6 seconds, is known as
Picture fluorescence. Despite this, some molecules require
10-4 seconds to undergo certain electron spin changes.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 25
The emission of light as a result of these changes is 3. Limits of detection – the detector’s sensitivity and
known as phosphorescence. the wavelengths it can pick up can drastically affect
readings
Basic Components 4. Quenching of fluorescence – occurs when the
picture excited state of the molecule loses some of that
1. Light source energy by interaction with another component of the
 Emits short wavelength high-energy excitation light reaction system transferring energy to the other.
 Gas-discharge lamp (mercury arc discharge lamp, 5. pH – changes in the pH can cause changes in the ionic
xenon arc tube) – most frequently used state of the molecules and there by alter the
 Incandescent tungsten lamp – seldom used substance’s fluorescing properties
 Mercury vapor lamp – commonly used in filter 6. Temperature – increased temperatures cause
fluorometry increase in molecular motion, causing more collisions,
 Xenon lamp – used in spectrofluorescence and thereby less fluorescence because of heat loss
2. Monochromators 7. Reagents/solvents – can either increase or
a. Primary filter (Excitation Monochromator) – placed decrease the substance’s ability to fluoresce
between the radiation source and the sample, 8. Glassware – scratched, dirty or unclear glassware
selects the desired wavelength that is best affects readings by blocking the light emitted by the
absorbed by the solution to be measured substance
b. Secondary filter (Emission Monochromator) – 9. Length of light exposure – increased exposure to
passes the longer wavelengths of fluorescent light light results in more excited molecules so increase in
preventing incident light from striking the fluorescence occurs
photodetector; either prism, diffraction grating or
filters Uses
3. Slit or attenuator – controls light intensity  Ideal in measuring concentrations when great
4. Cuvette – holds sample; can be glass, quartz or sensitivity is required.
silica cuvet  Porphyrin, magnesium, calcium, catecholamines,
5. Detector various drugs, and several enzymatic products are
 Placed at right angle to the sample cell commonly measured using fluorometry
 Either phototube or photomultiplier
6. Readout system ATOMIC EMISSION
SPECTROPHOTOMETRY/FLAME-EMISSION
Advantage PHOTOMETRY
 Very sensitive and specific. One thousand times more
sensitive than most spectrophotometric methods. Principle
 The measurement of emitted light when electrons in
Disadvantages an atom become excited by heat energy produced by
 Not all molecules fluoresce so some molecules that the flame
cannot fluoresce by themselves are added with side
chains such as the -NH2 group. Components
 Due to its high sensitivity, it is easily affected by picture
environmental changes, such as pH, temperature, 1. Gases – a mixture of hydrogen and oxygen gas
concentration, etc. (acetylene, propane or natural gas [methane])
2. Aspirator – pull sample into the atomizer
Factors Affecting Fluorescence Measurements 3. Atomizer or burner
1. Scattered light – can throw off the detector’s  Disperses the solution into the fine droplets so that
readings and give bad results the atom will absorb heat energy from the flame
2. Absorbance of sample – some samples have and get excited
better absorbance than others, causing an increase in  Sample viscosity, density and surface tension affect
fluorescence when it occurs droplets size and the rate of atomization

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 26
4. Flame or flame temperature will affect the sodium,
 Provide energy to break chemical bonds and to potassium, and lithium emissions proportionately. The
excite neutral atoms ration remains constant, thus compensating for
 If any atoms exist in the ionized form, the reducing possible fluctuations.
gases in the flame reduce metal ions to neutral,  Lithium fulfills the criteria and is commonly used as
ground state atoms an internal standard except when lithium is already
5. Monochromators present in serum, as is sometimes used as
 Isolate the emission wavelength characteristic for antidepressant agent. When lithium is to be
the substance being measured, instrument modifications are necessary.
 Frequently used monochromators: interference Some instruments reverse the K/Li ratio and use a
filter, cutoff filters, prisms or gratings K+ solution as internal standard so that Li+
 The number of interference cutoff filters needed is concentration = lithium signal/potassium signal.
dependent on the number of line spectra measured.  Other instruments use a fourth filter-photodetector
6. Detector with cesium as the internal standard.
 Measures the intensity of the emission signal by
converting light energy to a proportional electrical Use
current  Commonly used to measure sodium, potassium, and
 Usually consists of photomultiplier tubes lithium because these alkali metals are fairly easy to
7. Readout – displays the concentration of the element excite.
being assayed
8. Internal Standard Disadvantages
 Used to achieve stability or correct for changes in  Sensitivity is poor
temperature or aspiration rate that would affect  Temperature – affects the number of excited atoms
the emission signal present in the flame
 Also acts as a radiation buffer  Aspiration rate – has to be adjusted so that
sufficient sample is delivered to the flame for
Guidelines in Choosing Internal Standard proper sensitivity
1. The concentration of internal standard must be  Protein – may coat atomizer walls, affecting the
precisely the same in all samples and standards so ability of the atomizer to produce the fine mist
that the reference beam is constant. needed for introduction to the flame.
2. The amount of energy required to excite the internal  Concentration – if the sample introduced into the
standard must be close to that required to excite the flame is too highly concentrated, self-absorption
element being measured. occurs.
3. The emission lines of samples and internal standard
must be far enough apart so that they can be resolved ATOMIC ABSORPTION SPECTROPHOTOMETRY
by a monochromator and measurement can be
accomplished. Principle
4. Consist of a substance that is not normally found in  Measures the concentration of an element by
biological fluids detecting the absorption of light of a unique
5. The concentration of internal standard should be wavelength by atoms in the ground state, rather than
similar in magnitude to the unknowns so that a by molecule. The wavelength absorbed corresponds to
reasonable ratio is obtained the particular line spectrum for that element.

Notes Regarding Internal Standard Components

 For sodium or potassium measurements, lithium can picture
be added in equal amount to all standards and 1. Light source: Hollow Cathode Lamp
samples to act as internal standard. The readout is a  Supplies the electromagnetic radiation of a specific
ratio comparing the emission intensity for the internal wavelength
standard with that of the element being analyzed. The  Consists of evacuated gas-tight chamber containing
same amount of lithium is present in the blank, anode, cylindrical cathode and inert gas such as
sample, and standards. Any change in aspiration rate helium or argon.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 27
2. Atomizer or burner 6. Readout – presents the data in concentration,
 Break the chemical bonds and form free, unexcited, absorbance, % transmittance
ground state atoms.
 The flame is the sample cell in this instrument Advantages
 It is necessary to control flame size in order to  Provides good sensitivity and specificity
provide a constant light path.  Almost free from spectral interference
 Light from the hollow-cathode lamp passes through
the sample of ground-state atoms in the flame. The Disadvantages
amount of light absorbed is proportional to the  Chemical interference – incurred when the flame is
concentration. When a ground-state atom absorbs unable to dissociate the sample into the ground-state
light energy, an excited atom is produced. The atoms
excited atom then returns to the ground state,  Ionization interference – occurs when solvent
emitting light of the same energy as it is absorbed. contains high concentrations of salt
3. Mechanical rotating chopper – rotating disk that
alternatively passes and blocks the light at brief Uses
intervals modulating the light beam coming from  Used for detecting small amount of element when
hollow cathode lamp concentration is too small to accurately measure by
4. Monochromator the standard chemical means.
 Selects the wavelengths that are allowed to pass  Determine Ca, Mg, Co, Pb, Hg, Zn, Cr
the detector  Reference method for calcium
 Also serves to protect the photodetector from
excessive light emanating from flame emissions
 Uses prisms or gratings with narrow bandpass
5. Detector amplifier – detector is a photomultiplier
tube

ATOMIC EMISSION SPECTROPHOTOMETRY vs ATOMIC ABSORPTION SPECTROPHOTOMETRY

AES/FEP AAS
 No need for light source  Make use of light source
 Measure the amount of light emitted by excited atom  Measure the amount of light absorbed by ground state atom
 Absorption intensity is greatly influenced by temperature variation  Absorption intensity does not depend upon temperature
 Beer’s law is not obeyed  Beer’s law is obeyed over a wide range of concentration
 Analyte determination: Na, K, Li  Analyte determination: Ca, Mg, Co, Pb, Hg, Zn, Cr

CHROMATOGRAPHY Planar
 Refers to the ground of techniques used to separate 1. Paper Chromatography
complex mixtures into their components on the basis  The stationary phase is a very uniform absorbent
of different physical interaction between individual paper and the mobile phase is a suitable liquid
compounds and stationary phase of the system solvent or mixture of solvents.
 Used for separation of sugar and amino acids
Components 2. Thin-Layer Chromatography
1. Mobile phase – can be gas or liquid, which carries  A variant of column chromatography
the complex mixtures (sample
2. Stationary phase – can be solid or liquid supported COMPONENTS
on a solid, through which the mobile phase flows picture
3. Column – holding the stationary phase 1. Thin, uniform layer of sorbent coated on a
4. Eluate – separated components piece of glass (metal or rigid plastic) backing
plate
2 Forms of Chromatography  Most commonly used sorbent are as follows:
1. Planar – Alumina
2. Column – Silica gel

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 28
– Cellulose Column
– Cross-linked dextran
2. Solvent mixture – mobile phase HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
 Most commonly used solvent:  Once called High Pressure Liquid Chromatography
– Acetic acid which refers to the high pressure of 3000 psi or more
– Water-miscible organic solvents commonly required for proper operation
3. Glass tank with cover  A separation technique that permits rapid
fractionation and identification of molecules without
PRINCIPLE the destructive effects seen with gas chromatography.
 Each sample to be analyzed is applied as a spot near  Terms:
one edge of the plate.  Mobile Phase – to the solvent being continuously
 The plate is then place in a closed chamber containing applied to the column, or stationary phase
an appropriate solvent mixture (mobile phase).  Stationary Phase – the solid support contained
 The solvent migrates up the thin layer by capillary within the column
action, dissolving and carrying sample molecules.
 After the solvent reaches a predetermined height, the Principle
plate is removed.  The sample solution is injected into the mobile phase
 Spots may be identified by their color if spots are of the assay through the injector port.
colored.  As the sample solution flows with the mobile phase
 If not, they can be dried under UV light or by spraying through the stationary phase, the components of that
with color-producing agents. solution will migrate according to the non-covalent
 Each sample component Retention factor (Rf) is interactions of the compounds with the stationary
compared with the Rf of the standards. phase.
𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟  The chemical interactions of the stationary phase and
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑙𝑒𝑎𝑑𝑖𝑛𝑔 𝑒𝑑𝑔𝑒 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑
=
𝑡𝑜𝑡𝑎𝑙 𝑑𝑖𝑠𝑡𝑎𝑛𝑡 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑
the sample with the mobile phase, determines the
picture degree of migration and separation of the
 For example, if the sample component (a) travelled components contained in the sample.
1.7 cm from the base line while the solvent (b) had
travelled 5.0 cm, then the Rf value for the red dye is: Components
0.34 picture
1. Sample injectors – used to introduce the sample

ADVANTAGES via the injection port

 Simple and rapid 2. Pump/motor

 One of the most versatile methods  Forces the mobile phase through the column
 Maintains the constant volume or constant pressure

DISADVANTAGES flow
 In drug identification, it lacks reproducibility from one  Most widely used pump today is the mechanical

chromatography to the next. reciprocating pump

 Slight differences in the concentration of solvents, the 3. Columns – contains packing appropriate for

degree of equilibration with vapor phase in the particular separation

separation chamber and the term render the Rf value a. Guard Columns – protect the analytical column

are somewhat variable from one analysis to another. from contamination and damage
b. Normal-Phase Columns

USES  Column is filled with tiny silica particles (polar

 Semiquantitative screening test for substance abuse stationary phase), and the solvent is non-polar
because of simplicity and rapidity. (hexane, for example)
 Separation and identification of urine sugars, amino  Polar compounds in the mixture being passed

acids, drugs and other groups of compounds through the column will stick longer to the polar
 Often used to monitor the progress of organic silica than non-polar compounds will. The non-
reactions and to check the purity of products polar ones will therefore pass more quickly
through the column.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 29
c. Reversed-Phase Columns concentration of the compounds that produced the
 Has polar mobile phase (e.g., a mixture of water peaks.
and an alcohol such as methanol) and non-polar
stationary phase (such as hydrocarbon on the Uses
silica of the column)  Separation, identification, purification, quantification
 In this case, there will be a strong attraction of various compounds.
between the polar solvent and polar molecules in  Analysis of high-molecular-weight components such
the mixture being passed through the column. as proteins and peptide
There won’t be as much attraction between the
hydrocarbon chains attached to the silica (the Advantages
stationary phase) and the polar molecules in the  Efficient than the “conventional” chromatography
solution. Polar molecules in the mixture will  High selectivity and high sensitivity
spend most of their time moving with the  Widely applicable
solvent.  Only small sample required
 Non-polar compounds in the mixture will tend to  Nondestructive to sample fragile at high temperatures
form attractions with the hydrocarbon groups  Readily adapted to quantitative analysis
because of van der waals dispersion forces. They  Can accommodate nonvolatile and thermally unstable
therefore spend less time in solution in the compounds
solvent and this will slow them down on their  Generally applicable to inorganic ions
way through the column.  Easy automation of instrument operation and data
 That means that now it is the polar molecules analysis
that will travel through the column more quickly.  HPLC columns can be reused without repacking or
 Reversed phase HPLC is the most commonly used regeneration
form of HPLC.
4. Detector – monitors that eluate as it leaves the Disadvantages
column and ideally produce an electronic signal  Expensive equipment
proportional to the concentration of each separated  Give slower result than Gas Chromatography
component
5. Retention time GAS CHROMOTOGRAPHY
 It refers to the time taken for a particular  Terms:
compound to travel through the column to the  Stationary Phase
detector. This time is measured from the time at  May be a solid or a nonvolatile liquid coated on a
which the sample is injected to the point at which solid contained in a coiled column
the display shows a maximum peak height for that  Must be liquid at the elevated temperatures (up
compound. to 4000C) of the column, nonvolatile &
 Different compounds have different retention nonreactive with the samples or solvents moving
times. For a particular compound, the retention through the column
time will vary depending on:  Mobile Phase – inert carrier gas, moves and
 Pressure used (because that affects the flow rate pushed the analyte towards detector
of the solvent)
 Nature of the stationary phase Principle
 Exact composition of the solvent  The sample mixture is introduced into a heated
 Temperature of the column injector, carrier through a separating column by an
6. Recorders inert gas, and interact with stationary phase on the
 Record detector signal versus the time the mobile support material.
phase passed through the instrument  The compounds separate from another, come off the
 The graph is called a chromatogram support material, and are detected as a series of
 Retention time is used to identify compounds when peaks on a recorder when components leave the
compared with standard retention times run under column.
identical conditions. Peak is proportional to the

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 30
Components  The process where a substance divides itself
picture between two immiscible solvents because it is
1. Carrier gas more soluble in one than the other is known as
 Carries the vaporized sample through the column partition.
towards the detector 7. Retention time
 Has to be inert and can be nitrogen, helium or  Retention time is the time from sample injection
argon into the column to the maximum peak.
 Volatility and solubility in the liquid phase affect  The area under a peak is related to the amount of
the rate of flow of sample molecules through the gc that component present in the mixture.
column  Different compounds have different retention
2. Gas flow regulator – the mobile phase or carrier times. For a particular compound, the retention
gas flows through the instrument from a pressurized time will vary depending on:
tank a. Boiling point of the compound – high boiling
3. Sample – samples may be pure compounds, point means a long retention time
however, they are often prepared as dilute solutions b. Solubility in the liquid phase – high solubility in
due to the sensitivity of the detection methods the liquid phase means a high retention time
4. Column oven c. Temperature of the column – high column
 The injector is contained in an oven whose temperature shortens retention times for
temperature can be controlled. everything in the column
 It is hot enough so that all the sample boils and is 8. Detector
carried into the column as a gas by the helium (or  Only thermal conductivity (TC) and flame ionization
other carrier gas). detectors are discussed here because they are the
5. Column most stable
 Columns can be short, large diameter packed  TC detectors contain wires (filaments) that change
column or long, very small diameter capillary electrical resistance with change in temperature.
columns. Each has its own use and associated Helium, which has a high thermal conductivity, is
advantages and disadvantages. usually the carrier gas
 Liquid layer is coated on the walls of the column;  Flame ionization detectors are widely used in the
solid support coated with a liquid stationary phase clinical laboratory. They are more sensitive than TC
may in turn be coated on column walls detectors.
 Liquid stationary phase must be nonvolatile at the
temperatures used, must be thermally stable, and Advantages
must not react chemically with the solutes to be  Only small amount of analyte is required
separated  Its combination with ms is very sensitive and specific
6. Separation in drug screening and steroid identification
 One of three things might happen to a particular
molecule in the mixture injected into the column of Disadvantages
which none is necessarily permanent:  Any sample studied by this system had to be either
 It may condense on the stationary phase. volatile or capable of being converted to a volatile
 It may dissolve in the liquid on the surface of the material for analysis
stationary phase.  Best worked only with low molecular-weight
 It may remain in the gas phase. components
 A compound with a boiling point higher than the
temperature of the column will obviously tend to Uses
condense at the start of the column.  Used to separate, identity and quantitate mixtures of
 Similarly, some molecules may dissolve in the compounds that are volatile or can be made volatile
liquid stationary phase. Some compounds will be  Used in toxicology laboratories to screen and
more soluble in the liquid than others. The more quantitate drugs
soluble ones will spend more of their time  Used to measure steroids, fatty acids, alcohols, blood
absorbed into the stationary phase; the less soluble gases, anesthetics in blood, intermediates of
ones will spend more of their time in the gas.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 31
metabolism, vitamins, pesticide residues, and various  Gas Chromatography -Mass Spectrophotometry
amines including serotonin, histamine and tryptamine (GC-MS) – gold standard for drug testing.

