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Avi Sayag

Clinical Biochemistry

Table of contents

Topic 1: Reference range, requesting a test, sources of error, interpretation of results, specificity and sensitivity, predictive value…………………………………………………………………….4 Topic 2: Laboratory signs of cellular damage. Basis of clinical enzymology…………………7 Topic 3: Inborn errors of amino acid metabolism and lab diagnosis of CF……………………9 Topic 4: inborn errors of carbohydrate and lipid metabolism………………………………… Topic 5: Pathobiochemistry of inflammation…………………………………………………. Topic 6: Pathobiochemistry of plasma proteins………………………………………………. Topic 7: Biochemical effects of tumors………………………………………………………. Topic 8: Tumor markers in the diagnosis of malignant diseases……………………………… Topic 9: Iron metabolism, hemochromatosis, iron deficiency anemia……………………… Topic 10: Lab diagnosis of hemoglobinopathies……………………………………………… Topic 11: Lab diagnosis of hemolytic anemia……………………………………………… Topic 12: Lab diagnosis of megaloblastic anemia…………………………………………… Topic 13: Major lab characteristics of ALL and CLL………………………………………… Topic 14: Major lab characteristics of AML and CML………………………………………… Topic 15: Lab diagnostics of quantitative platelet disorders………………………………… Topic 16: Inheritance of ABO blood group system and its clinical significance……………… Topic 17: Inheritance and clinical significance of Rh blood group system…………………… Topic 18: Coagulopathies, lab control of anticoagulant treatment……………………………. Topic 19: Platelet function disorders………………………………………………………… Topic 20: Inherited thrombophilias…………………………………………………………… Topic 21: Acquired thrombophilias…………………………………………………………… Topic 22: Consumption coagulopathies………………………………………………………. Topic 23: Lab tests of glomerular and tubular functions……………………………………… Topic 24: Clinical biochemistry of acute and chronic renal failures; tubulopathies…………. Topic 25: Disturbances of acid-base balance…………………………………………………. Topic 26: Predominant water depletion; isoosmolar volume depletion……………………… Topic 27: Water and sodium excess; predominant sodium depletion………………………… Topic 28: Hypokalemia………………………………………………………………………. Topic 29: Hyperkalemia………………………………………………………………………. Topic 30: Pathogenesis of diabetes mellitus…………………………………………………. Topic 31: Lab diagnosis and management of diabetes mellitus………………………………. Topic 32: Acute metabolic complications of diabetes mellitus………………………………. Topic 33: Hypoglycemia………………………………………………………………………. Topic 34: Disorders and lab diagnosis of lipid metabolism………………………………… Topic 35: Risk factors of atherosclerosis………………………………………………………. Topic 36: Disturbances of uric acid metabolism……………………………………………… Topic 37: Lab diagnostics of acute MI………………………………………………………… Topic 38: Hypovitaminosis and hypervitaminosis……………………………………………. Topic 39: Lab diagnosis of hepatocellular damage; evaluation of liver function……………. Topic 40: Lab diagnosis of cholestasis and liver cirrhosis……………………………………. Topic 41: Pathobiochemistry and laboratory diagnostics of the gastrointestinal tract…………. Topic 42: Laboratory diagnosis of acute pancreatitis………………………………………… Topic 43: Clinical biochemistry of the hypothalamus and the pituitary………………………. Topic 44: Pathobiochemistry and laboratory diagnosis of hypothyroidism and hyperthyroidism Topic 45: Hypocalcemia………………………………………………………………………… Topic 46: Hypercalcemia……………………………………………………………………… Topic 47: Clinical biochemistry of disturbances of the adrenal cortex…………………………. Topic 48: Clinical biochemistry of disturbances of the adrenal medulla………………………. Topic 49: Clinical biochemistry of the reproductive system……………………………………. Topic 50: Lab procedure and diagnosis of bone and skeletal disorders…………………………. Topic 51: Lab procedures in the diagnosis of muscle disorders…………………………………. Topic 52: Clinical biochemistry at the extremes of age…………………………………………. Topic 53: Clinical biochemistry and lab diagnosis of porphyrias………………………………. Topic 54: Lab diagnostics of CNS diseases; lab investigation of the CSF………………………. Topic 55:

Avi Sayag

Clinical Biochemistry

Practical topics

Topic 1: Molecular genetic methods for the investigation of inherited diseases………….14 Topic 2: Determination of Hb and Htc……………………………………………………. Topic 3: Procedure of blood drawing, vacutainer tubes…………………………………… Topic 4:

Topic 5:

Topic 6: Principles of cell counting and differentials by hematology analyzers…………. Topic 7: Characterization of leukemic cells by morphology……………………………… Topic 8: Characterization of leukemic cells by cytochemistry and immunophenotyping…. Topic 9: Determination of ABO and Rh blood group…………………………………… Topic 10: Determination of PT……………………………………………………………. Topic 11: APTT assay……………………………………………………………………. Topic 12: Thrombin time, D-dimer determination………………………………………. Topic 13: Bleeding time, Detection of fibrin monomers………………………………… Topic 14: Lab methods for the determination of urea and creatinine…………………… Topic 15: Examination of urine (general and sediment analysis)…………………………. Topic 16: Measurement of serum sodium and potassium………………………………… Topic 17: Determination of glucose in serum; point of care tests…………………………. Topic 18:

Topic 19: Cholesterol, HDL, LDL assays………………………………………………… Topic 20: Triglyceride assay, visual test, lipoprotein electrophoresis…………………… Topic 21: Tests in the lab diagnosis of MI…………………………………………………. Topic 22: Assay of serum bilirubin. Detection of bilirubin and UBG in the urine………… Topic 23:

Topic 24: Tests in the investigation of bone metabolism………………………………… Topic 25: Principles of chromatography and its application in diagnostics……………….15

Avi Sayag

Clinical Biochemistry

Topic 1

Reference range, requesting a test, sources of error, interpretation of results, specificity and sensitivity, predictive value

In clinical lab investigation there are 3 phases:

1. The pre-analytical phase: requesting the test, preparing the patient, collecting the sample, transporting it and storing it.

2. Analytical phase (manual or automatic)

3. Post-analytical phase: calculation of the results, validation of the results, consultation, reporting, making the archive and interpreting the results.

Requesting a test:

The test request should include the following:

1. Patient's name, sex, date of birth and insurance number;

2. Ward/clinic/address;

3. The name of the requesting doctor and ways to contact him/her.

4. The diagnosis;

5. The name of the test requested;

6. The type of specimen;

7. Date and time of sampling;

8. Relevant treatment;

9. Indication of potentially hazardous samples;

Sources of error

In the pre-analytical phase, any one of the steps can lead to an error:

1. Requesting the test in an inappropriate manner (switching names of patients, e.g.). Errors made during collection of the specimens: when blood specimen is collected, some variables can influence the results, such as posture, venous stasis, hemolysis, the labeling of the container. Also, the chemical composition of urine can change during collection: destruction of glucose by bacteria; urea is converted to ammonia, the pH decreases and phosphate precipitates; urobilinogen and porphobilinogen are oxidized. Hemolysis interferes with the determination of CK, because RBCs release adenylate cyclase (not CK!). AC converts 2 ADPs to ATP + AMP, and the ATP carries the second reaction, leading to overestimation of CK levels.

2. During storage, blood specimens can change: glycolysis occurs in RBCs (glycolysis

in whole blood causes a 5% decrease in blood glucose level per hour), K + and LDH are released from RBCs (hemolysis during venipuncture can lead to pseudohyperkalemia – see topic 29), CO 2 is lost from the specimen, organic phosphate esters are hydrolyzed, and labile enzymes lose their activity. In the analytical phase, human errors or instrumental errors can occur. In this category, the error can be systemic: accuracy vs. inaccuracy; precision vs. imprecision; or random errors. The ideal analytical method is accurate, precise, sensitive and specific. Precise: the result is the same if the procedure is repeated. It is assessed by repeated analysis

(n=15-20):

Within batch variation: the variation between the results of repeated tests in the same "batch" (on the same day, for the same person, with the same specimen collected).is repeated. It is assessed by repeated analysis (n=15-20): Day-to-day variation Precision is expressed as coefficient

Day-to-day variation Precision is expressed as coefficient of variance: CV(%) = SDx100/mean. Therefore, if the SD is very small and approaches the mean, the CV(%) is close to 100% (= the test is very precise). Accurate : it gives the same result, that is, the deviation from the assigned value Accurate: it gives the same result, that is, the deviation from the assigned value ("true" value). Accuracy is given by: 100(mean-X t )/ X t

Avi Sayag

Clinical Biochemistry

Avi Sayag Clinical Biochemistry The mean value is the same in method A and B, but

The mean value is the same in method A and B, but the scatter about the mean is less in method A than in method B. Therefore, method A is said to be more precise. Both method C and D are equally precise, but in method D the mean value differs from the true value. Method C is more accurate.