MASS SPECTROMETRY ELECTROPHORESIS

 A powerful analytical technique used to quantify  The migration of charged solutes or particles in an
known materials, to identify unknown compounds electrical field
within a sample, and to elucidate the structure and  It separates proteins on the basis of their electrical
chemical properties of different molecules (molecular charge densities
weight). ▪ The basic principle of mass spectrometry
(MS) is to generate ions from either inorganic or Fundamental of Electrophoresis
organic compounds by any suitable method, to
separate these ions by their mass-to-charge ratio MOVEMENT OF CHARGED PARTICLES
(m/z) and to detect them qualitatively and  The positively charged particles move toward the
quantitatively by their respective m/z and abundance. positive electrode
picture  The negatively charged particles move toward the
 Atoms can be deflected by magnetic fields – provided positive electrode
the atom is first turned into an ion. Electrically  An amino acid as its pI will not migrate in either
charged particles are affected by a magnetic field direction.
although electrically neutral ones aren’t.  Factors that affect the rate of migration:
 Net charge of the particle
An Outline of What Happens in a Mass  Size and shape of the molecule
Spectrometer  Strength of the electric field
1. Step 1: Ionization  Chemical and physical properties of the supporting
 Mass spectrometers always work with positive medium
ions.  Temperature of the operation
 The atom is ionized by knocking one or more COMPONENTS OF ELECTROPHORESIS SYSTEM
electrons off to give a positive ion. picture
picture 1. Power supply
2. Stage 2: Acceleration  Either supplies constant current or constant voltage
 The positive ions are repelled away from the very  The migration rate can be kept constant by using a
positive ionization chamber and pass through three power supply with constant current.
slits, the final one of which is at 0 volts. The middle 2. Buffer
slit carries some intermediate voltage. All the ions  Two properties of buffer that affect the charge of
are accelerated into a finely focus beam. ampholytes
picture  An ampholyte is a molecule whose net charge can
3. Stage 3: Deflection be either positive or negative.
 The ions are then deflected by a magnetic field  pH
according to their masses. The lighter they are, the  If the buffer is more acidic than the isoelectric
more they are deflected. point (pH of a solution at which the net charge
 The amount of deflection also depends on the of a protein becomes zero) of the ampholyte,
number of positive charges on the ion – in other the ampholyte binds H+ ions, becomes
words, on how many electrons were knocked off in positively charged, and migrates toward the
the first stage. The more the ion is charged, the cathode.
more it gets deflected.  If the buffer is more basic than the pI, the
picture ampholyte loses H+ ions, becomes negatively
4. Stage 4: Detection charged, and migrates toward the anode.
 The beam of ions passing through the machine is  A particle without a net charge will not
detected electrically. migrate, remaining at the point of application.
picture  Ionic strength
 Note: Before a compound can be detected and  During electrophoresis, ions cluster around
quantified by MS, it must be separated by GC. migrating particles. The higher the ionic
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 32
concentration, the higher the size of the ionic 5. Sample
cloud and the lower the mobility of the  Serum is routinely diluted with buffer before
particle. electrophoresis
 The most widely used buffers are made of  Urine and csf are usually concentrated
monovalent ions because their ionic strength  Hemoglobin hemolysate is used without further
and molality are equal. concentration
3. Support medium
 Holds the sample and provides a path for the PROCEDURE
migration of the charged particles 1. The sample is soaked in hydrated support medium for
 May be a passive one, with little or no physical approximately 5 minutes.
interaction with the charged molecules other than 2. The support medium is put into the electrophoresis
support chamber, which was previously filled with the buffer
 In other systems, it plays an important role in the  Enough buffer must be added to the chamber to
separation process, either by serving as a sieve or maintain contact with the support.
by affecting the charge in the system 3. Electrophoresis is carried out by applying a constant
 The electrophoretic separation takes place on the voltage or constant current for a specific time.
support medium 4. The support is then removed and placed in a fixative
 After a sample is applied to the support material, or rapidly dried to prevent diffusion of the sample.
both ends of the material are placed in contact with 5. This is followed by staining the zone with appropriate
buffer solution to allow passage of the electric dye.
current  The uptake of dye by the sample is proportional to
 Kinds of support materials: sample concentration.
a. Cellulose Acetate – fractionation is due to 6. After excess dye is washed away, the supporting
molecular size medium may need to be placed in a clearing agent.
b. Agarose Gel – fractionation is due to electrical Otherwise, it is completely dried.
charge
c. Polyacrylamide Gel Electrophoresis DETECTION OF PROTEINS AFTER SEPARATION
(PAGE)  A key ingredient to the success of any electrophoretic
 Protein fraction is due to: technique is its ability to detect the separated
 Molecular size (major factor) – the larger proteins on the support medium
the molecule size, the slower its movement  Stains for visualization of fractions/bands:
through the gel (due to composition and  Amido black – all proteins
sieving effect of the gel)  Ponceau S – all proteins
 Molecular charge – the greater the  Oil Red O – lipids
molecular charge, the faster the protein  Sudan Black – lipids
moves  Fat Red 7B – lipids
 Yields more detailed patterns of proteins than  Coomassie Blue – CSF protein
agarose gel electrophoresis  Gold/silver stain – excellent for low-level detection
– PAGE separated serum proteins into 20 or of proteins (very sensitive)
more fractions rather than usual 5 fraction
separated by cellulose acetate or agarose. QUANTITATION OF PROTEIN FRACTIONS BY DENSITOMETRY
 It is widely used to study individual proteins  Measures the absorbance of stain – concentration of
(e.g. Isoenzymes) the dye and protein fraction
picture  It scans and quantitate electrophoretic pattern
4. Electrophoresis  Methods of choice for assessing the level of a given
 Component of the electrophoresis system where protein after electrophoresis and staining
the sample on the support medium is placed  Process: Most densitometric systems are based on
 Wiring of the chamber allows passage of electric colorimetry. The protein fractions are stained with a
current into one buffer chamber, where the electric material which produces visible bands of color on the
flows then goes across the support material into support medium. The strip is placed in a holder and is
the second buffer chamber slowly moved through a beam of light. As the light
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 33
interacts with the dye bound to the specific protein due to excess protein, an alternative mode of
fraction, a certain amount of light is absorbed, analysis such as ISE undiluted specimen can be
depending on the concentration of the protein and the employed to yield correct electrolyte activity.
amount of dye bound to it. Absorption of lights is
roughly proportional to concentration of dye (and to Amperometry
concentration of protein fraction). The readout from  Is the measurement of the current flow produced by
the system is a trace which relates the amount of light an oxidation reaction
absorption to the location of protein fractions (strip  Use: pO2, glucose, chloride, and peroxidase
chart). determinations

ELECTROCHEMISTRY Coulometry
 The measurement of current or voltage generated by  Is the measurement of the amount of electricity (in
the activity of a specific ion coulombs) at a fixed potential.
 Is an electrochemical titration in which the titrant is
Potentiometry electrochemically generated, and the endpoint id
 Is the measurement of electrical potential due to the detected by amperometry
activity of free ions (change in voltage indicates  It follows Faraday’s law
activity of each analyte)  Use: chloride test (CSF, serum and sweat)
 It is also the measurement of differences in voltage  Interference: bromide, cyanide and cysteine
(potential) at a constant current
 It follows the Nernst equation Voltammetry
 Concentration of ions in a solution can be calculated  The measurement of current after which a potential is
from the measured potential differences between the applied to an electrochemical cell
two electrodes.  It allows sample to be preconcentrated, thus utilizing
 Reference electrodes: calomel and silver-silver minimal analyte
chloride  Anodic stripping voltammetry – for lead and iron
 Use: pH and pCO2 tests testing
 Ion Selective Electrode (ISE)
– Is an electrochemical transducer capable of IMMUNOCHEMISTRY
responding to one given ion  Immunochemistry is an advanced area of
– It is very sensitive and selective for the ion it immunology. It deals with the chemical components
measures – it measures the activity of one ion and chemistry (chemical reactions) of immunological
much more than other ions present in the sample phenomena, that is of antibody and antigen.
– Its ionic selectivity depends on the  Immunochemical methods are processes utilizing the
membrane/barrier composition used highly specific affinity of an antibody for its antigen.
– ISE Membrane: It detects the distribution of a given protein or
 Glass aluminum silicate (sodium), valinomycin antigen in tissues or cells. The methods used for the
gel (potassium), organic liquid membrane ion immunochemical analysis are called Immunochemical
exchanges (calcium and lithium), gas and enzyme techniques; they are highly important in diagnostic
electrodes and clinical context, as now even normal cell with
– ISE analyzers measure the electrolyte dissolved in many proteins are altered in diseased state (in
the fluid phase of the sample in mmol/L of plasma cancer).
water
– ISE analyzers using undiluted samples are not CHEMILUMINESCENCE
subject to pseudohyponatremia caused by  It differs from fluorescence and phosphorescence in
hyperlipidemic samples that the emission of light is created from a chemical
– 2 types of ISE: or electrochemical reaction, and not from absorption
 Direct ISE (without sample dilution) of electromagnetic energy.
 Indirect ISE (with sample dilution)  Principle: The chemical reaction yields an
– Interference: protein coating the ISE membrane electrochemically excited compound that emits light
would cause a response error – if interference is as it returns to its ground state, or that transfers its
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 34
energy to another compound, which then produces – Applicable regulatory or certifying standards
emission  Procedure/policy
 Use: Immunoassays – Principle
 Photodetector: Photomultiplier tube – Testing personnel
 It is more sensitive than fluorescence – Specimen
 In this method, no excitation radiation is required, – Reagents, supplies, & equipment
and no monochromators are needed because the – Maintenance
chemiluminescence arises from one species – Power
 It involves the oxidation of an organic compound (e.g., – Calibration/calibration verification
dioxetane, luminol, acridinium ester) by an oxidant – Quality control
(hydrogen peroxide, hypochlorite, or oxygen). These – Patient testing procedure
oxidation reactions may occur in the presence of – Reference & therapeutic ranges
catalysts, such as enzymes, metal ions, and hemin. – Reporting results
 The excited product formed in the oxidation reaction – Data transfer
produce chemiluminescence on return to single state. – Limitations, notes
 A typical signal from a chemiluminescent compound – Proficiency testing
rises rapidly with time and reaches a maximum when – Quality improvement
reagent and analyte are completely mixed. – Troubleshooting
– Alternative method
SCINTILLATION COUNTING – References
 In scintillation counting, the sample is mixed with a  Training checklist
material that will fluoresce upon interaction with a – Start with checklist template.
particle emitted by radioactive decay. The scintillation – Must be clear & sufficiently detailed
counter quantifies the resulting flashes of light / light – Should document all operator training
pulses (called scintillations). The scintillations are – Use 2-copy system: 1 for employee’s file, 1 for POC
detected with the help of a photomultiplier tube that office.
gives rise to an equivalent electric pulse.  Recertification checklist
 Paper forms/logs
POINT-OF-CARE TESTING – Quality control log
 Those analytical patient-testing activities provided – Patient testing log
within the institution but performed outside the – Problem/corrective action log
physical facilities of the clinical laboratories – Quality improvement log

Validation POC APPLICATION

 Required to meet regulatory requirements and
ensures reliable test results POC Glucose
 Components of validation  Highest-volume POC test in most institutions; most
 Accuracy frequently used home POC
 Precision  Frequently used to monitor glucose level for diabetic
 Sensitivity & specificity patients
 Reportable range/linearity  Procedure
 Split-sample correlation vs. reference method 1. Small drop of blood, usually from capillary
 Methods and instruments should also be validated puncture, is applied to test strip.
2. Reaction occurs between blood & reagents in test
Implementation strip.
 Collect materials: 3. Meter measures reaction & converts it to
– Instrument manual(s) quantitative result.
– Package insert(s) for reagents & quality controls
– Materials safety data sheet (MSDS) POC Chemistries and Blood Gases
– Sample procedure & training materials from vendor  Measure potentiometric, amperometric,
– Other institution’s procedure for test conductometric changes via sensors
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 35
 Feature both non-disposable and disposable sensors Advantages of Automation
– Instruments with disposable sensors may be  Rapid results
configured for multi-specimen analysis.  Increase in the number of tests performed
– Some instruments use a single-sample disposable  Saves time and effort
cartridge that contains all of the system’s  Eliminate the need for more staff
components.  Economical
 Errors are reduced
POC Coagulation  Better precision and accuracy
 Most common: activated clotting time (ACT)  Minimize variation in results from one laboratory to
 Used for monitoring heparin therapy, to avoid another
bleeding or clotting
 Provides an in vitro, nonspecific measurement of History of Automated Analyzers
cellular & noncellular components of coagulation  First Automated Analyzer: “Autoanalyzer”
process  Introduced by Technicon in 1957
 A continuous-flow, single-channel, sequential batch
POC Hematology analyzer
 Institutions moving away from spun hematocrit test  Capable of providing a single test result on about
due to safety concerns & variation in test results 40 samples/hr
 Many now use system with disposable cuvets & an  Second Generation: Simultaneous Multiple Analyzer
analyzer.  Multiple channels working synchronously
– Cuvet cavity contains reagents deposited on inner  Produced 6–12 test results simultaneously at rate
walls. of 360–720 tests/hr
– Reagents hemolyze red cells when blood sample is  First Commercial Centrifugal Analyzer Introduced in
drawn into cavity by capillary action. 1970
– Released hemoglobin is converted to azide  Spin-off technology from NASA space research
methemoglobin.  Developed by Dr. Norman Anderson at Oak Ridge
– Cuvet is then placed in analyzer, where absorbance National Laboratory
is measured and hemoglobin level is calculated.  An alternative to continuous-flow technology
 Automatic Clinical Analyzer (ACA) Introduced by
POC Connectivity DuPont (now Siemens)
 Most significant recent development in POCT  First non-continuous flow, discrete analyzer
 Makes it possible to electronically document testing  First instrument to have random access capabilities
 Three components of connectivity:  Unique features: plastic test packs, positive patient
1. Device: Instrument itself can store data. identification, infrequent calibration
2. Data management: Instrument uploads data to  Thin Film Analysis Technology Introduced in 1976
workstation.  Kodak Ektachem Analyzer Produced in 1978
3. Interface: Data manager transmits test results to  First instrument to use microsample volumes and
information system. reagents on slides for dry chemistry analysis
 Newer systems manage results from multiple vendors  First instrument to incorporate computer
via a single integrated system. technology extensively into its design and use
 Discrete Analyzers Since 1980
AUTOMATION AND ROBOTICS  Ion-selective electrodes (ISEs), fiberoptics,
polychromatic analysis
Driving Forces toward More Automation and  Sophisticated computer hardware and software for
Robotics data handling
1. Human performances vary over the course of time  Astra analyzers (Beckman Coulter) and Hitachi
2. Repetitiveness of many laboratory tasks leads to analyzers (Roche Diagnostics)
boredom
3. To solve the problems of growing shortage of trained
lab personnel

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 36
THREE BASIC APPROACHES TO AUTOMATION Centrifugal Analyzer
 Uses the force generated by centrifugation to transfer
Continuous Flow Analyzer specimens and reagents and then contain the liquids
 All samples are carried through the same analysis in separate cuvettes for measurement at the
pathway. perimeter of a spinning rotor (1000rpm).
 All samples automatically pass from one step to  It uses acceleration and deceleration of the rotor to
another without waiting to bring the samples to the transfer the reagents and sample from one chamber
same stage of completion. to another. Centrifugal force (rotor) is also utilized for
 The reactions are not necessarily carried to equilibrium mixing of sample ang reagents.
since samples and standards are treated exactly alike.
 Liquids (reagents, diluents, and samples) are pumped ADVANTAGES
through a system of continuous tubing.  Batch analysis
 Samples are introduced in a sequential manner,  Maybe used for labs with high workload
following each other through the same network.
 A series of air bubbles at regular intervals serve as Discrete Sampling Analyzer
separating and cleaning media.  Each sample reaction is handled in a separate
picture compartment and does not come into contact with
another sample.
FEATURES  The samples and standards are handled on a batch
 Use of plastic tubes of different diameters and a basis and must be brought before proceeding to the
peristaltic pump for continuous pumping of samples next procedure.
and reagents. This maneuver replaces the pipetting  All reactions must be carried out until equilibrium is
steps in the manual procedures. reached.
 Introduction of air bubbles
 To separate the sample and reagent streams into FEATURES
segments  Employs a variety of syringe pipettes to aspirate and
 To separate one sample from the next dispense sample and reagents
 For continuous scrubbing of tubing  Most popular and versatile analyzers
 Prevents cross contamination or carry over by the  Can run multiple tests on one sample at a time or
previous specimen multiple samples one test at a time
 Removal of proteins by dialysis  Have random access capability that allows STAT
 Flow-through cuvettes in interference filter samples to be easily accessed
photometer, using a fixed reference light path.
 Recorded read-out DESIGNS OF AUTOMATED ANALYZER
 Modular design permitting interchanging of major  Batch testing – all samples are loaded at the same
parts. time and a single test is conducted on each sample
 Parallel testing – more than one test is analyzed
ADVANTAGES concurrently on a given specimen
 Resolves the major consideration of uniformity in  Sequential testing – multiple test analyzed one after
performance of tests because each sample follows the another on a given specimen
same reaction path  Random access testing – any test can be performed
 Assists the laboratory that needs to run many on any sample in any sequence
samples requiring the same procedure  Closed reagent system – operator can only use
manufacturer’s reagent
DISADVANTAGES  Open reagent system – a system other than
 Significant carryover problems manufacturer’s reagent can be used
 Wasteful use of continuously flowing reagents

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 37
GENERAL FUNCTION OF AUTOMATED ANALYZERS

IDENTIFICATION AND PREPARATION

Sample identification This is usually done by reading the bar code. This information can be entered manually.
Determine test(s) to perform The LIS communicate to the analyzer which test(s) have been ordered.
CHEMICAL REACTION
Reagent systems and delivery One or more reagents can be dispensed into the reaction cuvet.
Specimen measurement and delivery A small aliquot of the sample is introduced into the reaction cuvet.
Chemical reaction phase The sample and reagents are mixed and incubated.
DATA COLLECTION AND ANALYSIS
Measurement phase Optical readings may be initiated before or after all reagents have been added.
Signal processing and data handling The analyte concentration is estimated from a calibration curve that is stored in the analyzer.
Send result(s) to LIS The analyzer communicates results for the ordered tests to the LIS

TOTAL LABORATORY AUTOMATION  Walk-away capability – the ability of the operator

picture to program the instrument to perform other tasks
while the instrument processes the tests
Terminologies  Shelf life – the term used to define reagent stability
 Test repertoire – number of tests that can be before use.
performed on instrument.  Carry over – this occurs when a previous sample to
 Selective – only performs requested test. have a higher or lower result. This occurs in systems
 Dwell time – minimum time required to obtain that reuse cuvettes that are insufficiently washed
result after the initial sampling of the specimen. after each testing cycle.
 Turnaround Time – time interval from the time of  Maintenance time – the time the analyzer is not in
submission of a process to the time of the completion use.
of the process  Downtime – the time that an analyzer is
 Throughput – maximum number of samples or tests unavailable for testing because a periodic
that can be processed in an hour; the measure of maintenance or reasons pertaining to
speed of an analytical system. troubleshooting.
 Cost – labor maintenance, reagents, calibration,  Bar code – a means of providing positive sample
quality control, consumables and capital. identification.
 Test menu – a list of the analytes or tests that a  Linearity – the range over which patient results can
laboratory would to be able to provide for patient be reported without manipulating the sample (i.e.
testing. using a dilution). The linear range is generally defined
 Workload – the number of test results that are by the values of the highest and lowest calibrations
generated by a laboratory during a given time period. available for a particular instrument.

UNIT 6: CARBOHYDRATES
CARBOHYDRATE  Building blocks for many processes of metabolism
 Most abundant bio-organic substance on planet  The name carbohydrate literally means “hydrates of
 Major food source and energy supply of the body carbon”
 Stored primarily as liver and muscles glycogen  The term “sugar” is applied to carbohydrates soluble
 “Central ingredient for life” in water and sweet to taste

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 38
CLASSIFICATION OF CARBOHYDRATES

ALDOSE
depending in carboxyl group present
KETOSE

TRIOSE
MONOSACCHARIDE
TETROSE

depending on number of carbons present *PENTOSE GLUCOSE

reducing *HEXOSE FRUCTOSE

DISACCHARIDE
CARBOHYDRATE non-reducing HEPTOSE GALACTOSE
OLIGOSACCHARIDE TRISACCHARIDE

TETRASACCHARIDE

GLYCOGEN

POLYSACCHARIDE STARCH

CELLULOSE

Monosaccharides Oligosaccharides
1. Glucose 1. Sucrose
 Grape sugar, dextrose, blood sugar  Glucose + fructose
 Most abundant monosaccharide in nature and most  “Common table sugar”
important from human nutritional standpoint  Best known of the disaccharides
 Central, pivotal point of carbohydrate metabolism  Obtained from beets, sugar cane
 Use to assess total CHO use by the body when we  Provides a major portion of CHO intake for many
measure blood glucose level individual
2. Fructose  Most common Non-reducing sugar
 Levulose and fruit sugar 2. Lactose
 Sweetest-tasting of all sugar; sometimes used as a  Glucose + Galactose
dietary sugar because less is needed for the same  “Milk sugar”
amount of sweetness  Found in dairy products such as milk and cheese
 Formed from the glucose and from the breakdown 3. Maltose
of the sucrose  Glucose + Glucose
 Intermediate in the utilization of the  Malt sugar
monosaccharides  Good sources are cereals, wheat and malt products
3. Galactose  Produced whenever polysaccharide starch is
 It is synthesized from glucose in mammary glands hydrolyzed
for used in lactose
 Is a chemical markers that distinguish various Polysaccharides
types of blood – A, B, AB and O 1. Starch
 “Brain sugar” – it is a component of glycoproteins  Is a homopolysaccharide containing only glucose
found in nerve tissues monosaccharide units
 Must be converted to glucose before it can be used  Amylose (unbranched glucose polymer that
by the body accounts for 20%) and Amylopectin (branched
 Less significant from a metabolic point of view glucose polymer that accounts for 80%)
 Galactosemia – have difficulty in carrying out  Primary carbohydrates in the diet and is found in
this transformation most plant
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 39
2. Glycogen (animal starch)  The salivary glands in the oral cavity secrete saliva
 Polysaccharide containing only glucose units and it that coats the food particles.
has similar structure with that of amylopectin  Saliva contains the enzyme, salivary amylase
 Liver cells and muscle cells are the storage sites for (ptyalin).
glycogen in humans  This enzyme breaks the bonds between the
3. Cellulose monomeric sugar units of disaccharides,
 Structural component of plant cell wall with similar oligosaccharides, and starches.
structure in amylose  The salivary amylase breaks down amylose and
 Another polysaccharide in the plants amylopectin into smaller chains of glucose, called
 Not digested by humans, water insoluble, but it dextrins and maltose.
does provide the bulk for proper intestinal  Only 5% of starch is broken down by salivary
functioning (↑ concentrations of fiber) amylase due to limited exposure (this is a good thing
as more glucose in the mouth would lead to more
DIETARY INTAKE OF CARBOHYDRATES tooth decay).
 The only 2 types of CHO which contribute to human  When carbohydrates reach the stomach no further
nutrition: chemical breakdown occurs because the amylase
1. Starch enzyme does not function in the acidic conditions of
2. Disaccharides the stomach.
 But the mechanical breakdown is ongoing—the
CARBOHYDRATE DIGESTION AND METABOLISM strong peristaltic contractions of the stomach mix the
carbohydrates into the more uniform mixture of
Digestion and Absorption of Carbohydrates chyme.
 The goal of carbohydrate digestion is to break down  The chyme is gradually expelled into the upper part of
all disaccharides and complex carbohydrates into the small intestine.
monosaccharides for absorption, although not all are  Upon entry of the chyme into the small intestine, the
completely absorbed in the small intestine (e.g., pancreas releases pancreatic juice through a duct.
fiber).  This pancreatic juice contains the enzyme, pancreatic
 The mechanical and chemical digestion of amylase (amylopsin), which starts again the
carbohydrates begins in the mouth (Figure 6.1). breakdown of dextrins into shorter and shorter
 Chewing, also known as mastication, crumbles the carbohydrate chains (oligosaccharides &
carbohydrate foods into smaller and smaller pieces. disaccharides).

STARCH
salivary amylase
DEXTRINS and MALTOSE
pancreatic amylase
DISACCHARIDE

GLYCOLYSIS
GLUCOSE, GALACTOSE, and
FRUCTOSE GLYCOGENESIS

GLYCOGENOLYSIS

GLUCONEOGENESIS

LIPOLYSIS/GENESIS

Figure 6.1 Carbohydrate digestion

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 40
 Additionally, enzymes are secreted by the intestinal  The cells in the small intestine have membranes that
cells that line the villi (Figure 6.2). contain many transport proteins in order to get the
 These enzymes are sucrase, maltase, and lactase. monosaccharides and other nutrients into the blood
 Sucrase breaks sucrose into glucose and fructose where they can be distributed to the rest of the body.
molecules.  The first organ to receive glucose, fructose, and
 Lactase breaks the bond between galactose and galactose is the liver.
glucose.  The liver takes them up and converts galactose to
 Maltase breaks the bond between the two glucose glucose, breaks fructose into even smaller carbon-
units of maltose. containing units, and either stores glucose as
PICTURE glycogen or exports it back to the blood.
 How much glucose the liver exports to the blood is
 Once carbohydrates are chemically broken down into under hormonal control and you will soon discover
single sugar units they are then transported into the that even the glucose itself regulates its
inside of intestinal cells (Figure 6.3). concentrations in the blood.

Starch, Disaccharides, and other small CHO polymers

Monosaccharide Intestinal wall Bloodstream

Metabolism of Carbohydrates HEXOSE MONOPHOSPHATE SHUNT/PENTOSE PHOSPHATE

PATHWAY
GLYCOLYSIS OR EMBDEN-MEYERHOF PATHWAY  An alternative metabolic pathway by which glucose is
 The metabolic pathway by which glucose (C6 molecule) used to produce NADPH needed in lipid synthesis and
is converted into two molecules of pyruvate (C3 production of ribose-5-phosphate, a pentose
molecule), chemical energy in the form of ATP is derivative needed for the synthesis of nucleic acids.
produced and NADH-reduced coenzymes are produced.
 The conversion of glucose to pyruvate is an oxidation
process in which no molecular oxygen is utilized
(anaerobic pathway).

Figure 6.5. Hexose Monophosphate Shunt/Pentose Phosphate

Pathway

GLYCOGENESIS
 The metabolic pathway by which glycogen is
synthesized from glucose-6-phosphate.

Figure 6.6. Glycogenesis Pathway

Figure 6.4. Glycolysis or Embden-Meyerhof Pathway
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 41
DEFINITION OF TERMS (Pathways of Glucose Metabolism)  Decreasing intestinal entry into the cell,
 Glycolysis – metabolism of glucose molecule to increasing gluconeogenesis, glycogenolysis and
pyruvate or lactate for production of energy lipolysis (primary lipolytic hormone)
 Gluconeogenesis – formation of glucose-6-phosphate 5. Growth Hormone (somatotropin)
from noncarbohydrate sources  Decreasing entry of glucose into the cells,
 Glycogenolysis – breakdown of glycogen to glucose increasing glycogenolysis
for use as energy 6. Adrenocorticotropic Hormone (ACTH) – stimulates the
 Glycogenesis – conversion of glucose to glycogen for adrenal cortex to release cortisol
storage  Conversion of liver glycogen to glucose and
 Lipogenesis – conversion of carbohydrates to fatty promotes gluconeogenesis
acids 7. Thyroxin – increasing glycogenolysis,
 Lipolysis – decomposition of fat gluconeogenesis and intestinal absorption of glucose
8. Somatostatin – produced by δ-cells of the Islets of
HORMONAL REGULATION OF CARBOHYDRATES Langerhans
1. Insulin  Inhibition of insulin, glucagon, growth hormone and
 A 51 amino acid protein. other endocrine hormones
 The primary hormone responsible for the entry of
glucose into the cell (decreasing glucose in the CLINICAL SIGNIFICANCE OF CARBOHYDRATES
blood circulation).
 It is synthesized from a precursor known as pro- Inborn Errors of Carbohydrate Metabolism
insulin [single polypeptide containing alpha, beta  Inborn errors of carbohydrate metabolism are inborn
and C-peptide chain]. error of metabolism that affect the catabolism and
 C-peptide chain is removed by peptidases enzymes anabolism of carbohydrates.
to convert into active form insulin.  Carbohydrates account for a major portion of the
 It is synthesized by the ß-cells of islet of human diet. These carbohydrates are composed of
Langerhans in the pancreas. three principal monosaccharides: glucose, fructose
 When these cells detect an increase in body and galactose; in addition glycogen is the storage
glucose, they release insulin. form of carbohydrates in humans. The failure to
 Increases glycogenesis and glycolysis effectively use these molecules accounts for the
 Increases lipogenesis majority of the inborn errors of human carbohydrates
 Decreases glycogenolysis metabolism.
2. Glucagon 1. Galactosemia – congenital deficiency of one of
 A 29 amino acid hormone. three enzymes involved in galactose metabolism
 The primary hormone responsible for increasing 2. Essential Fructosuria – fructokinase deficiency
glucose levels (primary hyperglycemic hormone). 3. Hereditary Fructose Intolerance – defect in
 It is synthesized by the alpha cells of islet of Fructose 1,6-biphosphate aldolase B activity
Langerhans. 4. Fructose 1,6-biphosphate Deficiency – a defect in
 When these cells detect a decrease in body glucose, Fructose 1,6-biphosphate results in failure of
they release glucagon. hepatic glucose generation
 Increases glycogenolysis 5. Glycogen Storage Diseases – deficiency of enzymes
 Increases gluconeogenesis (primary involved in the metabolism of glycogen
gluconeogenetic hormone)
3. Epinephrine Hypoglycemia
 Produced by adrenal gland  Results from an imbalance between glucose utilization
 Increases plasma glucose by inhibiting insulin and production.
secretion, increasing glycogenolysis and  A diagnosis of hypoglycemia should not be made
promoting lipolysis unless a patient meets the criteria of Whipple’s Triad-
 Released during times of stress Low blood glucose concentration, typical signs and
4. Glucocorticoids symptoms alleviated by glucose administration
 Corstisol – released by the adrenal cortex  Low Blood sugar <50 mg/dL

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 42
 At about 50-55 mg/dL (2.8-3.0 mmol/L) observable KETOACIDOSIS
symptoms of hypoglycemia appear.  Dangerously high levels of ketone in the circulation; a
 Diagnostic test: 5-hour glucose tolerance test life-threatening condition
 Symptoms of Hypoglycemia:  Body does not make enough insulin due to an existing
1. Neurogenic – tremors, palpitations, anxiety, physiological condition (Type 1 DM – “Diabetic
diaphoresis ketoacidosis”)
2. Neuroglycopenic – dizziness, tingling, blurred  Occurs when the body starts breaking down fat at a
vision, confusion, behavioral changes rate that is too much fast, leading to raised levels of
ketones in the body, which causes the blood to
CLASSIFICATION OF HYPOGLYCEMIA become acidic.
1. Drug administration – insulin, alcohol, salicylates,  Without treatment, you could fall into a coma or die
sulfonamides, pentamidine
2. Critical illnesses – hepatic failure, sepsis, renal DIABETES MELLITUS
failure, cardiac failure, malnutrition  Diabetes mellitus is a group of diseases in which
3. Hormonal deficiency – epinephrine, glucagon, blood glucose levels are elevated due to deficiency in
cortisol, growth hormone insulin secretion or due to abnormal insulin action
4. Endogenous hyperinsulinism - pancreatic beta cell  Glucosuria occurs when the plasma glucose level
disorders (e.g. insulinoma) exceeds 180 mg/dL
5. Autoimmune hypoglycemia – insulin antibodies  The terms insulin-dependent diabetes mellitus (IDDM)
6. Non-beta cell tumors – leukemia, hepatoma, and noninsulin-dependent diabetes mellitus (NIDDM)
pheochromocytoma, lymphoma are obsolete. Since many individuals with type 2 DM
7. Hypoglycemia of infancy and childhood – eventually require insulin treatment for control of
galactosemia, GSD, Reye’s syndrome glycemia.
8. Alimentary (reactive) hypoglycemia – post-gastric  Age is not a criterion in the classification system.
surgery Although type 1 DM most commonly develops before
9. Idiopathic (functional) postprandial hypoglycemia the age of 30, an autoimmune beta cell destructive
process can develop at any age. It is estimated that
Hyperglycemia between 5 and 10% of individuals who develop DM
 An increase in blood glucose concentration after age 30 have type 1 DM. Likewise, type 2 DM
 It is toxic to beta cell function and impairs insulin more typically develops with increasing age but is
secretion now being diagnosed more frequently in children and
 Causes: stress, severe infection, dehydration or young adults, particularly in obese adolescents.
pregnancy, pancreatectomy, hemochromatosis, insulin  DM is classified on the basis of the pathogenic process
deficiency or abnormal insulin receptor that leads to hyperglycemia, as opposed to earlier
 Laboratory Findings: criteria such as age of onset or type of therapy
1. Increase glucose in plasma and urine  The two broad categories of DM are designated type 1
2. Increase urine specific gravity and type 2.
3. Ketones in serum and urine (ketosis/ketoacidosis)  Etiological Classification of Diabetes Mellitus
4. Decreased blood and urine pH 1. Type 1 Diabetes
5. Electrolyte imbalance (decreased Na and HCO3, 2. Type 2 Diabetes
increased K) 3. Gestational Diabetes Mellitus
4. Other Specific Types of Diabetes
KETOSIS
 A metabolic state characterized by raised levels of Type 1 Diabetes Mellitus
ketone bodies in the body tissues
 Caused by excessive synthesis of Acetyl CoA ETIOLOGY OF TYPE 1 DM
 Frequent finding in patients with severe, uncontrolled  This form of diabetes is immune-mediated in over
DM 90% of cases and idiopathic in less than 10%.
 Can be reversed by insulin administration

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 43
 Immune-mediated hyperglycemia. This results in a loss of glucose as
– The rate of pancreatic B cell destruction is quite well as free water and electrolytes in the urine.
variable, being rapid in some individuals and slow  Thirst (Polydipsia) – consequence of the
in others. hyperosmolar state, as is blurred vision, which often
– Approximately one-third of the disease develops as the lenses are exposed to hyperosmolar
susceptibility in immune mediated type is due to fluids.
genes and two-thirds to environmental factors.  Weight loss – despite normal or increased appetite
 Idiopathic (polyphagia) is a common feature of type 1 when it
– Less than 10% of subjects have no evidence of develops sub acutely; the weight loss is initially due
pancreatic B cell autoimmunity to explain their to depletion of water, glycogen, and triglycerides;
insulinopenia and ketoacidosis. thereafter, reduced muscle mass occurs as amino
– This subgroup has been classified as "idiopathic acids are diverted to form glucose and ketone bodies.
type 1 diabetes" and designated as "type 1B”.  Lowered plasma volume – produces symptoms of
– Although only a minority of patients with type 1 postural hypotension; total body potassium loss and
diabetes fall into this group, most of these are of the general catabolism of muscle protein contribute to
Asian or African origin. the weakness.
 Paresthesia – abnormal dermal sensation (e.g., a
PATHOPHYSIOLOGY OF TYPE 1 DM tingling, prickling, chilling, burning, or numb sensation
 Auto Immune Destruction on the skin) with no apparent physical cause.
 Pathologically, the pancreatic islets are infiltrated  Ketoacidosis
with lymphocytes (in a process termed insulitis).
 After all beta cells are destroyed, the inflammatory Type 2 Diabetes Mellitus
process abates, the islets become atrophic.
 The autoimmune destruction of pancreatic β-cells ETIOLOGY OF TYPE 2 DM
leads to a deficiency of insulin secretion.  Type 2 Diabetes Mellitus is a disorder that is
 It is this loss of insulin secretion that leads to the characterized by high blood glucose in the context of
metabolic derangements associated with Type 1 insulin resistance and relative insulin deficiency.
DM.  Circulating endogenous insulin is sufficient to prevent
 In addition to the loss of insulin secretion, the ketoacidosis but is inadequate to prevent
function of pancreatic α-cells is also abnormal. hyperglycemia in the face of increased needs owing to
 There is excessive secretion of glucagon in Type 1 tissue insensitivity (insulin resistance).
DM patients.  Genetic and environmental factors combine to cause
 Normally, hyperglycemia leads to reduced glucagon both the insulin resistance and the beta cell loss.
secretion.  The disease is polygenic and multifactorial since in
 However, in patients with Type 1 DM, glucagon addition to genetic susceptibility, environmental
secretion is not suppressed by hyperglycemia. factors (such as obesity, nutrition, and physical
 The resultant inappropriately elevated glucagon activity) modulate the phenotype.
levels exacerbate the metabolic defects due to  The mechanisms by which these genetic alterations
insulin deficiency. increase the susceptibility to type 2 diabetes are not
clear.
GENETIC CONSIDERATION
 Children of diabetic parents are at increased lifetime PATHOPHYSIOLOGY OF TYPE 2 DM
risk for developing type 1 diabetes.  Type 2 DM is characterized by impaired insulin
 A child whose mother has type 1 diabetes has a 3% secretion, insulin resistance, excessive hepatic
risk of developing the disease and a 6% risk if the glucose production, and abnormal fat metabolism.
child’s father has it.  In the early stages of the disorder, glucose tolerance
remains near-normal, despite insulin resistance,
CLINICAL MANIFESTATIONS OF TYPE 1 DM because the pancreatic beta cells compensate by
 Polyuria – Increased urination is a consequence of increasing insulin output.
osmotic diuresis secondary to sustained  As insulin resistance and compensatory
hyperinsulinemia progress, the pancreatic islets in
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 44
certain individuals are unable to sustain the  Habitual physical inactivity
hyperinsulinemia state.  Race/ethnicity (e.g., African American, Latino, Native
 IGT, characterized by elevations in postprandial American, Asian American, Pacific Islander)
glucose, then develops.  Previously identified IFG or IGT
 A further decline in insulin secretion and an increase  History of GDM or delivery of baby >4 kg (>9 lb)
in hepatic glucose production lead to overt diabetes  Hypertension (blood pressure >140/90 mmHg)
with fasting hyperglycemia. Ultimately, beta cell  HDL cholesterol level <35 mg/dL (0.90 mmol/L)
failure may ensue. and/or a triglyceride level >250 mg/dL (2.82 mmol/L)
Insert pic  Polycystic ovary syndrome or acanthosis nigricans
 Insulin Resistance  History of vascular disease
– Insulin resistance is a state in which a given
concentration of insulin produces a less-than- CLINICAL MANIFESTATIONS OF TYPE 2 DM
expected biological effect  While many patients with type 2 diabetes present
– Causes of Insulin Resistance: with increased urination and thirst, many others have
1. Pre-receptor an insidious onset of hyperglycemia and are
 Abnormal insulin (mutations) asymptomatic initially. This is particularly true in
 Anti-insulin antibodies obese patients, whose diabetes may be detected only
2. Receptor after Glycosuria or hyperglycemia is noted during
 Decreased number of receptors (mainly, routine laboratory studies.
failure to activate tyrosine kinase)  Occasionally, type 2 patients may present with
 Reduced binding of insulin evidence of neuropathic or cardiovascular
 Insulin receptor mutations complications
 Insulin receptor – blocking antibodies  Chronic skin infections are common. Generalized
3. Post-receptor pruritus and symptoms of vaginitis are frequently the
 Defective signal transduction initial complaints of women.
 Mutations of GLUT4  Diabetes should be suspected in women with chronic
4. Combinations of defects Candida vulvovaginitis as well as in those who have
 Obesity is associated mainly with post delivered large babies (> 9 lbs, or 4.1 kg) or have
receptor abnormality and is also associated had polyhydramnios, preeclampsia, or unexplained
with a decreased number of insulin receptors. fetal losses.
 Obesity is the most common cause of insulin  Mild hypertension is often present in obese diabetics.
resistance.  Eruptive xanthomas
5. Aging
 This may cause insulin resistance through a TYPE 1 DM vs TYPE 2 DM
decreased production of GLUT- 4 transporters. Pathogenesis β-cell destruction Insulin resistance
6. Increase production of insulin antagonists
Incidence Rate 10-15% 90-95%
Onset Any; childhood/teens Any; over 40 y/o
 A number of disorders are associated with
Genetic, obesity,
increased production of insulin antagonists, Risk Factors Genetic, autoimmune
lifestyle
such as - Cushing syndrome; Acromegaly; and, C-peptide
Decreased/undetectable Detectable
Stress states, such as trauma, surgery, Levels
diabetes ketoacidosis, severe infection, Autoantibodies (–
Pre-diabetes Autoantibodies (+)
uremia, and liver cirrhosis. )
Develop
7. Medications
Symptoms Develop abruptly gradually /
 Include glucocorticoids (Cushing syndrome), asymptomatic
cyclosporine, niacin, and protease inhibitors. Common, poorly
Ketosis Rare
8. Human immunodeficiency virus (HIV) controlled
Medication Insulin Oral agents
RISK FACTORS FOR TYPE 2 DM
 Family history of diabetes (i.e., parent or sibling with Gestational Diabetes Mellitus
type 2 diabetes)  Glucose intolerance during pregnancy
 Obesity (BMI >25 kg/m2)  Due to metabolic and hormonal changes