The last source of variation is the biological one. The variation can be within-individual or between-individual. The diet of a person, one's posture, the drug the person takes, etc. all influence the results and variation can occur from test to test for the same person. Age, race and sex can make the results different for different individuals.

After we have requested the test and considered all possible errors on the way, we have finally received the results and have to interpret them. Reference interval: the interval between two reference limits, including the limits. It is designed as the central interval of values bounded by the lower and upper reference limit at certain designated percentiles, usually at percentile 2.5 and 97.5 (that is, it is supposed to include 95% of the reference population). When choosing the reference population, we have to select for the most appropriate ones, and exclude those who consume alcohol for example (unless we wish to establish reference range

for alcoholics), obese people, drug abusers, fasting people or non-fasting, etc. Also, we have to select the group we wish to establish reference range for. Therefore, we partition the population according to sex, race, age, blood group, stage of menstrual cycle, pregnancy, exercise, fasting/non-fasting and so forth. As the reference limit refers to the mean value of the sampled population, there are other limits as well:

1. Medical decision limit (optimal cut-off values): in glucose levels, for example, DM is defined as the level of glycemia at which diabetes-specific complications occur rather than on deviations from a population-based mean.

2. Risk limit: for example, the risk limit for cholesterol is 5.8 mmol/L (a reference range does not exist, as cholesterol level exceeds the desired value in the majority of the population).

3. Panic value - results from a specimen that must be reported immediately to a

clinician, i.e., of such severity as to mandate urgent therapy. For example, glucose levels < 2.6 mmol/L or > 26.9 mmol/L, Na + < 120 mmol/L or > 158 mmol/L. When considering a lab test, it is important to know its sensitivity and specificity. Sensitivity is the incidence of true positive results or the ability of the method to measure low concentrations of the analyte. It is given by the formula:

TP

Sensitivity = ×100

TP + FN

Specificity is the incidence of true negative results, and the test is not subject to interference by other substances. It is given by the formula:

Specificity =

When judging the positive or negative results, the predictive value should be considered.

TN

TN + FP

×100

Avi Sayag

Clinical Biochemistry

TP

The predictive value of positive results = ×100 . This is the proportion of patients

TP + FP

with positive results who are correctly diagnosed. It reflects the probability that a positive result reflects the underlying condition being tested for.

TN

The predictive value of negative results = ×100 . This is the proportion of patients

TN + FN

with negative test results who are correctly diagnosed. For example, the predictive negative value of a low result of CRP in CSF ruling out bacterial meningitis is 97%. That is, if a person gets a low CRP result in CSF examination, he has a 97% chance that he really does not have a bacterial meningitis.

Myoglobin lacks cardiac specificity as a marker. That is, if the myoglobin is elevated 4-8 hours following the onset of pain, but the ECG shows no sign of cardiac condition, then more cardiac-specific markers should be sought for. However, if myoglobin is not elevated 4-8 hours following the onset of pain, myocardial necrosis can be excluded. That is, myoglobin is sensitive. It is the only early marker measured (4-6 hours), despite its specificity.

Avi Sayag

Clinical Biochemistry

Topic 2

Laboratory signs of cellular damage. Basis of clinical enzymology

Cellular damage can be caused by physical agents, genetic defects, hypoxia, chemical agents, nutritional imbalance, immunological reactions, infections and aging. As the cell injury progresses, such as in MI, the ion pumps are the first to fail, leading to ion leakage to the ECF. Then, metabolites leak out (e.g. lactate, adenosine) and finally, as the membrane is damaged, macromolecules leak out (enzymes and proteins). Ions and metabolites drain to the intravascular space and less to the interstitial space, as opposed to macromolecules that drain to the interstitial space and slowly drained through the lymphatic system. There are 2 main groups of enzymes in the plasma:

1. Enzymes released from cells as a result of leakage or cell death. These enzymes have no known function in the blood. Serum enzymes in health are derived from the metabolic breakdown and turnover of cells and tissues.