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 45
 After childbirth, the individual generally returns to 6. Infections
normal metabolism.  Congenital rubella
 However, there is an increased chance that type 2  Cytomegalovirus
diabetes mellitus may develop later in life.  Others
7. Uncommon Forms of Immune-Mediated
Other Specific Types of Diabetes Diabetes
1. Genetic Defects of Beta Cell Function  “Stiff-man” syndrome
 Chromosome 12, HNF-1α (MODY3)  Anti-insulin receptor antibodies
 Chromosome 7, glucokinase (MODY2)  Others
 Chromosome 20, HNF-4α (MODY1) 8. Other Genetic Syndromes Sometimes
 Chromosome 13, insulin promoter factor-1 (IPF-1; Associated with Diabetes
MODY4)  Down syndrome
 Chromosome 17, HNF-1β (MODY5)  Klinefelter’s syndrome
 Chromosome 2, NeuroD1 (MODY6)  Turner’s syndrome
 Mitochondrial DNA  Wolfram’s syndrome
 Others  Friedreich’s ataxia
2. Genetic Defects in Insulin Action  Huntington’s chorea
 Type A insulin resistance  Laurence-Moon-Biedl syndrome
 Leprechaunism  Myotonic dystrophy
 Rabson-Mendenhall syndrome  Porphyria
 Lipoatrophic diabetes  Prader-Willi syndrome
 Others  Others
3. Diseases of the Exocrine Pancreas
 Pancreatitis COMPLICATIONS OF DIABETES MELLITUS
 Trauma/pancreatectomy
 Neoplasia Acute Complication of DM
 Cystic fibrosis 1. Diabetic ketoacidosis
 Hemochromatosis 2. Hyperosmolar non-ketotic coma
 Fibrocalculous pancreatopathy 3. Lactic acidosis
 Others 4. Hypoglycemia
4. Endocrinopathies
 Acromegaly DIABETIC KETOACIDOSIS
 Cushing’s syndrome  Diabetic Ketoacidosis (DKA) is a state of inadequate
 Glucagonoma insulin levels resulting in high blood sugar and
 Pheochromocytoma accumulation of organic acids and ketones in the
 Hyperthyroidism blood.
 Somatostatinoma  It is a potentially life-threatening complication in
 Aldosteronoma patients with diabetes mellitus.
 Others  It happens predominantly in type 1 diabetes mellitus,
5. Drug or Chemical Induced but it can also occur in type 2 diabetes mellitus under
 Vacor certain circumstances.
 Pentamidine
 Nicotinic acid Clinical Manifestations:
 Glucocorticoids  Nausea and vomiting
 Thyroid hormone  Pronounced thirst
 Diazoxide  Excessive urine production and abdominal pain that
 β-Adrenergic agonists may be severe.
 Thiazides  Hyperglycemia is always present.
 Dilantin  In severe DKA, breathing becomes labored and of a
 γ-Interferon deep, gasping character (a state referred to as
 Others "Kussmaul respiration").
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 46
 The abdomen may be tender to the point that an  Potassium levels can fluctuate severely during the
acute abdomen may be suspected, such as acute treatment of DKA because insulin decreases
pancreatitis, appendicitis or gastrointestinal potassium levels in the blood by redistributing it
perforation. into cells.
 Coffee ground vomiting (vomiting of altered blood)  Serum potassium levels are initially often mildly
occurs in a minority of patients; this tends to raised even though total body potassium is
originate from erosions of the esophagus. depleted. Hypokalemia often follows treatment.
 In severe DKA, there may be confusion, lethargy,  This increases the risk of irregularities in the heart
stupor or even coma (a marked decrease in the rate. Therefore, continuous observation of the
level of consciousness). heart rate is recommended, as well as repeated
 On physical examination there is usually clinical measurement of the potassium levels and addition
evidence of dehydration, such as a dry mouth and of potassium to the intravenous fluids once levels
decreased skin turgor. fall below 5.3 mmol/l.
 If the dehydration is profound enough to cause a  If potassium levels fall below 3.3 mmol/l, insulin
decrease in the circulating blood volume, administration may need to be interrupted to allow
tachycardia (a fast heart rate) and low blood correction of the hypokalemia.
pressure may be observed.
 Often, a "ketotic" odor is present, which is often HYPEROSMOLAR HYPERGLYCEMIC STATE (HHS)
described as "fruity"  Hyperosmolar hyperglycemic state (HHS) is a
complication of diabetes mellitus in which high blood
Diagnosis: sugar results in high osmolarity without significant
 Diabetic Ketoacidosis may be diagnosed when the ketoacidosis. Symptoms include signs of dehydration,
combination of hyperglycemia (high blood sugars), weakness, leg cramps, vision problems, and an
ketones on urinalysis and acidosis are altered level of consciousness.
demonstrated.  ◦ HHS occurs in elderly individuals with type 2 DM,
 Arterial blood gas measurement is usually with a several week history of polyuria, weight loss,
performed to demonstrate the acidosis and diminished oral intake that culminates in mental
 Urea and creatinine estimations (measures of confusion, lethargy, or coma.
kidney function, which may be impaired in DKA as a
result of dehydration) and electrolytes. LACTIC ACIDOSIS
 Markers of infection (complete blood count, C-  Lactic acidosis occurs in hypoxic individuals and is due
reactive protein) and acute pancreatitis (amylase to an excessive production of lactate by peripheral
and lipase) may be measured. tissues.
 Given the need to exclude infection, chest  It is characterized by extreme metabolic acidosis.
radiography and urinalysis are usually performed.  There is high anion gap (higher than normal levels of
 If cerebral edema is suspected because of acid in the blood) with low or absent ketones and high
confusion, recurrent vomiting or other symptoms, lactate levels.
computed tomography may be performed to assess  Treatment: Sodium bicarbonate is needed to correct
its severity and to exclude other causes such as the acidosis
stroke.
HYPOGLYCEMIA
Management:  Hypoglycemia caused by excess insulin is the most
 The main aims in the treatment of diabetic common complication of insulin therapy, occurring in
ketoacidosis are replacing the lost fluids and more than 90 % of the patients.
electrolytes while suppressing the high blood  In normal individuals, hypoglycemia triggers a
sugars and ketone production with insulin. compensatory secretion of counter regulatory
 Fluid replacement- The amount of fluid depends on hormones, most notably glucagon and epinephrine,
the estimated degree of dehydration. which promote hepatic production of glucose.
 Insulin is usually given continuously. However, patients with type 1 diabetes also develop
 Sodium bicarbonate solution is administered to a deficiency of glucagon secretion.
rapidly improve the acid levels in the blood.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 47
 These patients thus rely on epinephrine secretion to  The earliest detectable change in the course of
prevent severe hypoglycemia. diabetic nephropathy is a thickening in the
 However, as the disease progresses, type 1 diabetes glomerulus.
patients show diabetic autonomic neuropathy and  At this stage, the kidney may start allowing more
impaired ability to secrete epinephrine in response to albumin than normal in the urine (microalbuminuria).
hypoglycemia.  Once macroalbuminuria develops, blood pressure
 The combined deficiency of glucagon and epinephrine rises slightly, and the pathologic changes are likely to
secretion creates a condition sometimes called be irreversible
“Hypoglycemia unawareness”. Insert pic
 Thus, patients with long standing diabetes are
particularly vulnerable to hypoglycemia Neurological Complications (Neuropathy)

Chronic Complications of DM DIABETIC NEUROPATHY

 Chronic complications can be divided into vascular and  Diabetic neuropathy occurs in ~50% of individuals
nonvascular complications. with long-standing type 1 and type 2 DM.
 The vascular complications of DM are further  Peripheral Neuropathy - This type usually affects the
subdivided into microvascular and macrovascular feet and legs. Rare cases affect the arms, abdomen,
 Vascular Complications: and back.
1. Microvascular Complications:  Autonomic Neuropathy - This type usually affects the
a. Ocular Complications (Retinopathy) digestive system, especially the stomach. It can also
b. Renal Complications (Nephropathy) affect the blood vessels, urinary system, and sex
c. Neurological Complications (Neuropathy) organs.
2. Macrovascular Complications:  Proximal Neuropathy - This type causes pain (usually
a. Coronary Artery Disease (CAD) on one side) in the thighs, hips, or buttocks. It can also
b. Peripheral Arterial Disease (PAD) lead to weakness in the legs.
c. Cerebrovascular Disease  Focal Neuropathy - This type can appear suddenly and
 Nonvascular Complications: affect specific nerves, most often in the head, torso,
 Gastroparesis, infections, and skin changes. Long- or leg. It causes muscle weakness or pain.
standing diabetes may be associated with hearing Insert pic
loss
DIABETIC GANGRENE
MICROVASCULAR COMPLICATIONS  The factors responsible for its development, in
addition to peripheral vascular disease, are small
Ocular Complications (Retinopathy) vessel disease, peripheral neuropathy with loss of
 Diabetic retinopathy both pain sensation and neurogenic inflammatory
Insert pic responses, and secondary infection.
 Diabetic cataract  The peripheral sensory neuropathy interferes with
Insert pic normal protective mechanisms and allows the patient
 Glaucoma to sustain major or repeated minor trauma to the
Insert pic foot, often without knowledge of the injury.
 Blindness  Peripheral artery disease (PAD) and poor wound
healing impede resolution of minor breaks in the skin,
Renal Complications (Nephropathy) allowing them to enlarge and to become infected.
 Diabetic nephropathy (nephropatia diabetica), also  Approximately 15% of individuals with DM develop a
known as Kimmelstiel-Wilson syndrome, and foot ulcer (great toe or MTP areas are most common),
intercapillary glomerulonephritis, is a progressive and a significant subset will ultimately undergo
kidney disease amputation (14–24% risk with that ulcer or
 It is the principal cause of ESRD (End Stage Renal subsequent ulceration).
Disease) in the western world. Insert pic

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 48
MACROVASCULAR COMPLICATIONS  Diagnostic value: ≥ 200 mg/dL (≥11.1 mmol/L) plus
Atherosclerosis symptoms of diabetes
 A disease in which plaque builds up inside your
arteries. Plaque is made up of fat, cholesterol, ORAL GLUCOSE TOLERANCE TEST
calcium, and other substances found in the blood.  The patient is given a standard glucose load and the
Over time, plaque hardens and narrows your arteries. ability of the patient to handle the glucose load is
This limits the flow of oxygen-rich blood to your monitored by measuring blood glucose levels versus
organs and other parts of your body. time.
 Atherosclerosis can lead to serious problems,  This is done to diagnose Diabetes Mellitus and
including heart attack, stroke, or even death. impaired glucose tolerance on patients whose fasting
glucose is elevated, but not above 140 mg/dL.
NONVASCULAR COMPLICATIONS
 Chronic pyogenic infections of the skin may occur, 24-HOUR POST PRANDIAL BLOOD SUGAR
especially in poorly controlled diabetic patients  Normal: <200 mg/dL
 Fungal infections are also very common in diabetics  Glucose testing 2 hours after a real meal
 It causes vulvovaginitis in most chronically  A variation of this test is to use a standardized load of
uncontrolled diabetic women with persistent solution containing 75 g glucose which will be
glucosuria and is a frequent cause of pruritus. administered to the patient and a specimen for the
plasma glucose measurement is drawn 2 hours later
ROLE OF LABORATORY IN DIAGNOSIS OF  If the level is ≥ 200 mg/dL and is confined on a
PATIENTS WITH GLUCOSE METABOLIC subsequent day by either an increased random or
ALTERATIONS fasting glucose level, the patient is diagnosed with
diabetes
Methods of Glucose Measurement
 Glucose can be measured from serum, plasma, or GLYCATED HEMOGLOBIN
whole blood.  Other names: HbA1c, glycohemoglobin, glycosylated
 Today, most glucose measurements are performed on hemoglobin, and fast hemoglobin
serum or plasma.  Glycosylated hemoglobin refers to the compound of
 The glucose concentration in whole blood is hemoglobin formed when glucose reacts with the
approximately 11% lower than the glucose amino group of hemoglobin
concentration plasma. – The glucose molecules attach nonenzymatically to
 Serum or plasma must be refrigerated and separated the hemoglobin molecule in a ketoamine structure
from the cells within 1 h to prevent substantial loss of to form a ketoamine
glucose by the cellular fraction, particularly if the – The rate of formation is directly proportional to the
white blood cell count is elevated. plasma glucose concentrations
Insert pic
FASTING BLOOD SUGAR  Because the average red blood cell lives
 It is obtained in the morning after an approximately approximately 120 days, glycosylated hemoglobin
8- to 10-hours fast (not longer than 16 hours). Fasting level at any one time reflects the average blood
plasma glucose values have a diurnal variation with glucose level over the previous 2-3 months
the mean FBG higher in the morning than in the  HbA1c is a reliable method of monitoring long-term
afternoon. Diabetes in patients tested in the diabetes control rather than random plasma glucose
afternoon may be missed because of this variation. (FBS) wherein values are unaffected by the day-to-day
 Normal: < 100 mg/dL; Impaired/Pre-diabetes: 100- changes in glucose concentration, degree of exercise,
125 mg/dL; DM: ≥ 126 mg/dL or recent food ingestion.
 Normal values range from 4% to 6.4%
RANDOM BLOOD SUGAR
 A blood sugar test taken from a non-fasting subject. FRUCTOSAMINE
This test assumes a recent meal and therefore has  Refers to glycosylated albumin and other protein
higher reference values than the fasting glucose test.  Gives a clear picture of more short-term glucose
levels
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 49
 2-3 weeks – albumin  Maternal history of diabetes or gestational
 2-3 days – other proteins diabetes mellitus
 All adults >45 years old should have fasting blood
MICROALBUMINURIA glucose measured every 3 years, unless already
 DM causes progressive changes to the kidneys and diagnosed with diabetes.
ultimately results in diabetic renal nephropathy. This  Testing should be earlier or more frequent w/ these
complication progress over years and may be delayed risk factors:
by aggressive glycemic control. An early sign that  Overweight tendencies (BMI ≥25 kg/m2)
nephropathy is occurring is an increase in urinary  Habitual physical inactivity
albumin.  Family history of diabetes in a first-degree relative
 Microalbumin measurements are useful to assist in  High-risk minority population (African American,
diagnosis at an early stage of nephropathy and Latino)
before development of proteinuria  History of gestational diabetes or delivering baby
 Microalbumin concentrations are between 20 and 300 >9 lb
mg/day. Proteinuria is typically > 0.5 g/day (>500  Hypertension (≥140/90)
mg/day).
 A patient is determined to have microalbuminuria Criteria for Diagnosis of Diabetes Mellitus (ADA,
when 2-3 specimens collected within a 6-month period 2010)
are abnormal. 1. HbA1c ≥ 6.5%
2. Fasting plasma glucose ≥ 126 mg/dL (≥7.0 mmol/L)
SELF-MONITORING OF BLOOD GLUCOSE (SMBG) 3. 2-hour plasma glucose ≥ 200 mg/dL (≥11.1 mmol/L)
 Performed to monitor and maintain glucose levels as during an OGTT
close to normal as possible 4. Random plasma glucose ≥ 200 mg/dL (≥11.1
– For type 1 DM, the recommendation of use is 3-4 mmol/L) plus symptoms of diabetes
times/day
 Most of the monitors employ the glucose oxidase  High on at least two occasions is diagnostic diabetes
methodology although hexokinase systems do exist  If a patient has discordant results from 2 different
 Uses whole blood which is 15% lower than plasma tests, then the test for which the result is above the
determinations diagnostic cut point should be repeated on a different
day.
Criteria for Testing for Prediabetes and
Diabetes
 Criteria for type 2 diabetes testing in children,
beginning at age 10 or at onset of puberty & with
follow-up testing every 2 years.
 Family history (first- or second-degree) of type 2
diabetes
 Race/ethnicity (African American, Latino, Native
American)
 Signs of insulin resistance

UNIT 7: LIPIDS
INTRODUCTION because of their association with coronary heart
 Lipids and lipoproteins, which are central to the disease (CHD).
energy metabolism of the body, have become  Numerous epidemiologic studies have demonstrated
increasingly important in clinical practice, primarily that, especially in affluent countries with high fat
consumption, there is a clear association between the
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 50
blood lipid levels and the development of and as a result they’re usually solid at room
atherosclerosis. temperature and the longer the saturated
 Decades of basic research have also contributed to fatty acid chain, the more likely it will be
knowledge about the nature of the lipoproteins and solid at room temperature
their lipid and protein constituents, as well as their
role in the pathogenesis of the atherosclerotic
process.

LIPID CHEMISTRY
 Fats – essential part of a healthy diet.
– Contribute to the taste and texture of food, like the
smoothness in guacamole and the flakiness of a b. Unsaturated Fatty Acids – double bond
croissant.  It has not saturated with hydrogen atoms
– Major source of energy, a critical component of cells
(for every double bond there are two fewer
and tissues. hydrogen atoms).
– Help essential absorb vitamins and can be
 Double bond causes a kink in the molecule,
converted into other molecules like prostaglandins so the fats don’t pack together as nicely as
which help cells communicate with each other. saturated fats, as a result, they are usually
liquid at room temperature.

– Have a three-carbon backbone called glycerol, as

well as fatty acid chains.
 Unsaturated fats can be further classified
 Fatty acid chain – string of carbon and hydrogen
atoms according to the number of their double
– When an –OH group from a glycerol molecule binds
bonds.
i. Monounsaturated Fatty Acid – single
to an “H” atom from the fatty acid, an H2O or water
molecule gets released and the two molecules link double bond
ii. Polyunsaturated Fatty Acid – two or
up.
– If this happens once, the result is a
more double bonds
monoglyceride (1 glycerol, 1 fatty acid); if it
happens twice, it’s a diglyceride (1 glycerol, 2
fatty acid); and three times makes a triglyceride
(1 glycerol, 3 fatty acid).
– Types of Fatty Acid Chains:
1. According to length (number of carbons)
a. Short chain fatty acid (2-5 carbons)
b. Medium chain fatty acid (6-12 carbons)
 They can also be classified according to the
c. Long chain fatty acid (13 or more carbons)
2. According to the bonds connecting the carbons in
location/position of the double bond.
– The methyl end is called “omega”
the chain
– Count the number of carbons until the
a. Saturated Fatty Acid – single bond (one bond
between two carbon atoms) first double bond
 It has as many hydrogen atoms as possible
it has saturated with them.
 Fats with saturated fatty acids are nice and
straight so they pack together really well,
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 51
cows and pigs, which is why trans fats can be found
naturally in meat and dairy products.
– Trans fats are also created through the partial
hydrogenation of liquid oils, a process that makes
them solid.
– Partially hydrogenated oils have been largely
removed from foods in North America and Europe
because trans fats have been associated with
coronary heart disease.
– Well some foods have more than one type of fat
than another; the truth is all foods are made up of a
 Looking at the double bond of this unsaturated fat,
blend of fatty acids.
 Fats play a very important role throughout the body.
like most unsaturated fats, it’s got a cis configuration
 They have a great number of health benefits and
but some fats are in a trans configuration.
 Cis configuration
those benefits can vary by the type of we eat.
 Polyunsaturated fats are precursor for hormone
 The two functional groups are on the same side
of the double bonded carbons and the fatty acid like molecules called prostaglandins that stimulate
chain naturally bends. endothelial cells that line blood vessels to release
 A molecule that bends doesn’t pack tightly
nitric oxide.
 Nitric oxide is a vasodilator so that decreases
together so it’s a lot more fluid (think about
cooking oils, which are liquid at room resistance to blood flow and in turn lowers blood
temperature). pressure.
 Polyunsaturated fatty acids also help reduce the
 Trans configuration
 The functional groups are on opposite sides of
total and LDL cholesterol and that’s linked to lower
the double bonded carbons which keeps the rates of cardiovascular disease like heart attacks
chain straighter and easier to pack. and strokes.
 Long chain Omega-3 fatty acids like DHA and EPA
can both help to lower plasma triglyceride which
also protects against cardiovascular disease.
 DHA is important in the development of eyes and
brains of young infants.
 Healthy diet emphasizes on mono and
polyunsaturated fats over saturated.
 Trans fats result from a process called partial  Trans fats are associated with increased risk of
hydrogenation. cardiovascular diseases.
– In just plain old hydrogenation, hydrogens are  Improvements to our diet and health can be achieved
added to cis-fats to get rid all the double bonds. by focusing more on the type of fat we eat and less on
– Partial hydrogenation on the other hand, refers to the amount.
adding hydrogens to most but not all double bonds.
CLASSIFICATION OF LIPIDS

Triglycerides
 Triglycerides (TGs, also called neutral fats,
– Partial hydrogenation is a process that happens triacylglycerols, or triacylglycerides) are a common,
naturally in the digestive tract of some animals like
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 52
simple type of lipid consisting of three long-chain  Forms of phospholipid: lecithin (70%), sphingomyelin
fatty acids esterified to glycerol (20%), cephalin (10%)
 Exogenous triglycerides originate from food, while  Originate from the liver and intestine
endogenous triglycerides are formed in the liver.
 Triglycerides are transported through the body by FUNCTIONS
chylomicrons and VLDL (very-low-density lipoprotein). 1. Integral part of cell membranes
 Triglycerides make up more than 95% of lipids in the 2. Lung surfactant (lecithin is a major component of lung
diet. surfactant)
3. Play a role in mitochondrial metabolism
Cholesterol 4. Participate in blood coagulation
 Almost exclusively synthesized in the intestine and 5. Part of lipoprotein (lipid transport)
liver (although all cells have the capability except 6. Essential component of bile
erythrocytes)
 Not readily catabolized by most cells and therefore LIPOPROTEINS
does not serve as a source of fuel  Transporter of fats
 Low-density lipoprotein (LDL) is the primary carrier of  Defined as lipid-filled particles that have an outer
cholesterol. membrane consisting of a monolayer of special
protein called apolipoproteins interspersed with the
FORMS OF CHOLESTEROL polar lipids.
1. Free Cholesterol (unesterified form)  The size of the lipoprotein particles correlates with its
 Major component of cell membranes lipid content.
 1/3 in blood circulation  More lipid = larger size
2. Cholesteryl Ester (esterified form)  More protein = denser/heavier
 Not found on the surface of lipid layers but are
instead in the center of lipid drops along with
triglycerides
 2/3 in blood circulation

FUNCTIONS
1. Provide structure for cell membrane
2. Used to manufacture and repair cell membranes
3. Used to synthesize Vitamin D and steroid hormones
4. Used to synthesize bile salts

Phospholipids
 Structurally similar to triglycerides; the glycerol
backbone and two fatty acid ester are present but the
third ester is a group containing phosphate;
amphipathic lipid

CLASSIFICATION OF LIPOPROTEINS
%TAG %CHOLESTEROL APOLIPOPROTEIN CONTENT LPE
Chylomicrons 86 3 AI, B-48, CI, CII, CIII, E Origin
VLDL 55 12 B-100, CI, CII, CIII, E Pre-beta
IDL 23 29 B-100, E Pre-beta/Beta
LDL 6 42 B-100 Beta
HDL 3 15 AI, AII, CI, CII, CIII, E Alpha
Lp(a) (LDL) (LDL) (a), B-100 Pre-beta

CHYLOMICRONS  Carry no charge (no migration)

 Largest and the least dense of all lipoproteins  Produced by the intestine

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 53
 Role: delivery of dietary lipids to hepatic and HIGH DENSITY LIPOPROTEINS (HDL)
peripheral cells  The smallest, most dense and the fastest moving
 Because of their large size they refract light and fraction because it contains the most protein
account for turbidity of postprandial plasma  Synthesized in liver and intestine
 Because they are light, they float on the top of stored  Role: transports cholesterol from the tissues and
plasma and form the creamy layer which is only return it to the liver (“good cholesterol”)
characteristic for chylomicrons
ABBERANT LIPOPROTEINS
VERY-LOW DENSITY LIPOPROTEINS (VLDL) 1. Lipoprotein (a) – termed as “sinking pre-beta
 By density separation, are a little heavier than lipoprotein” because it migrates to pre-beta area;
chylomicrons competes with plasminogen (lyses fibrin clots) for
 Migrate to the pre-beta area binding sites thereby promoting clots along arterial
 Synthesized by the liver wall that will not be dissolved
 Role: transport endogenous TAG from the liver to 2. Floating Beta-Lipoprotein – appears as a broad beta
peripheral tissues band
 Also refract light and account for turbidity in lipemic 3. Lipoprotein X – only lipoprotein to migrate toward
plasma but don’t form creamy layer because they are the cathode (–)
heavier/denser
 Appearance of Hyperlipidemia using Standing Plasma APOLIPOPROTEINS
Test  Functions:
– Done only if plasma/serum is turbid or milky. 1. Serves as transport proteins for lipids
– 2 ml plasma/serum is used, placed overnight in 2. Help maintain structural integrity of lipid-protein
clear test tube and refrigerate, observe for result. complex
– Result: 3. Aid in the delivery of lipids via recognition of cell
 Chylomicrons: (+) floating “cream” layer, (–) surface receptors
turbidity 4. Mediate lipid exchange from one lipoprotein
 VLDL: (–) float, (+) turbid fraction to another
 VLDL & Chylomicrons: (+) float, (+) turbid 5. Activate or inhibit enzymes involved in lipid
metabolism
INTERMEDIATE DENSITY LIPOPROTEINS (IDL)  Types:
 Remnant of VLDL circulating in plasma after half of 1. Apo A – primary protein found in HDL; index of
VLDL triglyceride has been transferred to adipose or the amount of the antiatherogenic HDL present in
muscle cells plasma
 Most of IDL undergoes further delipidation, it a. A-I – activates lecithin-cholesterol
transfers to HDL all its apolipoprotein except Apo B acyltransferase (LCAT); enzyme that catalyze
and thus become LDL (small percentage of IDL binds to reverse cholesterol transport
liver cells where it is degraded) b. A-II – activates hepatic lipase; has structural
role in HDL
LOW DENSITY LIPOPROTEINS (LDL) c. A-III – transfers cholesteryl ester from HDL to
 Arises in plasma from IDL which now contains mostly other lipoproteins in exchange for TAG
cholesterol 2. Apo B – principal protein in LDL, VLDL,
 Migrates to the beta region chylomicrons; absent in HDL
 Role: delivers cholesterol it carries to other tissues a. B-100 (larger) – from the liver; found in LDL
for use as structural component of new cell (95%) & VLDL (25%); a ligand for the LDL
membranes, as precursor of steroid hormones, or for receptor
storage as cholesteryl esters b. B-48 (smaller) – from the intestine; exclusively
 Termed as “bad cholesterol” found in chylomicrons’ structural role
 Readily taken up by cells (macrophage) and this, in 3. Apo C – protein that interacts with enzyme during
part, accounts for the reason that elevated levels transport
promote atherosclerosis a. C-I – activates LCAT in vitro
b. C-II – activates lipoprotein lipase

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 54
c. C-III – inhibits lipoprotein lipase
4. Apo D – involve in transfer of cholesteryl ester
from HDL to VLDL and LDL
5. Apo E – important to identify the chylomicron
remnant in the liver for endocytosis and breakdown
 Interacts significantly with receptor in the liver
for lipoprotein clearance, lipolysis, and
cholesterol esterification

Lipid Metabolism  The micelles glide through the watery environment of

 Four Major Pathways Involved in Lipoprotein the intestinal lumen and reach the enterocytes in the
Metabolism intestinal wall.
1. Lipid Digestion & Absorption  When they get into the enterocytes, the micelles
2. Exogenous Pathway release the fatty acids and monoglycerides which
3. Endogenous Pathway diffuses into the enterocyte.
4. Reverse Cholesterol Transport Pathway  Inside the enterocytes, the fatty acids and
monoglycerides reassemble into triglycerides, and
LIPID DIGESTION AND ABSORPTION these get packed into a larger structure called a
chylomicron.
 The chylomicron has lipids and proteins so it’s a
lipoprotein.
 It has an outer membrane with phospholipids and
proteins and a hydrophobic core that has
triglycerides, cholesterol and fat-soluble vitamins
(ADEK) the chylomicrons then leave the enterocytes,
but it’s too large to get into the endothelial cells so
instead it enters a nearby lymphatic capillary called
the lacteal.
 When you eat a food like peanut butter, which has
about 75% of its calories from fats, the body goes
through a set of steps to digest and absorb the fatty
acids.
 Triglycerides are hydrophobic, so when mixed with
water they will form large globules of fat, like what
you see when you pour oil into water.
 Enzymes, called lipase, in the saliva and in the
stomach are secreted by pancreas to break down the  From there the chylomicron floats in the lymph and
globules of fat into free fatty acids and flows into the thoracic duct and then gets dumped into
monoglycerides. the blood – essentially bypassing the portal vein.
 But working on the surface of a globule is inefficient,
 Once in the blood, the chylomicron will be broken
so to speed things up bile salts produced by the liver down by enzymes into fatty acids and monoglycerides
breaks the large droplets of fat into smaller droplets again for absorption of cells such as in peripheral
which increases the surface area for the lipases to tissues like muscle which use them for energy as well
work. as adipose tissue which can store them.
 Once the triglycerides are broken down into
 After delivering the triglycerides, the chylomicron
monoglycerides and free fatty acids they self- shrinks in size and eventually gets engulfed by the
assemble to form into micelles (aggregate of liver.
surfactant molecules) which have a hydrophobic
interior and a hydrophilic or water loving exterior.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 55
EXOGENOUS PATHWAY  Apo C-II in VLDL activates LPL which hydrolyses half
1. Immediately after the chylomicrons enter circulation, of TAG in VLDL
they interact with proteoglycans, such as heparan  There is also dissociation and transfer of
sulfate on the surface of capillaries in various tissues lipoproteins and phospholipids to other lipoprotein
such as skeletal muscle, heart and adipose tissue. particles
 The proteoglycans on capillaries promote the  During this process, VLDL is converted to IDL
binding of lipoprotein lipase (LPL), which 3. IDL still interacts with LPL until the majority of TAG
hydrolyzes triglyceride on chylomicrons. has been transferred to adipocytes. The cholesterol-
 The free fatty acids and glycerol generated by the rich, triglyceride-poor lipoprotein is now called LDL.
hydrolysis of LPL can be taken up by cells and used 4. LDL is the major lipoprotein responsible for the
as a source of energy. delivery of endogenous cholesterol to peripheral
2. During the lipolysis of chylomicrons, there is a cells.
transfer of lipid and apolipoproteins onto HDL.
Chylomicrons are converted within a few hours after a REVERSE CHOLESTEROL TRANSPORT PATHWAY
meal into a chylomicron remnant.  Mechanism by which HDL removes cholesterol from
3. Chylomicron remnant is taken up by the liver through the peripheral tissue and brings it back to the liver to
interaction of APO E with specific remnant receptors be used up and be excreted as bile
on the surface of liver cells.
4. Once in the liver, the lysosomal enzymes break down SUMMARY
the remnant particles to release free fatty acids, free
cholesterols, and amino acids.
 Both bile acids and free cholesterol are directly
excreted into the bile but not all of the excreted
cholesterol and bile salt exits the body.
 Approximately half of the excreted biliary
cholesterol is reabsorbed by intestine, with the
remainder appearing in the stool as fecal neutral
steroids.
 Almost all of the bile acids are reabsorbed and LIPID AND LIPOPROTEIN DISORDERS
reused by the liver for bile production. 1. Fredrickson Classification of Dyslipidemia
2. Abetalipoproteinemia
ENDOGENOUS PATHWAY 3. Hypobetalipoproteinemia
1. The majority of triglycerides in the liver that are 4. Tangier Disease
packaged into VLDL are derived from the diet after 5. Lecithin-Cholesterol Acyltransferase Deficiency
recirculation from adipose tissue. 6. Arteriosclerosis
2. VLDL particles, once secreted into the circulation,
undergo a lipolytic process similar to that of
chylomicrons.

FREDRICKSON CLASSIFICATION OF DYSLIPIDEMIA

TYPE REFRIGERATION LPE LPs
I positive, clear normal inc TAG (chylos)
IIa negative, clear inc B band inc chole (LDL)
IIb negative, cloudy inc B, pre-B inc TAG, chole (LDL, VLDL)
III occasional, cloudy inc pre-B inc TAG chole (IDL, VLDL)
IV negative, cloudy inc a-2 inc TAG (VLDL)
V positive, cloudy inc a-2 inc TAG, chole (chylos, VLDL)

TYPE I HYPERLIPOPROTEINEMIA (FAMILIAL  Recessive inheritance

HYPERCHYLOMICRONEMIA)  Normal serum cholesterol
 Apo C-II deficiency or lipoprotein lipase deficiency  Increased triglyceride primarily in chylomicrons
 Manifests early in life, usually at infancy
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 56
 Patients with this condition do not develop premature of survivors of myocardial infarctions) and
coronary disease, implying that chylomicrons peripheral vascular disease.
themselves are not atherogenic (not associated with
high risk CHD but is potentially life threatening TYPE IV HYPERLIPOPROTEINEMIA (FAMILIAL
because it can cause acute and recurrent pancreatitis) HYPERTRIGLYCERIDEMIA)
 Features include:  An overproduction of VLDL with impaired catabolism
 Acute pancreatitis  Increased endogenous TAG without increase in
 Hyperinsulinism cholesterol (increased VLDLs)
 Eruptive xanthoma – these are characteristic of  Autosomal dominant inheritance
severe hypertriglyceridemia - yellow papules on  Exacerbated by weight gain, estrogen, alcohol or
the extensor surfaces of back, arms, buttocks and increase carbohydrate diets
legs
 Lipemia retinalis may occur – retinal arteries and TYPE V HYPERLIPOPROTEINEMIA (FAMILIAL COMBINED
veins appearing white on fundoscopy HYPERTRIGLYCERIDEMIA)
 Apo C-II deficiency or lipoprotein lipase deficiency
TYPE IIa HYPERLIPOPROTEINEMIA (FAMILIAL  Similar to Type I but with high VLDL ( due to
HYPERCHOLESTEROLEMIA) overproduction) in addition to chylomicrons
 Autosomal dominant disorder caused by one several  Onset is usually in adulthood
mutations in the LDL-receptor resulting in a defective  Raised triglyceride (chylomicrons and VLDL)
receptor that cannot bind or clear LDL from the  Normal or slightly raised plasma cholesterol
circulation  Clinical features may include:
 High LDL levels  Eruptive xanthoma
 Gene frequency 1/500  Lipemia retinalis
 Presentation is with early onset ischemic heart  Hyperinsulinism
disease  Hepatosplenomegaly may occur
 Untreated life expectancy is 20 years
Abetalipoproteinemia
TYPE IIb HYPERLIPOPROTEINEMIA (FAMILIAL COMBINED  Basses-Kornzweig Syndrome
HYPERLIPOPROTEINEMIA)  Failure to synthesize apo B; autosomal recessive
 Decreased LDL-receptor expression with excessive disorder;
production of Apo B  VLDL, LDL, and chylomicrons are absent from plasma,
 Similar with type IIa except VLDL is also increased cholesterol and triglycerides are low
 Raised plasma cholesterol (raised LDL)  Patients have retarded growth, progressive
 Raised triglyceride (raised VLDL) degeneration of CNS, poor absorption of fat
 Polygenic inheritance  Acanthocytes are characteristically visible on PBS
 There is an increased risk of ischemic heart disease
Hypobetalipoproteinemia
TYPE III HYPERLIPOPROTEINEMIA (FAMILIAL  Disorder that impairs the body's ability to absorb and
DYSBETALIPOPROTEINEMIA) transport fats. This condition is characterized by low
 Characterized by raised triglyceride (raised VLDL) and levels of LDL and total cholesterol.
raised cholesterol levels (due to abnormally high  Severity of signs and symptoms experienced by
concentration of IDL and chylomicron remnants) as a people with vary widely. The most mildly affected
result of defective catabolism due to defective APO E individuals have few problems with absorbing fats
isoforms (presence of very rare APO E2/2) from the diet and no related signs and symptoms.
 Inherited as an autosomal recessive trait. Many individuals develop an abnormal buildup of fats
 This disease may present in early adult life with in the liver called hepatic steatosis or fatty liver. In
possible other features such as: more severely affected individuals, fatty liver may
 Yellow palmar creases, palmer xanthomas and progress to chronic liver disease (cirrhosis).
tuberoeruptive xanthomas Individuals with severe cases have greater difficulty
 Increased risk of coronary artery disease (this absorbing fats as well as fat-soluble vitamins such as
condition may be found in approximately 3% vitamin E and vitamin A. This difficulty in fat
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 57
absorption leads to excess fat in the feces  Milder form of LCAT deficiency = Fish-eye disease
(steatorrhea). In childhood, these digestive problems
can result in an inability to grow or gain weight at the Arteriosclerosis
expected rate (failure to thrive).  Deposition of lipids, mainly in the form of esterified
 Decreased risk for cardiovascular disease cholesterol, in the walls of the arteries.
 This lipid deposition starts with thin layers called
Tangier Disease fatty streaks.
 A rare familial deficiency of HDL caused by defects in  The fatty streaks develop over time into plaques,
gene for the ABCA1 transporter which partially block blood flow.
 Associated with an abnormal orange or yellow  Peripheral Vascular Disease (PVD) – plaque formation
discoloration of the tonsils and pharynx develops in arteries of arms or legs
 Poses an increased risk for coronary heart disease  Coronary Artery Disease (CAD) – develops in the
vessels of the heart; associated with angina and
Lecithin-Cholesterol Acyltransferase Deficiency Myocardial infarction
 Due to mutation in LCAT gene  Cerebrovascular Disease (CVD) –vessels of the brain;
 HDL-cholesterol levels are typically less than 10 associated with stroke
mg/dL

UNIT 8: PROTEINS
INTRODUCTION  Made up of carbohydrate, hydrogen, oxygen, nitrogen
and sulfur; the 16% nitrogen makes it different from
Proteins carbohydrates and lipids
 Macromolecules made of amino acids, with each
amino acid being linked to another via a peptide Amino Acids
bond.  Building blocks of proteins
 All proteins are composed of the same 20 amino acids