2. Enzymes with clearly defined actions in the blood (e.g. coagulation factors, ACE).

When measuring an enzyme, what influences the value obtained?

1. The rate of release from cells: 4 factors affect the rate

a. Hypoxia/anoxia/drugs membrane permeability leakage

b. Cell necrosis release of mitochondrial enzymes

c. Increased synthesis (e.g. in bile duct obstruction, synthesis of enzymes (ALP, GGT) is induced)

d. Duct obstruction (e.g. liver and pancreas)

2. The volume of distribution of the enzyme in the ECF

3. The rate of removal from the plasma (catabolism in RES or excretion in bile and urine). The clearance from the plasma also depends on the half life of the enzyme.

For example, CK has a 1/2 life of 1.4 days and GPT - 6.3 days.

4. The presence of factors in the plasma that may affect the method of assay (i.e.

inhibitors or activators of enzyme activity). Enzymes can have isoenzymes and isoforms:

Isoenzymes: enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different K M values), or different regulatory properties. The existence of isoenzymes permits the fine- tuning of metabolism to meet the particular needs of a given tissue or developmental stage (e.g. LDH). In many cases, they are coded for by homologous genes that have diverged over time. Alleloenzymes represent enzymes from different alleles of the same gene, and isoenzymes represent enzymes from different genes that process or catalyse the same reaction. An example of an isozyme is glucokinase, a variant of hexokinase which is not inhibited by G6P. Its different regulatory features and lower affinity for glucose (compared to other hexokinases), allows it to serve different functions in cells of specific organs, such as control of insulin release by the beta cells of the pancreas, or initiation of glycogen synthesis

by liver cells. Both of these processes must only occur when glucose is abundant, or problems occur. Isoenzymes can be organelle specific (enzymes of the mitochondria e.g.) or tissue specific. Isoforms: different forms of the same enzyme, which are not the result of genetic causes. Isoforms can be formed by post-translational modification on the protein component or on the non-protein component as well as by aggregation of enzyme molecules (CK-MM, CK-MB, CK-BB). We can use several techniques to differentiate isoenzymes and isoforms:

1. Zone electrophoresis

2. Isoelectric focusing

3. Differences in catalytic properties

4. Immunochemical methods

5. Selective inactivation

6. Ion exchange chromatography

These factors are important in making the choice of enzyme activity measurement:

Sensitivity, selectivity, time-course of elevation and technical errors.

Avi Sayag

Clinical Biochemistry

The international unit of enzyme activity: the amount of enzyme which, under given assay conditions, will catalyze the conversion of 1 µmol of substrate per minute.

Enzyme assays have not been standardized. Therefore, the reference range may change from lab to lab. The given assay conditions include:

1. The nature and concentration of the substrate

2. The direction of the reaction

3. The temperature

4. The pH, concentration and nature of the buffer

5. The presence of inhibitors and activators

Katal = mol/sec 1 nkatal = 16.7 IU (international unit)

Avi Sayag

Clinical Biochemistry

Topic 3

Inborn errors of amino acid metabolism and lab diagnosis of CF

Amino acids (AA) are supplied to the body by diet (essential AA) and by de novo synthesis. As proteins are synthesized in the liver and in other tissues, the AA pool decreases. Deamination (degradation) of AA in the liver and kidney, as well as transamination, also decrease the AA pool of the body. Under normal conditions, the AA level in the plasma changes during the day (diurnal variation). In the urine, AA are present: glycine>alanine>serine>glutamine>histidine. During pregnancy it changes to histidine>phenylalanine>lysine>tyrosine. Cells have more AA than the plasma, and the plasma level of AA is greater than that of the CSF. In the liver, AA and α-ketoglutarate contribute to the synthesis of liver and plasma proteins, purines, pyrimidines, porphyrines, hormones, etc. Also, they contribute to gluconeogenesis and the formation of urea. In the kidney, AA are filtered and completely reabsorbed by active transport systems (one for

neutral AA, one for basic AA (lysine, arginine and histidine), one for proline, hydroxyproline and glycine, and one for dicarboxylic AA (aspartate and glutamate) ). Homocysteine and cystathionine are not efficiently reabsorbed. Aminoaciduria: high blood levels of AA that result in significant renal excretion. This can be primary (genetic) or secondary (liver/kidney disorders). There are 3 types:

1. Overflow aminoaciduria due to plasma AA level that exceeds tubular capacity. In this case the plasma AA level is high.