AMINO ACIDS
ESSENTIAL NONESSENTIAL CONDITIONALLY ESSENTIAL
Not synthesized by the body but is provided Usually not essential, except in
Synthesized by the body
by dietary intake time of illness and stress
arginine
Histidine (His) alanine cysteine (oxidized form:
Phenylalanine (Phe)
Isoleucine (Ile) asparagine cysteine)
Threonine (Thr)
Leucine (Leu) aspartate/aspartic acid glutamine
Tryptophan (Trp)
Lysine (Lys) glutamate/glutamic acid glycine
Valine (Val)
Methionine (Met) serine proline
tyrosine

PROTEIN INTAKE AND METABOLISM  Acid environment (pH 2-3) helps to unfold proteins for
1. Digestion of protein enzyme attack
2. Intestinal absorption of amino acids  Pepsin (in the stomach) begins the process of protein
3. Metabolism of amino acids digestion by cleaving amide linkages to form smaller
4. Formation of new proteins (Anabolism) peptides
5. Catabolism of proteins  Enzymes responsible for breakdown of proteins into
6. Excretion of protein small peptides: trypsin, elastase, chymotrypsin,
carboxypeptidase
Digestion of Protein  Smaller molecules are then hydrolyzed to single
 Protein digestion begins in the stomach and is amino acids by aminopeptidases
completed in the small intestine
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 58
Intestinal Absorption of Amino Acids Excretion of Protein
 Amino acids are then rapidly absorbed from the  The glomerulus (filtering apparatus of the kidney) has
intestine into the portal blood and subsequently cutoff of about 70,000 MW.
become part of the body pool of amino acids.  Compounds larger than the cutoff size are not filtered
and excreted
Metabolism of Amino Acids  Compounds with low MW pass through the glomerulus
 Fate of amino acids in the amino acid pool: and excreted in the urine
 Primarily used for the synthesis of body proteins  A few proteins can be lost in this manner (e.g.,
 Synthesis of nonprotein nitrogen containing albumin: 65,000 MW and amylase: 40,000-50,000 MW)
compounds
Classification of Proteins
Formation of New Proteins (Anabolism) 1. Simple Proteins – polypeptides composed of only
 Protein synthesis is regulated by chromosomes amino acids
contained in the nucleus of each cell 2. Conjugated Proteins – composed of protein
 The amino acid sequence for each protein and the (apoprotein) and nonprotein (prosthetic group)
amount of protein produced are determined by the components; prosthetic groups are commonly metal,
genetic code each individual possesses lipid, and carbohydrate in nature.
 Hormones that are involved in promoting synthesis: a. Metalloproteins – protein with metal prosthetic
thyroxin, growth hormone, insulin, testosterone group (e.g., ceruloplasmin)
 Hormones which have catabolic effects on proteins: b. Lipoproteins – protein with a lipid prosthetic
Glucagon and Cortisol group
c. Glycoproteins – p rotein with 10%-40%
Catabolism of Protein carbohydrates attached (e.g., haptoglobin)
 The disintegration of protein occurs in the digestive d. Mucoproteins – protein with >40%
tracts, kidneys, and particularly, the liver. carbohydrates attached (e.g., mucin)
 During catabolism, proteins are hydrolyzed to their e. Nucleoproteins – p rotein with DNA or RNA
constituent amino acids; the amino acids are nucleic acids attached (e.g., chromatin)
deaminated, producing ammonia and ketoacids.
– The ammonia is converted to urea by the PROTEIN FUNCTIONS
hepatocytes and excreted in the urine 1. Energy Production – protein can be broken down into
– The ketoacids are oxidized by means of citric acid amino acids that can be used in the citric acid cycle to
cycles and converted to glucose or fat. produce energy.
 A balance exists between protein anabolism and 2. Maintenance of water distribution between cells and
catabolism (nitrogen balance) tissue
 nitrogen balance  nitrogen intake = nitrogen 3. Buffer – maintain pH which is due to the amphoteric
excreted nature of proteins
 Negative Nitrogen Balance 4. Transporter – binding of proteins allows movement of
 Protein catabolism exceeds protein anabolism other molecules in the circulation
(nitrogen excreted exceeds that of ingested) 5. Antibodies – proteins that protect the bodies against
 Occurs in conditions in which there is excessive “foreign” invaders
tissue destruction, such as burns, wasting 6. Structural proteins – provide cellular or body support
diseases, continual high fevers, or starvation (e.g., collagen, keratin)
 Positive Nitrogen Balance 7. Enzymes – catalyst that accelerate chemical reactions
 Protein anabolism is greater than protein 8. Hormones – messenger proteins that help coordinate
catabolism (nitrogen intake is greater than the certain bodily functions.
loss of nitrogen; there is an increase in the total
body pool of protein) PROTEIN FRACTIONS (ELECTROPHORESIS)
 Found during growth, pregnancy, and repair  Modern understanding of the protein composition of
processes serum and plasma derives from the electrophoretic
techniques introduced by Tiselius.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 59
 He separated proteins dissolved in an electrolyte 1. alpha-1-antitrypsin (AAT)
solution by application of an electric current through a  Major protein in alpha1-globulin zone; an acute
U-shaped quartz tube that held the protein solution. phase reactant & a protease inhibitor
 At pH 8.6, four serum protein fractions, designated  Most important function the inhibition of the
albumin, α, β, and γ, were identified and quantified protease neutrophil elastase
optically by change in refractive index at the  Increased in: inflammatory reactions, pregnancy
boundaries among these bands. and contraceptive use
 Protein fractions:  Decreased in: pulmonary emphysema, juvenile
1. Pre-albumin fraction (Transthyretin) hepatic cirrhosis
2. Albumin fraction 2. alpha-1-fetoprotein (AFP)
3. Alpha-1-globulin  Synthesized in the developing embryo and fetus
4. Alpha-2-globulin and then by the parenchymal cells of the liver.
5. Beta globulin Normally, adult levels are very low.
6. Gamma globulin  Has no known function in normal adults; in
normal fetuses, AFP binds the hormone estradiol.
Pre-Albumin Fraction (Transthyretin)  Principal fetal protein
 Migrates ahead of albumin and accounts for  Maternal serum AFP is measured between 15
approximately 4% of protein in the CSF and 20 weeks of gestation
 Usually not visible on routine serum protein  Increased in amniotic fluid and maternal
electrophoresis serum: neural tube defects, fetal distress,
 Indicator of nutritional status and is one of the spina bifida
proteins that transports thyroid hormones  Decreased level: Down’s syndrome, trisomy
 Increased in: steroids therapy, alcoholism, chronic 18
renal failure  In adults, used as tumor marker for liver cancer
 Decreased in: acute phase reaction (inflammation), and certain gonadal tumors in adults
malignancy, poor nutrition (protein malnutrition), 3. alpha-1-acid glycoprotein (α1-
tissue necrosis or liver disease AAG/Orosomucoid)
 Acute-phase reactant produced in the liver
Albumin Fraction  Binds to basic drugs (lidocaine)
 53-65% of the total protein (3.5-5.0 g/dL)  Increased in inflammatory disorders such as
 Synthesized in the liver rheumatoid arthritis, pregnancy, cancer,
 Largest single contributor to the total protein level in pneumonia and other conditions associated with
the body cell proliferation
 Migrates farthest on electrophoresis  Decreased in nephrotic syndrome
 Regulator of blood osmotic pressure and binds many 4. alpha-1-antichymotrypsin (α1-ACT)
analytes for transport in blood circulation  An acute phase reactant & a protease inhibitor
 Increased serum albumin does not occur naturally but produced in the liver
may be seen in dehydration (relative increase)  Deficiency is associated with liver disease
 Decreased serum level is seen in: liver disorders  Mutations have been identified in patients with
(decreased production), protein malnutrition, muscle Parkinson disease and chronic obstructive
wasting diseases, severe burns, renal disease, pulmonary disease
pregnancy, starvation, inflammation 5. alpha-1-lipoprotein – transports lipids (HDL)
6. inter-α-trypsin inhibitor (ITI) – protease
 Analbuminemia – absence of albumin (abnormality inhibitor
of genetic origin resulting from autosomal recessive 7. Gc-globulin (Group Specific Component;
trait) Vitamin D-Binding Protein)
 Bisalbuminemia – two bans in the albumin region  Transports vitamin D and binds actin
or a band which is broader than usual  Increased in the third trimester of pregnancy,
estrogen oral contraceptive therapy
Alpha-1-Globulin
 2.5-5% of total protein (0.1-0.4g/dL)

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 60
 Decreased in severe liver disease, protein-losing  Transport iron to its storage sites to form ferritin
syndromes  Also carries iron to cells, such as bone marrow
which synthesizes hemoglobin and other iron-
Alpha-2-Globulin containing compounds
 7-13% of total protein (0.3-0.8 g/dL)  Increased in iron deficiency anemia and
1. alpha-2-macroglobulin pregnancy
 One of the largest non-immunoglobulin proteins  Decreased in infections, liver disease,
in plasma produced by hepatocytes malnutrition, protein-losing disorders,
 Protease inhibitor; inhibits proteases such as inflammation
trypsin, pepsin, and plasmin 5. Hemopexin
 Also contributes more than one-fourth of the  Functions: remove circulating heme, ferriheme
thrombin inhibition normally present in the blood and porphyrins
 10x fold increased in nephrotic syndrome  Increased in diabetes mellitus, Duchenne-type
 Increased in diabetes, liver disease, use of muscular dystrophy, some malignancies
contraceptives and pregnancy (especially melanoma)
 Decreased in prostatic cancer and acute  Decreased in hemolytic disorders and
pancreatitis administration of diphenylhydantoin
2. haptoglobulin 6. Complement system
 Synthesized in the hepatocytes and, to the small  A collective term for several proteins that
extent, in cells of the reticuloendothelial system participate in the immune reaction and serve as
 Acute-phase reactant a link to the inflammatory responses
 Responsible for binding free hemoglobin  Increased in inflammatory states
 Increased in inflammatory conditions, burns,  Decreased in malnutrition, lupus erythematosus,
trauma, nephrotic syndrome & disseminated intravascular coagulopathies
 Decreased in intravascular hemolysis and liver (DIC)
disease 7. Fibrinogen
3. ceruloplasmin  One of the largest proteins in blood plasma
 Copper-containing protein (approx. 90% of synthesized in the liver; acute phase reactant
serum copper is bound in ceruloplasmin);  Classified as glycoprotein because it has a
 Copper transport, coagulation, angiogenesis, considerable carbohydrate content
ferroxidase activity, and defense against  Function: Precursor of fibrin clot; form a fibrin
oxidative stress clot when activated by thrombin
 Acute-phase reactant; synthesized in the liver  Seen only in plasma and not in serum
 Increased in pregnancy, inflammatory processes,  Increased in inflammation, pneumonia,
malignancies, intake of oral estrogen and oral rheumatoid fever, pregnancy and use of birth
contraceptives control pills
 Decreased in wilson’s disease, malnutrition,  Decreased in values generally reflect extensive
malabsorption, severe liver disease, and coagulation, liver disease and
menke’s kinky hair syndrome hypofibrinogenemia
8. C-Reactive Protein (CRP)
Beta Globulin  Synthesized in the liver and appears in the blood
 8-14% (0.6-1.1 g/dL) of patients with diverse inflammatory diseases
1. Pre-beta-Lipoproteins – transports lipids  Highly sensitive acute phase reactants (One of
(primarily VLDL triglyceride) the first acute phase proteins to rise in response
2. Beta-Lipoproteins – transports lipids (primarily to inflammation)
LDL cholesterol)  Motivates phagocytosis in inflammatory disease
3. B2-microglobulin (B2M) – component of  Increased in acute rheumatic fever, bacterial
leukocyte antigen (HLA) molecules infections, myocardial infarctions, rheumatoid
4. Transferrin (siderophilin) arthritis, carcinomatosis, gout and viral
 Major beta globulin infections.
 Major function: binds and transport iron

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 61
Gamma Globulin 5. IgE
 12-22% (0.5-1.7 g/dL)  Associated with allergic and anaphylactic
 Synthesized in the plasma cells wherein synthesis is reactions
stimulated by an immune response to foreign  Increased in allergic and anaphylactic reactions
particles and microorganisms. and during parasitic infections
 Five major classes:
1. IgG Miscellaneous Proteins
 Migrates throughout the gamma region 1. Fibronectin
 Most abundant class of antibodies found in blood  Plasma fibronectin: used as a nutritional marker
plasma and lymph  Fetal fibronectin (fFn): used to help predict the
 Can cross placenta short-term risk of premature delivery
 Act on bacteria, fungi, viruses, and foreign 2. Amyloid
particles  A protein polysaccharide complex produced and
 Increased in liver disease, infections, and deposited in tissue during some chronic infections,
collagen disease malignancies, and rheumatologic disorders
 Decreased is associated with an increased 3. Cystatin C
susceptibility of infections and monoclonal  A protein polysaccharide complex produced and
gammopathy deposited in tissue during some chronic infections,
2. IgA malignancies, and rheumatologic disorders
 Migrates between betta and gamma peaks
 Levels increase after birth Acute Phase Reactants
 Provides antibody protection in body secretions 1. Alpha-1-antitrypsin
 Protects mucous membranes; found in secretions, 2. alpha-1-acid glycoprotein
saliva & tears 3. Haptoglobin
 Increased in: liver disease, infections and 4. Ceruloplasmin
autoimmune diseases 5. Complement proteins
 Decreased in: inhibited protein synthesis, ataxia- 6. Fibrinogen
telangiectasia, hereditary immunodeficiency 7. CRP
disorders 8. Immunoglobulin
3. IgM
 Migrates closer to the beta region Negative Acute Phase Reactant
 Cannot cross the placenta; it is made by the fetus 1. Prealbumin
 First antibody that appears in response to 2. Albumin
antigenic stimulation and is present in b cells 3. Transferrin
 Increased in toxoplasmosis, cmv, rubella,
herpes, syphilis, various bacterial and fungal Protease Inhibitors
diseases, and waldenström macroglobulinemia 1. Alpha-1-antitrypsin
 Decreased in renal diseases associated with 2. Aplha-1-antichymotrypsin
protein loss and immunodeficiency disorders 3. Inter-alpha-trypsin inhibitor
4. IgD 4. Alpha-2 macroglobulin
 May help regulate B-cell function. 5. Cystatin C
 Increased in infections, liver disease, connective-
tissue disorders

CLINICAL APPLICATION OF SERUM PROTEIN ELECTROPHORESIS

CONDITIONS ALBUMIN ALPHA1 ALPHA2 BETA GAMMA
Acute inflammation N, D I I N, D
Chronic inflammation N, D I I N, I I
Hepatitis D D D I
Acute cirrhosis D D Beta-gamma bridge
Renal protein loss D I N, D
Hypogammaglobulinemia D
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 62
Hypergammaglobulinemia D I
Alpha-1 antitrypsin deficiency D
Diabetes mellitus D I I
Macroglobulinemia D I I
Myeloma D I
Gastrointestinal loss (protein losing enteropathies D D D D D
Acute thermal injury (burns) D D I N D
LEGEND: N - Normal; D – Decrease; I – Increase

 Total protein is usually elevated

 Abnormal increase of IgG and IgA will produce a
sharp peak in the gamma-globulin region
 Frequent occurrence of Bence Jones Proteins in
the urine
2. Waldenstrom’s macroglobulinemia
 The cancer cells in people with WM are similar to
those of multiple myeloma and non-Hodgkin
lymphoma (NHL). Multiple myeloma is considered
a cancer of plasma cells, and non-Hodgkin
lymphoma is a cancer of lymphocytes. WM cells
have features of both plasma cells and
lymphocytes.
 Characterized by increased IgM
3. Cryoglobulinemia (some cases, i.e., *types I and
II)
 Medical condition in which the blood contains
large amounts of pathological cold sensitive
antibodies called cryoglobulins – proteins
(mostly immunoglobulins themselves) that
become insoluble at reduced temperatures.
 Proteins that reversibly precipitate in serum left
overnight in the refrigerator
Monoclonal Gammopathies  “Cryoglobin/Cryoglobulin” refers to a physical
 A sharp peak in the gamma region due to an increase
property not a distinct protein.
in single class of immunoglobulin (plasma cells make
too many copies of one antibody) Polyclonal Gammopathy
 This increase of plasma cells results in a single
 Increase in concentration of more than 2
homogenous spike (“M protein”) in the beta-gamma immunoglobulin
region  Peak is broader and diffused
 When M protein is present, there is usually a decrease
 Usually all three major immunoglobulins (IgG, IgA and
in normal immunoglobulins IgM) are increased in variable concentrations
 High M proteins levels and decreased levels of other
 See in viral hepatitis, sarcoidosis, rheumatoid
immunoglobulins may be associated with a malignant arthritis, chronic infection, chronic liver disease,
clinical course Subacute bacterial endocarditis, AIDS
 Causes of monoclonal gammopathies include:
1. Multiple Myeloma
Nephrotic Syndrome
 Cancer that forms in a type of white blood cell
 Nephrotic syndrome is a kidney disorder that causes
called plasma cell. Plasma cells help you fight your body to pass too much protein in your urine.
infections by making antibodies that recognize  Nephrotic syndrome is a primary glomerular disease
and attack germs. Multiple myeloma causes characterized by hypertension, proteinuria, edema,
cancer cells to accumulate in the bone marrow, hypoalbuminemia, albuminuria, and hyperlipidemia.
where they crowd out healthy blood cells.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 63
 It can be caused by diabetes mellitus, connective rest of your body. Eventually, the disease can cause
tissue disease, glomerular disease, and circulatory permanent damage or deterioration of the nerves.
disease.  Basic defect is the breakdown of the myelin sheath
that covers the nerve fibers, leading to a number of
URINE PROTEIN ELECTROPHORESIS neurological complications
 Urine from a healthy individual  CSF is the primary material for lab testing in multiple
 Low level of total protein sclerosis
 A small amount of albumin, with small amount of  Oligoclonal bands (multiple bands, two or more bands)
other low-molecular-weight-proteins in the gamma-globulin regions are frequently seen
 Urine protein electrophoresis is done for the (usually IgG)
identification of Bence Jones Proteins in patients with  85-95% of patient with MS have oligoclonal bands
suspected multiple myeloma
 It should always be done in conjunction with serum HEMOGLOBIN ELECTROPHORESIS AND
electrophoresis for comparison HEMOGLOBINOPATHIES

CEREBROSPINAL FLUID PROTEIN Hemoglobin Structure and Function

ELECTROPHORESIS  With porphyrin ring and atom at the center
 Predominant fraction is albumin (just like in serum);  Hb is composed of 4 polypeptide chains 2 alpha and 2
other fractions are usually blurred beta chains
 The major difference between serum and CSF is the  3 fractions of Hb in the normal RBC:
presence of a very clear prealbumin fraction in CSF Hb A 2 alpha, 2 beta 95%
 CSF protein electrophoresis is done to observe the Hb A2 2 alpha, 2 delta 3.5%
Hb F 2 alpha, 2 gamma 2%
abnormal immunoglobulin bands seen in multiple
sclerosis
Hemoglobin Electrophoresis Patterns
1. At pH 8.6 (on either celullos acetate or agarose)
Multiple Sclerosis Cathode (–) Anode (+)
 Multiple sclerosis (MS) is a potentially disabling Origin A2 S F A
disease of the brain and spinal cord (central nervous C D
system) E
 In MS, the immune system attacks the protective 2. At pH 6.2 (on citrate agar)
sheath (myelin) that covers nerve fibers and causes Anode (+) Cathode (–)
C S Origin A F
communication problems between your brain and the A2
D

UNIT 9: NON-PROTEIN NITROGEN COMPOUNDS

INTRODUCTION
 The determination of non-protein nitrogenous NON-PROTEIN NITROGEN
substances in the blood has traditionally been used to  Determination of Total Urinary Nitrogen to assess:
monitor renal function. Nitrogen Balance for nutritional management
 Non-protein nitrogen (NPN) originated in the early  Present in concentration of mg/or dL or less because
days of clinical chemistry when analytic methodology of ready clearance into the urine
required removal of protein from the sample before  Consist of all nitrogen-containing substances of the
analysis. blood with the exception of protein
 The biochemistry, clinical utility, and analytical  Measurement of these substances has traditionally
methods for measurement of the NPN compounds been used to monitor renal function
urea, uric acid, creatinine, creatine, and ammonia are
presented in this unit.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 64
COMPONENTS OF NON-PROTEIN NITROGEN

CLINICALLY SIGNIFICANT NONPROTEIN NITROGEN COMPOUNDS

APPROXIMATE PLASMA APPROXIMATE URINE CONCENTRATION
COMPOUND
CONCETRATION (% OF TOTAL NPN) (% OF EXCRETED NITROGEN)
Urea 45-50 86.0
Amino acids 25 —
Uric acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 —
Ammonia 0.2 2.8

UREA (NH2CONH2)  Uremia/Uremic Syndrome – very high levels of

 Major excretory/ waste product of protein metabolism plasma urea accompanied by renal failure
in the liver. 1. Pre-renal Changes
 Formed In the liver from amino group (-NH2) and free a. Decreased blood flow to kidneys – reduction in
ammonia formed during protein catabolism via Urea blood flow delivers less urea to the kidney and
Cycle; amino acid serve as form of energy or stored therefore less urea is filtered
as fatty acid or glycogen.  Congestive heart failure
 Hepatic enzyme converts ammonia from amino acids  Shock, hemorrhage
to urea.  Dehydration
 Filtered from plasma by the glomerulus.  Other factors resulting in significant marked
 90% are excreted by the kidneys. decreased volume
 Concentration of urea in the blood is determined by: b. Level of Protein metabolism
 Protein diet  High protein diet
 Rate of protein catabolism  Increased protein catabolism
 Renal function/perfusion – Fever
 Principal excretory form of nitrogen: Urea – Major illness
 Used for calculation of Nitrogen balance: Urinary urea. – Stress
 Biochemistry: Oxidation of protein produces energy or – Burns
stored as fat and glycogen, the nitrogen is converted – Corticosteroid therapy
to urea and excreted as a waste product. – GI bleeding
2. Renal Charges
Blood Urea Nitrogen (BUN)  Acute/chronic renal failure
 Term is used when referring to urea measurement  Glomerulonephritis
 Historically, assay for urea were based on nitrogen  Tubular necrosis
content  Other intrinsic renal diseases
 Determination urinary concentration is of value in the 3. Post-renal Changes
assessment of nitrogen balance for nutritional  Due to obstruction of urine flow by tumors of the
management bladder or prostate stones, severe infection
 Abnormal serum urea levels may be due to prerenal,
renal, or postrenal disorders. CONDITIONS WITH DECREASED LEVELS OF PLASMA UREA
 Clinical Application: used to evaluate renal function  Decreased protein intake
 Poor nutrition
Disease Conditions  High fluid intake, overhydration
 Severe liver disease
CONDITIONS WITH INCREASED LEVELS OF PLASMA UREA  Severe vomiting and diarrhea
 Azotemia – denotes increase in serum nonprotein  Increased protein synthesis such as late pregnancy
nitrogenous compounds, primarily urea, but may and infancy
include other NPN substances as well; elevated urea
in blood

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 65
BUN/Creatinine Ratio CREATINE/CREATININE
 Aids in the differentiation of the cause of abnormal
urea concentration Creatine
 Normal: 10:1 or 20:1  Produced by the liver and pancreas from arginine,
 Higher elevation in BUN than creatinine glycine and methionine
– Caused by prerenal conditions wherein creatinine  Enters the bloodstream and distribute in cells
remains normal specially in the muscle where it is converted to
– Seen in CHF, increased protein breakdown phosphocreatine
 High BUN/creatinine ratio with an elevated creatinine  Increased in muscular dystrophy or poliomyelitis,
– Usually seen in post renal conditions hyperthyroidism, trauma
 Low BUN/creatinine ratio – Seen increase in urine and plasma creatine but
– Observed in conditions associated with decreased normal creatinine level
urea production – Plasma creatine levels are not elevated in renal
– Seen in low protein intake, acute tubular necrosis, disease
severe liver disease
 Reference Method: isotope dilution mass spectrometry DISEASE CONDITIONS
(IDMS)  Elevated in muscle disease
– Muscular dystrophy
– Poliomyelitis
– Hyperthyroidism
– Trauma (plasma creatine and urinary creatine
elevated, normal plasma creatinine)
 Measurement of creatine kinase is used typically for
the diagnosis of muscle disease because analytic
Figure 9.1. Enzymatic assay for urea methods for creatine are not readily available in most
clinical laboratories
– Plasma creatine concentration is not elevated in
renal disease

Creatinine
 Waste product of creatine formed during normal
muscle metabolism
 Released into the circulation at a relatively constant
rate that has been shown to be proportional to an
individual’s muscle mass
 Readily filtered by glomerulus, not reabsorbed by
tubules
 Small amount is secreted by the kidney tubules at
high serum concentrations
 Plasma creatinine concentration is a function of
relative muscle mass, the rate of creatinine turnover,
and renal function
 More specific, but less sensitive monitor of renal
function compared to bun
 In contrast to urea, it is not affected by diet, not
reabsorbed once secreted, formed at constant rate
depending on muscle mass
Figure 9.2. The urea cycle pathway  Used to determine completeness of 24-hours
collection
 Formed from the oxidation of creatine by creatine
hydrolase or addition of a strong acid or alkali.
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 66
 Jaffe reaction is used to measure creatinine; it is the Renal Clearance and Glomerular Filtration Rate
reaction of creatinine with alkaline picrate to form a  Creatinine Clearance (CrCl) – measure of the amount
colored compound of creatinine eliminated from the blood by the kidneys
 GFR – used to gauge renal function
 Note: creatine phosphate is converted to creatine in  Volume of plasma filtered (V by the glomerulus per
the synthesis of ATP from ADP (creatinine phosphate) unit of time (t)
catalyzed by enzyme creatine kinase/CK  Formula:
 Creatine are IRREVERSIBLY converted to creatinine in 𝐺𝐹𝑅 = 𝑈𝑐𝑟 𝑉𝑢 × 1.73
𝑃𝑐𝑟 𝑡 𝐴
small amounts which is excreted by the kidneys. 𝑈𝑟𝑖𝑛𝑎𝑟𝑦 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 (𝑚𝑔/𝑑𝐿 0 × 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝑙/𝑚𝑖𝑛)
𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 (𝑚𝑔𝑑𝐿) 6
 Precursor of creatinine is creatine
– For fresh urine specimen should be adjusted to pH  Specimen of Choice: 24h urine
7.0 so measurement of creatinine will be possible.  Inulin Clearance Test: gold standard test for GFR
 Daily excretion of creatinine is constant except in measurement
crushing injury and degenerative disease which
results to massive damage to muscle PREDICTION EQUATION FOR PATIENTS WITH CHRONIC
KIDNEY DISEASE
 Serum Creatinine is a specific but not a sensitive for 1. Serum creatinine
the measurement of Glomerular Function (60% of 2. Height
filtration capacity of the kidneys is lost when serum 3. Weight
creatinine becomes elevated. 4. Age

 Cystatin C – superior marker than serum creatinine to Formula: Cockcroft – Gault

measure GFR; substance of choice for evaluating GFR: 𝐶𝑐𝑟 (𝑚𝑙/𝑚𝑖𝑛)
(140 − 𝐴𝑔𝑒) × 𝑊𝑒𝑖𝑔ℎ𝑡
Plasma creatinine and Cystatin C =
72 × 𝑆𝑒𝑟𝑢𝑚 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 × (0.85 𝑖𝑓 𝑓𝑒𝑚𝑎𝑙𝑒)

DISEASE CONDITIONS MODIFICATION OF DIET IN RENAL DISEASE EQUATION

 Increase values are seen in:  Modified Schwartz Equation – developed to calculate
– Significantly decreased glomerular filtration eGFR in pediatric population
– Urinary tract obstruction  Formula:
– Muscular disease or injury (Duchenne’s Muscular
Dystrophy) 𝑒𝐺𝐹𝑅 (𝑚𝐿/𝑚𝑖𝑛/1.73 𝑚2) =
(0.41 ℎ𝑒𝑖𝑔ℎ𝑡)
𝑆𝑒𝑟𝑢𝑚 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒
CLINICAL APPLICATION
 To determine the sufficiency of kidney function
 Note: Height is measured in cm and Scr is serum
 The severity of kidney damage
(plasma) creatinine concentration in mg/dL. The
 To monitor the progression of kidney disease
equation can be used to calculate eGFR for children
 Measure completeness of 24 - hour urine
<18 years old.
𝑒𝐺𝐹𝑅 = 186 × 𝑝𝑙𝑎𝑠𝑚𝑎 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 − 1.154 × 𝐴𝑔𝑒
− 0.203 (𝑚𝑎𝑙𝑒)
× 0.742 (𝑖𝑓 𝑓𝑒𝑚𝑎𝑙𝑒)
× 1.210 (𝑖𝑓 𝑏𝑙𝑎𝑐𝑘)

IMPORTANCE
 Detect the onset of renal insufficiency
 Adjust drug dosages for drugs excreted by the kidney
 Evaluate therapies instituted for patients with chronic
renal diseases
 Document eligibility for Medicare reimbursement in
end-stage renal diseases
Figure 9.3. Interconcersion of creatine, creatine phosphate, and
creatinine
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 67
 Accrue points for patients awaiting cadaveric kidney – Glycogen storage disease type I (Glucose-6-
transplants phosphatase deficiency)
 Creatinine Clearance values decrease by 6.5 – Fructose intolerance (Fructose-1-phosphate
ml/min/1.73m2 decade. aldolase deficiency)
– Treatment of myeloproliferative disease with
URIC ACID cytotoxic drugs
 The product of catabolism of the purine nucleic acids – Hemolytic and proliferative processes
 Oxidation of proteins yields urea along with co2, h2o, – Purine rich diet
and inorganic acids. Catecholamine are oxidized , – Increased tissue catabolism or starvation
forming vanillylmandelic acid (vma) and homovanillic – Toxemia of pregnancy
acid (hva) – Lactic acidosis
 Filtered by the glomerulus and secreted by the distal – Chronic renal disease
tubules into the urine, most is reabsorbed in the – Drugs and poisons
proximal tubules and reused – Renal failure
 Insoluble in blood and can deposited in joints and
tissues causing inflammation  Increased production; raised serum levels:
 Produced even in the absence of dietary purine intake  Idiopathic mechanism assoc. with primary gout
 Synthesized in the liver catalyzed by enzyme  Excessive dietary purines (organs, legumes)
(xanthine oxidase)  Cytolytic Treatment of malignancies, esp. in
 In the kidney from the blood it is partially filtered, leukemia and lymphomas
reabsorbed and secreted. In low purine diet: daily  Polycythemia
excretion is 0.5 g and normal diet it is 1g.  Myeloid metaplasia
 Present in plasma as monosodium urate  Psoriasis
 Concentration >6.8 mg/dl results to saturated plasma  Sickle cell anemia
then urate crystals precipitate in tissues  Decreased excretion; raised serum levels:
 Measured to confirm diagnosis and monitor for  Alcohol ingestion
treatment of gout  Thiazide diuretics
 Lactic acidosis (competition in the binding sites in
Clinical Application the renal tubules)
 To confirm diagnosis and monitor treatment of gout  Aspirin doses <2 g/day
 To Prevent uric acid nephropathy (chemotherapeutic  Ketoacidosis (especially in children)
treatment)  Renal failure, any cause
 To assess inherited disorders of purine metabolism  Toxemia of pregnancy
 To detect kidney dysfunction and assist in the  Chronic renal disease
diagnosis of renal calculi
 Gout – a disease found primarily in men; usually  Decreased concentration
diagnosed between 30 and 50 years of age; formed – Liver disease
renal calculi – Defective tubular reabsorption (Fanconi syndrome)
 Hyperuricemia – result of overproduction of uric – Chemotherapy with azathioprine or 6-
acid ( purine-rich diet, drugs, and alcohol ) mercaptopurine
 Hypouricemia – less common than hyperuricemia – Overtreatment with allopurinol
and usually secondary to severe liver disease or – Pagets
defective tubular reabsorption. – Xanthine oxidase disease

Disease Conditions  Increased excretion; lower serum levels:

 Increased concentration  Probenecid, sulfinpyrazone, aspirin doses above
– Lesch-Nyhan syndrome (hypoxanthine-guanine 4 g/day
phosphoribosyltransferase - HGPRT)  Corticosteroids and ACTH
– Phosphoribosylpyrophosphate synthetase  Coumarin anticoagulants
deficiency  Estrogens

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 68
 Decreased production; lower serum levels:  Consumed by the parenchymal cells of the liver in the
 Allopurinol production of urea
 Excreted as ammonium by the kidney and acts t buffer
urine
 Used to monitor the progress of several clinical
condition
 Usually measured in order to evaluate hepatic coma
 Hepatic coma
 Caused by accumulation of ammonia in the brain
as a result of liver failure.
 The ammonia increases central nervous system
pH and is coupled to glutamate, a central
nervous system neurotransmitter, forming
glutamine.
 Blood and cerebrospinal fluid ammonia levels
are used to distinguish encephalopathy caused
by cirrhosis or other liver disease from
nonhepatic causes to monitor patients with
hepatic coma
 Ammonia is neurotoxic

Synthesis
 Bacteria in GIT breakdown CHON and release
ammonia.
 A normal liver removes ammonia from the portal
circulation and discards it in the form of urea which is
excreted in the urine.

Disease Conditions
 Liver failure – in severe liver disease when there’s
significant collateral circulation or when parenchymal
liver cell function is severely impaired, ammonia is
not removed from circulation and blood concentration
increases
 Reye’s syndrome – occurs most commonly in children
and can be fatal; the disease is preceded by a viral
AMMONIA infection, often treated with aspirin
 The product of amino acid deamination during protein  Inherited deficiencies of urea cycle enzymes
metabolism
 Anaerobic metabolic reactions occur in skeletal muscle
during exercise

UNIT 10: RENAL FUNCTION TEST

INTRODUCTION
 Kidney function tests are used in the assessment of renal disease, water balance, and acid-base disorders and in
situations of trauma, head injury, surgery, and infectious disease.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 69
ANATOMY OF RENAL (KIDNEY) 3. Loop of Henle – composed of thin descending limb
(medulla), ascending limb (both medulla and the
cortex)
4. Distal Convoluted Tubule – located in the cortex
5. Collecting Duct – formed of two or more DCT;
collection of urine

 Each kidney contains approximately 1 million

nephrons

Figure 10.1. Anatomy of the kidney

Kidney
 Paired, bean-shaped organs that is located in
retroperitoneal either side of the spinal column.
 Urine formation
 Fluid and electrolyte balance
 Regulation of acid-base balance
 Excretion of the waste products of protein metabolism
 Excretion of drugs and toxins
 Secretion of Hormones of renin, erythropoietin, 1,25-
Dihydroxy vitamin D3, and prostaglandins.

Parts of Kidney
1. Two Regions:
a. Outer region – called as “Cortex”.
b. Inner region – called as “Medulla”
2. Pelvis – basin-liked cavity at the upper end of the
ureter into which newly formed urine passes.
3. Bilateral ureters – thick walled canals (connected in
Figure 10.2. Representation of a nephron and its blood supply
the kidneys to the urinary bladder.
THREE BASIC RENAL PROCESSES
1. Glomerular Filtration
Parts of Nephron
2. Tubular Reabsorption
1. Glomerulus
3. Tubular Secretion
 Also known as Bowman’s capsule
 A capillary tuft surrounded by the expanded end of
a renal tubule.
 Filtration of incoming blood (1,200 – 1,500 mL
received each minute)
 Albumin, plasma proteins, cellular elements and
protein bound Figure 10.3. Renal processed of filtration, reabsorption, and
– Afferent arteriole secretion
– Efferent arteriole
 Filters out of 125 to 130 mL (protein free, cell-free  The figure above illustrates how three different
fluid) substances are variably processed by the Nephron
 Glomerular filtration rate (GFR) – the volume of and description of how specific substances are
blood per filtered per minute regulated to maintain homeostasis.
2. Proximal Convoluted – located in the cortex  Substance A: Filtered and secreted, but not
reabsorbed
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 70
 Substance B: Filtered and a portion reabsorbed DISTAL CONVOLUTED TUBULE
 Substance C: Filtered and completely reabsorbed  Shorter than the proximal tubule, it has two or three
coils that connect to a collecting duct.
Glomerular Filtration  For electrolyte and acid-base homeostasis
 Filtration of incoming blood (1,200 – 1,500 mL  Hormonal Control: Antidiuretic hormone (ADH) and
received each minute) aldosterone
 Albumin, plasma proteins, cellular elements and  ADH –peptide hormone secreted by the posterior
protein bound pituitary
– Afferent arteriole  Aldosterone
– Efferent arteriole  Produced by the adrenal cortex under the
 Filters out of 125 to 130 mL (protein free, cell-free influence of the renin-angiotensin mechanism
fluid)  Stimulates sodium reabsorption in the distal
 Glomerular filtration rate (GFR) – the volume of blood tubules and potassium and hydrogen ion
per filtered per minute secretion

Tubular Reabsorption COLLECTING DUCT

1. Proximal Convoluted Tubule (PCT)  The final site for either concentrating or diluting urine.
2. Loop of Henle  Reabsorbed water, sodium, chloride and urea
3. Distal Convoluted Tubule  Urea – for maintaining the hyperosmolality of the
4. Collecting Duct renal medulla

PROXIMAL CONVOLUTED TUBULE WATER, ELECTROLYTE, AND ACID-BASE

 Receives the now cell free and essentially protein- HOMEOSTASIS
free blood.
 It contains waste products that are toxic to the body Water
above a certain concentration, and a substance that  The contribution of water in the kidneys is to balance
are valuable to the body. the body through water loss or water conservation
 It returns the bulk of valuable substance back to the which is regulated by the hormone ADH.
blood circulation.  States of dehydration – the renal tubules reabsorb
 Water, sodium, and chloride (75%) water at their maximal rate, resulting in production of
 Glucose (100% up to the renal threshold) a small amount of maximally concentrated urine (high
 Amino acids, vitamins, and proteins and amounts of urine osmolality, 1,200 mOsm/L)
urea, uric acid, and ions (magnesium, calcium,  States of water excess – the tubules reabsorb water
potassium and bicarbonate) at only a minimal rate, resulting in excretion of a
 Tubular reabsorption – process when the substances large volume of extremely dilute urine (low urine
move from the tubular lumen to the peritubular osmolality, down to 50 mOsm/L)
capillary plasma
 Tubular secretion – secrete products of kidney tubular Electrolyte Balance
cell metabolism of (hydrogen ions, and drugs ex.  Sodium – primary extracellular cation in the human
Penicillin) body and excreted through the kidneys. (Renin-
 Two ways: angiotensin-aldosterone hormonal system is the
1. Describes the movement of substances from major mechanism for the control of sodium balance).
peritubular capillary plasma to the tubular lumen  Potassium – main intracellular cation in the body.
2. Describes when tubule cells secrete products of  Chloride – principal extracellular anion and is
their own cellular metabolism into the filtrate in the involved in the maintenance of extracellular fluid
tubular lumen. balance.
 Phosphate – occurs in higher concentration in the
LOOP OF HENLE intracellular than in the extracellular fluid
 A hairpin-like loop between the proximal tubule and environments. Either a protein bound or a non-
the distal convoluted tubule. (sodium and chloride are protein bound form, homeostatic balance under the
actively and passively reabsorbed) control of parathyroid hormone (PTH).
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 71
 Calcium – the second most predominant intracellular  Produced by the kidneys increase renal blood flow,
cation (inorganic messenger in the cell). sodium and water excretion, and renin release
 Magnesium – major intracellular cation (enzymatic  Act to oppose renal vasoconstriction due to
cofactor) angiotensin and norepinephrine.