2. Renal aminoaciduria due to a defect in the renal transport. In this case the plasma level of AA is normal.

3. No-threshold aminoaciduria due to excessive level of AA in the plasma, but with renal excretion, plasma AA level remains normal.

Effects of enzyme defects: (a) Product D is synthesized from A by a series of reactions catalyzed by enzymes a, b and c. Enzyme c' catalyzes the formation of a small amount of product E in a minor pathway. (b) In the absence of the enzyme c, no D is synthesized. (c) If the conversion of C to D is blocked, the concentration of the intermediate C, and the possibly other precursors, may increase. (d) Increased formation of E may occur if the concentration of C increases and conversion of C to D is blocked.

of C increases and conversion of C to D is blocked. There are 6 disorders that

There are 6 disorders that should be mentioned (there are more than 6 inherited AA disorders, but these are the ones addressed in the course):

1. Hyperphenylalaninemia (and phenylketonuria)

2. Tyrosinemia

3. Alkaptonuria

4. Cystinuria

5. Homocystinuria

Avi Sayag

Clinical Biochemistry

1. Hyperphenylalaninemias (HPA)

HPA results from impaired conversion of Phe to tyrosine.

HPA results from impaired conversion of Phe to tyrosine. The most common and clinically important is

The most common and clinically important is phenylketonuria (PKU). PKU is an autosomal recessive disorder. There are several HPAs that are not PKU and are called non-PKU HPAs. HPA is defined as a plasma phenylalanine concentration >120µM. PKU is characterized by plasma phenylalanine >1000µM and non-PKU hyperphenylalaninemias have plasma phenylalanine amounts that are <1000µM. PKU is caused by mutation in the phenylalanine hydroxylase gene (PAH). The HPAs are disorders of phenylalanine hydroxylation. Because the reaction catalyzed by PAH involves tetrahydrobiopterin (BH4) as a co-factor, the HPAs can result from defects in any of the several genes required for synthesis and recycling of BH4. Removal of excess phenylalanine normally proceeds via the tyrosine biosynthesis reaction and then via tyrosine catabolism. The first reaction in this process is the PAH catalyzed hydroxylation of phenylalanine. There are 5 types of HPA, of which 3 are PKU (types I and II are worth mentioning: type I: a defect in Phe hydroxylase, and type II: a defect in dihydropteridine reductase). The accumulation of Phe inhibits the transport of other AA required for protein or neurotransmitter synthesis, reduces synthesis and increases degradation of myelin, and leads to inadequate formation of norepinephrine and serotonin. Phe is a competitive inhibitor of tyrosinase, a key enzyme in the pathway of melanin synthesis, and accounts for the hyperpigmentation of hair and skin. Untreated children with classic PKU are normal at birth, but fail to attain early development milestones, develop microcephaly, and demonstrate progressive impairment of cerebral function. Hyperactivity, seizures and mental retardation are major clinical problems later in life. To prevent mental retardation, diagnosis and initiation of dietary treatment of classic PKU must occur before the child is 3 weeks of age. Lab tests:

Screening: Guthrie test (on the 6 th -10 th day of life) Specific diagnosis: determination of plasma Phe and Tyr by HPLC (see following practical topic 25) Monitoring: determination of plasma Phe by HPLC Prenatal diagnosis: DNA analysis (see following practical topic 1) Treatment consists of diet low in Phe and supplemented with Tyr. (Gene therapy is being developed). Guthrie test: A drop of blood is usually obtained by pricking the heel of a newborn infant on the 6 th or 7 th day of life. A small disk of the filter paper is punched out and placed on an agar gel plate containing Bacillus subtilis and B-2-thienylalanine. Each gel holds 60-80 disks. The agar gel is able to support bacterial growth but the B-2-thienylalanine inhibits bacterial growth. However, in the presence of extra phenylalanine leached from the impregnated filter paper disk, the inhibition is overcome and the bacteria grow. Within a day the bacterial growth surrounding the paper disk is visible to the eye. The amount of growth, measured as the diameter of the colony, is roughly proportional to the amount of phenylalanine in the serum.