Acid-Base Balance  B2-Microglobulin (B2-M)

 Regeneration of Bicarbonate ions  A small, non-glycosylated peptide (molecular
 Excretion of Metabolic Acids weight, 11,800 Da) found on the surface of most
 Reaction with Ammonia (NH3) nucleated cells.
 Reaction with Monohydrogen Phosphate  Evaluate kidney damage and to distinguish
between disorders that affect the glomeruli and the
KIDNEY SUBSTANCES SYNTHESIS renal tubules
 Renin  Myoglobin
 Erythropoietin  Low molecular-weight protein (16, 900 Da)
 1,25-dihydroxy vitamin D3  Associated with acute skeletal and cardiac muscle
 Prostaglandins injury.
 To bind and transport oxygen from the plasma
Renin membrane to the mitochondria in muscle cells
 The initial component of the renin-angiotensin-  Blood levels, increase with severe muscle injury.
aldosterone system  Rhabdomyolysis – release from skeletal muscle is
 Produced by the juxtaglomerular cells of the renal sufficient to overload the proximal tubules and
medulla (fluid volume or blood pressure decreases) cause acute renal failure
 It catalyzes the synthesis of angiotensin by cleavage  Microalbumin
of the circulating plasma precursor angiotensinogen.  Important for the management of patients with
– Angiotensin is converted to angiotensin II diabetes mellitus
(angiotensin-converting enzyme)  Microalbuminuria – describes small amounts of
– Angiotensin II – powerful vasoconstrictor that albumin in the urine
increases blood pressure and stimulates release of  Neutrophil Gelatinase-Associated Lipocalin
aldosterone from the adrenal cortex.  A 25-kDa protein expressed by neutrophils and
epithelial cells (proximal tubule.)
Erythropoietin  Upregulated in the presence of renal ischemia,
 A single-chain polypeptide produced by cells close to tubule injury, and nephrotoxicity.
the proximal tubules  Can be measured in plasma and in urine (elevated
 Hypoxia produces increased serum concentrations within 2-6 hours) of acute kidney injury.
within 2 hours.
 Acts on the erythroid progenitor cells in the bone DISEASES RELATED TO RENAL FUNCTION
marrow (increase the number of red blood cells (RBCs) 1. Acute Glomerulonephritis
 Chronic renal, erythropoietin is reduced. 2. Chronic Glomerulonephritis
3. Nephrotic Syndrome
1,25-dihydroxy vitamin D3 4. Tubular Disease
 It is form in the site of the kidneys. 5. Renal Calculi or Kidney Stones
 Vitamin D is one of three major hormones that 6. Renal Kidney Injury
determine phosphate and calcium balance and bone 7. Chronic Kidney Disease
calcification in the human body.
 Chronic renal, often associated with osteomalacia Types of Kidney Stones
(inadequate bone calcification, adult form of rickets) 1. Calcium oxalate
 Hyperparathyroidism
Prostaglandins  Hugh urine calcium
 A group of potent cyclic fatty acids formed from  Vitamin D toxicity
essential (dietary) fatty acids, primarily arachidonic  Sarcoidosis
acid.  Osteoporosis

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 72
2. Magnesium ammonium phosphate  Cause:
 Infectious processes  Cardiovascular system failure
3. Calcium phosphate  Consequent hypovolemia
 Excess alkali consumption 2. Intrinsic AKI
4. Uric acid  The defect involves the kidney
 Gout  Cause:
 High levels of uric acid in blood and urine  Acute tubular necrosis
5. Cystine  Vascular obstructions/inflammations
 Inherited cystinuria  Glomerulonephritis
3. Post renal AKI
Renal Kidney Injury  The defect lies in the urinary tract after it exits the
 Acute Kidney Injury (AKI) kidney
 Sharp decline in renal function as a result of an  Cause:
acute toxic or hypoxic insult to the kidneys  Lower urinary tract obstruction
 Most common and serious condition  Rupture of the urinary bladder

THREE TYPES OF AKI Chronic Kidney Disease – a clinical syndrome that

1. Prerenal AKI occurs there is gradual decline in renal function over
 The defect lies in the blood supply before it reaches time
the kidney

SYSTEMATIC CLASSIFICATION OF CHRONIC KIDNEY DISEASE

STAGES
STAGE DESCRIPTION GFR (ml/min per 1.73 m3)
At increased risk* 60 (with risk factors)
1 Kidney damage with normal or GFR >90
2 Kidney damage with normal or GFR 60-89
3 Moderate GFR 30-59
4 Severe GFR 15-29
5 Kidney failure <15
Glomerular filtration rate (GFR); chronic kidney disease (CKD)
*At risk patients should be screened. Stages 1-5 llustrate the progression of CKD.

ETIOLOGY OF CHRONIC RENAL FAILURE 4. Inflammatory diseases

1. Renal circulatory diseases  Ex: Tuberculosis, chronic pyelonephritis
 Ex: Renal vein thrombosis, malignant hypertension 5. Renal obstruction
2. Primary glomerular diseases  Ex: Prostatic enlargement, calculi
 Ex: SLE (systemic lupus erythematosus, chronic 6. Congenital renal deformity
glomerulonephritis  Ex: Polycystic kidneys, renal hypoplasia
3. Renal sequelae to metabolic disease 7. Miscellaneous conditions
 Ex: Gout, diabetes mellitus, amyloidosis  Ex: Radiation nephritis

UNIT 11: LIVER FUNCTION TEST

INTRODUCTION
 The liver is a very large and complex organ responsible for performing vital tasks that impact all body systems.
 Complex functions:
1. Metabolism of carbohydrates (CHO) , lipids, proteins (CHON), and bilirubin
2. Detoxification of harmful substances
3. Storage of essential compounds

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 73
4. Excretion of substances to prevent harm
 The liver is unique in the sense that it is a relatively resilient organ that can regenerate cells that have been destroyed
by some-short term injury or disease or have been removed.
 If the liver is damaged repeatedly over a long period of time, it may undergo irreversible changes that permanently
interfere with its essential functions.
 If the liver becomes completely nonfunctional for any reason, death will occur within approximately 24h due to
hypoglycemia.

GROSS ANATOMY OF THE LIVER

 Liver is a large and complex organ weighing approximately 1.2-1.5 kg in the healthy adult.
 Located beneath and attached to the diaphragm, is protected by the lower rib cage, and is held in place by ligamentous
attachments.
 Divided unequally into two lobes by the falciform ligament.
– Right lobe is larger than the left lobe.
 It has two sources of blood supply:
1. Hepatic artery – a branch of the aorta, supplies oxygen-rich blood from the heart to the liver and responsible for
providing approximately 25% of the total blood supply to the liver
2. Portal vein – supplies nutrient-rich blood (collected as food is digested) from the digestive tract, and it is responsible
for providing approximately 75% of the total blood supply to the liver
 Sinusoid
– Blood flow to the central canal (central vein) of each lobule.
– It is through the central canal that blood leaves the liver.

– Approximately 1,500 mL of blood passes through the liver per minute.

– Liver drained by a collecting system of veins that empties into the hepatic veins and ultimately into the inferior
vena cava.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 74

 Excretory system of the liver begins at the bile canaliculi.

– Bile canaliculi – small spaces between the hepatocytes that form intrahepatic ducts, where excretory products
of the cell can drain.
 Lobules
– Functional units of the liver
– Responsible for all metabolic and excretory functions performed by the liver
– Each have a six-sided structure with a centrally located vein (central vein) with portal triads at each of the corners
– Portal triads contain:
 Hepatic artery
 Portal vein
 Bile duct
 Two major cell types of liver:
1. Hepatocytes
2. Kupffer cells

FOUR MAJOR FUNCTION OF THE LIVER

1. Excretion/secretion
2. Metabolism
3. Detoxification
4. Storage

Excretory and Secretory

 One of the most important functions of the liver
 The excretion of endogenous and exogenous substances into the bile or urine as the major heme waste products
 The liver is the only organ that has the capacity to rid the body of heme waste products.
 Body produces approximately 3L of bile per day and excretes 1 L of what is produced.
– Bilirubin – the principal pigment in bile and it is derived from the breakdown of red blood cells.
 The heme portion of hemoglobin is converted to bilirubin in 2-3h
 The bilirubin is bound by albumin and transported to the liver.
 Unconjugated/indirect bilirubin – form of bilirubin is insoluble in water and cannot removed from the body
 Conjugated/direct bilirubin – is the form of bilirubin in water soluble and is able to be secreted from the
hepatocyte into the bile canaliculi.
 Uridyldiphosphate glucoronyl transferase (UDPGT) – enzyme that is presence in the conjugation
(esterification) of bilirubin
 Urobilinogen – a colorless product
 Urobilin (stercobilin) – orange-colored product of urobilinogen and excreted in the feces; gives stool a brown
color
– Approximately 200-300 mg of bilirubin is produced per day.

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 75

Metabolism
 The liver is responsible for metabolizing many biological compounds including CHO, lipids, and CHON.
 The metabolism of CHO is one of the most important functions of the liver.
 Three things that liver can do when the CHO are ingested and absorbed:
1. Use the glucose for its own cellular energy requirements
2. Circulate the glucose for use at the peripheral tissues
3. Store glucose as glycogen (principal storage form of glucose) within the liver itself or within the tissues

 Liver is the major player in maintaining stable glucose concentrations due to its ability to store glucose as glycogen
(glycogenesis) and degrade glycogen (glycogenolysis).

Detoxification
 The liver serves as a gatekeeper between substances absorbed by the gastrointestinal tract and into systemic
circulation.
 It allows substances to reach the systemic circulation and can serve as the barrier to prevent toxic or harmful substances
from reaching systemic circulation.
 Two mechanism for detoxification:
1. Foreign materials (drugs and poisons)
2. Metabolic products (bilirubin and ammonia)

DISEASE RELATED TO THE LIVER

1. Jaundice
2. Cirrhosis
3. Tumors
4. Reye syndrome
5. Alcohol-induced liver injury

Jaundice
 Comes from the French word jaune means “Yellow”
 One of the oldest known pathologic conditions
 Jaundice or icterus – used to describe the yellow discoloration of the skin, eyes and mucous membranes.
 Icterus – most commonly used in the clinical laboratory to refer to a serum or plasma sample (due to elevated bilirubin
level).
 Types of Jaundice:
1. Prehepatic Jaundice
 Occurs when the problem causing the jaundice occurs prior to liver metabolism.
 Most commonly caused by an increased amount of bilirubin (seen in acute and chronic hemolytic anemia)
 It refers to as unconjugated hyperbilirubinemia.
2. Hepatic – occurs when the problem causing the jaundice resides in the liver (intrinsic liver defect or disease)
a. Gilbert Syndrome
 Benign autosomal recessive hereditary disorder
 Results from a genetic mutation in the gene (UGT1A1) that produces UDPGT (enzyme for bilirubin metabolism)
b. Crigler-Najjar Syndrome
 More severe and dangerous form of hyperbilirubinemia
 A syndrome of chronic nonhemolytic unconjugated hyperbilirubinemia
 Type I – complete absence of enzymatic bilirubin conjugation
 Type II – mutation causing a severe deficiency of the enzyme responsible for bilirubin conjugation
c. Dubin-Johnson
 Characterized as conjugated hyperbilirubinemia

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 76
 Rare autosomal recessive inherited disorder caused by a deficiency of the canalicular multidrug
resistance/multispecific organic anionic transporter protein (MDR2/cMOAT).
 The appearance of dark-stained granules on a liver biopsy sample.
d. Rotor Syndrome
 Clinically similar to Dubin-Johnson syndrome but the defect is not known.
 Hypothesized to be due to a reduction in the concentration or activity of intracellular binding proteins (ligandin)
 Relatively benign condition and carries an excellent prognosis
e. Jaundice of Newborn
 Deficiency in the enzyme glucoronyl transferase
 One of the last liver functions to be activated in prenatal life
 Kernicterus – cell damage caused by excessive levels of bilirubin in the blood during infancy
3. Post Hepatic Jaundice
 Results from biliary obstructive disease
 Usually from physical obstructions (gallstones or tumors) that prevent the flow of conjugated bilirubin into the bile
canaliculi

CHANGES IN CONCENTRATION OF BILIRUBIN IN THOSE WITH JAUNDICE

SERUM
TYPES OF JAUNDICE TOTAL BILIRUBIN CONJUGATED BILIRUBIN UNCONJUGATED BILIRUBIN
Prehepatic   
Hepatic
Gilbert’s disease   
Crigler-Najjar syndrome   
Dubin-Johnson   
Rotor syndrome   
Jaundice of newborn  
Posthepatic   

Cirrhosis
 Clinical condition in which scar tissue replaces normal, healthy liver tissue

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 77
 Sign and symptoms: fatigue, nausea, unintended weight loss, jaundice, bleeding from the gastrointestinal tract,
intense itching and swelling in the legs and abdomen
 Other causes: Chronic hepatitis B (HBV), Hepatic C (HCV), and Hepatitis D virus (HDV) infection, autoimmune hepatitis,
inherited disorders (ex. a1-antitrypsin deficiency, Wilson disease, hemochromatosis, and galactosemia), nonalcoholic
steatohepatitis, blocked bile duct, drugs, toxins, and infections

Tumors
 Cancers of the liver are classified as primary or metastatic
 Primary liver cancer – begins in the liver cells
 Metastatic cancer – occurs when tumors from other parts of the body spread (metastasize) to the liver
 Commonly spread to the liver include colon, lung, and breast cancer
 Can be classified as benign or malignant
 Benign tumors of the liver (hepatocellular adenoma: condition occurs in females of child-bearing age) and
(hemangiomas: masses of blood vessels with no known etiology)
 Malignant tumors, hepatocellular carcinoma (HCC) (also known as hepatocarcinoma, and hepatoma) and bile duct
carcinoma

Reye Syndrome
 Used to describe a group of disorders caused by infectious, metabolic, toxic, or drug-induced disease found almost
exclusively in children.
 Often preceded by a viral syndrome (varicella, gastroenteritis, or an upper respiratory tract infection such as influenza)
 Acute illness characterized by non-inflammatory encephalopathy and fatty degeneration of the liver

Alcohol-Induced Liver Injury

 Excessive consumption of alcohol
 Categorized into three stages:
1. Alcoholic fatty liver
 Slight elevated in AST, ALT and GGT
 On biopsy, fatty infiltrates are noted in the vacuoles of the liver
 Affect young to middle-aged people with a history of moderate alcohol consumption
2. Alcoholic hepatitis
 Presents with common signs and symptoms : fever, ascites, proximal damaged
 Moderately elevated AST, ALT, GGT, alkaline phosphatase (ALP) and elevated in total bilirubin >5 mg/dL
3. Alcoholic cirrhosis
 Most severe stage
 Common in males than females
 Symptoms: weight loss, weakness, hepatomegaly, splenomegaly, jaundice, ascites, fever, malnutrition and
edema.
 Increased liver function tests (AST, ALT, GGT, ALP and total bilirubin); decreased albumin and prolonged prothrombin
time.
 Liver biopsy – only method for definitive diagnosis

 Delta Bilirubin – the third fraction of bilirubin; conjugated bilirubin that is covalently bound to albumin

ENZYMES
1. Aminotransferases
2. Phosphatases

Aminotransferases
 AST
 Formerly referred to as serum glutamic oxaloacetic transaminase (SGOT)

TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 78
 Found in the heart, skeletal muscle and kidney
 ALT
 Formerly referred to as serum glutamic pyruvic transaminase (SGPT)
 Found mainly in the liver
 Specific marker
 Conditions:
 Acute (viral hepatitis)
 Drug
 Toxin induced liver necrosis
 Hepatic ischemia
 Organ dysfunction or failure (acute myocardial infarction, renal infarction, progressive muscular dystrophy
 Secondary liver diseases (infectious mononucleosis, diabetic ketoacidosis, and hyperthyroidism)

Phosphatases
 Alkaline phosphatase (ALP)
 Alkaline phosphatase
 Widely distributed in all tissues
 Highest activity seen in the liver, bone, intestine, kidney, and placenta
 May be seen in increased concentration (bone diseases, pregnancy, and childhood growth)
 5’Nucleotidase (5NT)
 Responsible for catalyzing the hydrolysis of nucleoside-5’phosphate esters.
 Elevated in hepatobiliary disease
 Useful in differentiating ALP elevations due to the liver form other conditions
 Gamma glutamyltransferase (GGT)
 Found in high concentrations in the kidney, liver, pancreas, intestine, and prostate but not in bone
 Seen in biliary obstruction
 Hepatic microsomal enzyme
 Ingestion of alcohol or certain drugs ( barbiturates, tricyclic antidepressants, and anticonvulsants)
 Useful if jaundice is absent for the confirmation of hepatic neoplasms
 Lactate dehydrogenase (LD)
 Enzyme with a very wide distribution through-out the body
 Released into circulation when the body are damaged or destroyed
 Serving as a general nonspecific marker of cellular injury
 Moderate elevated in acute viral hepatitis and cirrhosis
 High serum levels may be found in metastatic carcinoma of the liver

 Hepatic coma – caused by severe viral hepatitis or by intoxication

HEPATITIS
 Presence of inflammation in the liver tissue
 Include viral, bacterial and parasitic infections and non-infections (radiation, drugs, chemical, and autoimmune diseases
and toxins
 Symptoms:
 Jaundice
 Dark urine
 Fatigue
 Nausea
 Vomiting
 Abdominal pain

Types of Hepatitis
TPMEB 2020
 MEDT 13 CLINICAL CHEMISTRY I 79
1. Hepatitis A Virus (HAV)
 Known as infections hepatitis or short incubation hepatitis
 Most common form of viral hepatitis worldwide
 Caused by nonenveloped RNA virus of the Picornavirus family
 Fecal-oral route
 Symptoms: fever, malaise, anorexia, nausea, abdominal discomfort, dark urine and jaundice
2. Hepatitis B Virus (HBV)
 Serum hepatitis or long incubation hepatitis
 Cause both acute and chronic hepatitis
 Most ubiquitous of the hepatitis viruses (DNA nucleotide)
 Detected in all body fluids : blood, feces, urine, saliva, semen, tears, and breast milk
 Three major routes: parenteral, perinatal and sexual
 Sharing of drug needles
 Sharing of body fluids
 Children born to mother who are positive in hepatitis B surface antigen (HBsAg)
3. Hepatitis C Virus (HCV)
 Originally “non-A non –B hepatitis
 Caused by a virus with an RNA genome that is member of the Flaviviridae family
 Transmitted parenterally (Sexual and fecal-oral routes)
4. Hepatitis D Virus (HDV)
 Unique subviral satellite virus infections
 Small, defective RNA containing virus that cannot replicate independently but rather requires the HBsAg of HBV for
replication
 Modes of transmission is similar in HBV
5. Hepatitis E Virus (HEV)
 Nonenveloped RNA virus
 Only 27-34 nm in diameter
 Sole member of the genus Hepevirus in the family Hepeviridae
 Fecal-oral route and waterborne epidemics
 Most severe in pregnant women

TPMEB 2020