of growth, measured as the diameter of the colony, is roughly proportional to the amount of
of growth, measured as the diameter of the colony, is roughly proportional to the amount of
of growth, measured as the diameter of the colony, is roughly proportional to the amount of
of growth, measured as the diameter of the colony, is roughly proportional to the amount of

Avi Sayag

2. Tyrosinemia

Clinical Biochemistry

There are three types of tyrosinemia, each with distinctive symptoms and caused by the deficiency of a different enzyme. Type I tyrosinemia is the most severe form of this disorder and is caused by a shortage of the enzyme fumarylacetoacetate hydrolase. Fumarylacetoacetate hydrolase is the last in a series of five enzymes needed to break down tyrosine. Symptoms of type I tyrosinemia usually appear in the first few months of life and include failure to gain weight and grow at the expected rate (failure to thrive), diarrhea, vomiting, jaundice, cabbagelike odor, and increased tendency to bleed (particularly nosebleeds). Type I tyrosinemia can lead to liver and kidney failure, problems affecting the nervous system, and an increased risk of liver cancer. Worldwide, type I tyrosinemia affects about 1 person in 100,000. Type II tyrosinemia is caused by a deficiency of the enzyme tyrosine aminotransferase Tyrosine aminotransferase is the first in a series of five enzymes that converts tyrosine to smaller molecules, which are excreted by the kidneys or used in reactions that produce energy. This form of the disorder can affect the eyes, skin, and mental development. Symptoms often begin in early childhood and include excessive tearing, photophobia, eye pain and redness, and painful skin lesions on the palms and soles. About half of individuals with type II tyrosinemia are also mentally retarded. Type II tyrosinemia occurs in fewer than 1 in 250,000 individuals. Type III tyrosinemia is a rare disorder caused by a deficiency of the enzyme 4- hydroxyphenylpyruvate dioxygenase. This enzyme is abundant in the liver, and smaller amounts are found in the kidneys. It is one of a series of enzymes needed to break down tyrosine. Specifically, 4-hydroxyphenylpyruvate dioxygenase converts a tyrosine byproduct called 4-hydroxyphenylpyruvate to homogentisic acid. Characteristic features of type III tyrosinemia include mild mental retardation, seizures, and periodic loss of balance and coordination (intermittent ataxia). Type III tyrosinemia is very rare; only a few cases have been reported.

is very rare; only a few cases have been reported. Alkaptonuria is an autosomal recessive condition

Alkaptonuria is an autosomal recessive condition that is due to a defect in the enzyme homogenistic acid oxidase, which participates in the degradation of tyrosine. As a result, a toxic tyrosine byproduct called homogentisic acid (or alkapton) accumulates in the blood and is excreted in urine in large amounts. Excessive homogentisic acid causes damage to cartilage (HGA binds to collagen). The presentation also includes pigmentation in the ears and degenerative arthritis in middle age.

3. Cystinuria

Cystinuria is an AR disorder caused by defective transporters in the apical brush border of proximal renal tubule and small intestinal cells. It is characterized by impaired reabsorption and excessive urinary excretion of the dibasic AA lysine, ornithine and cystine. Because cystine is poorly soluble, its excess excretion predisposes to the formation of kidney stones, which are responsible for the signs and symptoms of the disorder. Lab tests:

Screening: cystine stones in the urinefor the signs and symptoms of the disorder. Lab tests: Specific diagnosis: AA detection in the

Specific diagnosis: AA detection in the urine by HPLC Treatment includes intake of large amount of water, alkalinizing the urine and administration of penicillamine (captopril) which chelates cystine.which are responsible for the signs and symptoms of the disorder. Lab tests: Screening: cystine stones

Avi Sayag

4. Homocystinurias

Clinical Biochemistry

Homocystinurias are 7 biochemically and clinically distinct disorders characterized by increased concentration of homocystine in blood and urine. The most common one is the classic homocystinuria, which results from reduced activity of cystathionine β-synthase (the enzyme that condenses homocysteine with serine to form cystathionine). Symptoms include vascular complications during the first decade of life and are the major cause of morbidity and mortality (due to homocysteine). Other symptoms include defects in collagen metabolism due to cystine, and some patients develop marfanoid habitus and radiologic evidence of osteoporosis. Other rare causes include a deficiency in MTHFR and MTHF transferase. Lab tests:

Screening: Guthrie testa deficiency in MTHFR and MTHF transferase. Lab tests: Specific diagnosis: determination of homocystine and

Specific diagnosis: determination of homocystine and methionine by HPLC. False negative results can occur if the diagnosis is made before 3 days of life, and false positive result can occur in case of premature enzymes, excessive protein intake, tyrosinemia and hepatitis.and MTHF transferase. Lab tests: Screening: Guthrie test 5. Urea cycle defects Excessive ammonia generated from

5. Urea cycle defects

Excessive ammonia generated from protein nitrogen is removed by the urea cycle, a process mediated by several enzymes and transporters. Complete absence of any of these enzymes usually causes severe hyperammonemia in newborns, while milder variants can be seen in adults. The accumulation of ammonia and glutamine leads to brain edema and direct neuronal toxicity. These disorders are AR, except for ornithine transcarbamylase deficiency, which is X-linked.

Any of the 5 enzymes can be deficient: 1. CPS: Carbamyl-Phosphate synthetase 2. OTC: Ornithine
Any
of
the
5
enzymes
can
be
deficient:
1. CPS: Carbamyl-Phosphate
synthetase
2. OTC:
Ornithine
transcarbamylase
3. ASS:
Argininosuccinate
synthetase
4. ASL: Argininosuccinase
5. ARG: Arginase

Symptoms include lethargy, vomiting, liver failure and coma. Lab tests:

Screening: detection of plasma ammonialethargy, vomiting, liver failure and coma. Lab tests: Specific diagnosis: detection of plasma and urine AA/enzyme

Specific diagnosis: detection of plasma and urine AA/enzyme detection.Arginase Symptoms include lethargy, vomiting, liver failure and coma. Lab tests: Screening: detection of plasma ammonia

Avi Sayag

Lab diagnosis of CF

Clinical Biochemistry

1. Sweat test: useful in the severe form of CF. Sweat is collected by pilocarpine

iontophoresis, and the concentration of chloride is determined:

a. Borderline values: 40-59 mM

b. Elevated values: >60 mM

2. The presence of IgG against Pseudomonas aeruginosa

3. Immunoreactive trypsinogen (used in newborn screening programs)

4. Tests of exocrine pancreatic functions:

a. Indirect: presence of enzymes in the stool and the fat levels; the level of

carotene in the serum;

b. Direct: analysis of the pancreatic secretion before and after stimulation with secretin and CCK (cholecystokinin)

5. DNA based CFTR mutation analysis: the tested mutations cover almost 60-95% of diagnosed CF cases. There are 4 classes of mutations:

a. Class I: caused mainly by premature termination due to splicing defects, frameshifts due to small deletions or insertions; non-sense mutations. The CFTR can be reduced or completely absent.

b. Class II: defective protein synthesis; misfolded protein; no transport to the membrane.

c. Class III: defective regulation; mutant proteins reach the membrane, but the function of the channel is altered.

d. Class IV: defective conduction. The channel is correctly processed and delivered to the membrane, but it generates reduced Cl - current. The rate of ion flow is reduced or the amount of time the channel is open is reduced.

6. ARMS, PCR-OLA, oligonucleotide hybridization assay

a. PCR-ARMS – Amplification Refractory Mutation System (also called: allele specific oligonucleotide hybridization): a method of PCR which distinguishes between alleles that differ in even a single nucleotide substitution. In this procedure the allele specific oligonucleotides (ASO) are immobilized on a nitrocellulose membrane and hybridized with a labeled PCR product spanning the variant nucleotide site. Discrimination between the 2 alleles is based on the fact that in a specific hybridization temperature the perfectly matched hybrid is more stable than the mismatched. After hybridization and washing, the detection is mainly colorimetric: if the PCR product is labeled with biotin, e.g. the detection is based on the strepatavidin-alkaline phosphatase conjugate enzyme reaction with a substrate. By using 2 ASOs it is possible to determine the wild type, heterozygous or homozygous.

b. PCR-OLA: PCR followed by oligonucleotide ligation assay (OLA) is a molecular method that can be used for the detection of nucleotide sequence variants. PCR-OLA is based on the enzymatic ligation of two oligonucleotides that anneal next to each other onto the PCR-amplified target DNA. Even a single-nucleotide mismatch between the oligonucleotides and the template precludes the ligation.

c. 2 CF mutations + an abnormal sweat test are diagnostic of CF!

7. Nasal potential difference: this test measures the potential difference between the apical and the basal surface of the nasal mucosa in the presence or absence of an

epithelial sodium channel antagonist (amiloride, e.g.). A decrease in the potential difference is much higher in CF patients than in healthy ones.

8. Bronchoalveolar lavage (lavage=washing a hollow organ): in this procedure there is a high percentage of neutrophils in the lavage fluid among CF patients (normally, neutrophils comprise 3% of the lavage fluid, whereas in CF patients it reaches 50%). P. aeruginosa may also be present.

9. Prenatal diagnosis is performed when family anamnesis suggests CF:

a. Immunoreactive trypsin in amniotic fluid

b. DNA tests (chromosome 7q31.2)

Avi Sayag

Clinical Biochemistry

Molecular genetic methods for the investigation of inherited diseases (Practical topic 1)

There 4 steps in genetic diagnosis of inherited diseases:

1. Isolation of genomic DNA: by traditional method or spin column purification

2. Determination of the concentration and purity of the genomic DNA

3. Storage of DNA

4. Mutation detection: by Southern blot or PCR. Following these, electrophoresis, restriction digestion, hybridization probes, hybridization with allele specific oligonucleotides and multiplex ligation-dependent probe amplification (MPLA) can be used.

1. Isolation of genomic DNA

The sample is taken from the buffy coat from peripheral blood anticoagulated by citrate or

EDTA. In the traditional method:

a. Cell harvesting by SDS 1

b. Proteolysis with a special enzyme

c. Multiple extractions by phenol, chloroform, isoamyl alcohol to dissolve hydrophobic contaminants and remove proteins.

d. DNA is precipitated by 96-100% ethanol

e. DNA is washed by 70% ethanol

f. DNA is dissolved in TRIS buffer

Spin column method: this method is based on the phenomenon that DNA/RNA binds selectively to silica in high salt concentration. Cell lysates are loaded onto a spin column containing silica membrane, and then several washing steps follow. This method is fast but expensive.

2. Determination of DNA purity

DNA yield is determined by measuring the concentration of DNA by absorbance at 260nm. As the peak absorbance of DNA is at 260nm, that of proteins is at 280nm. Thus, purity is

determined by calculating the ratio of absorbance at 260nm to that at 280nm. Pure DNA has a ratio of 1.7-1.9. Ratio less than 1.7 indicates protein contamination.

3. Storage

DNA can be stored at 4ºC for months, at -20ºC for years, and at -70ºC for at least 10 years.

4. Mutation detection

Southern blot:

a.

Restriction digestion: using enzymes that recognize specific base compositions that cleave the DNA at these sites. If the recognition sequence changes (for example due to a point mutation), the enzyme cannot cleave the DNA.

b.

Electrophoresis: separation according to size through an agarose gel or acrylamide gel. The detection is based on the intercalation of the fluorescent dye (ethidium bromide).

c.

Denaturation

d.

Transference to nylon membrane by capillary, vacuum or electric transfer.

e.

Immobilization by UV light

f.

Hybridization with a labeled probe

g.

Washing off the unbound probes

h.

Detection (depending on the labeling)

1 SDS – Sodiun Dodecyl Sulfate. This compound disrupts non-covalent bonds in proteins, denatures them, and causes the molecule to lose its native shape. Also, anions of SDS bind to the main peptide chain at a ratio of 1 SDS for every 2 AA residues. This imparts a negative charge on the protein that is proportional to the mass of that protein. This new negative charge is significantly greater than the original charge of that protein. The electrostatic repulsion created by binding of SDS causes proteins to unfold into a rod-like shape, thereby eliminating differences in shape as factor for separation in the gel.

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Clinical Biochemistry

PCR: amplification of a well-defined DNA fragment. The reaction consists of cycles:

a. Denaturation at 95ºC to separate the strands of the DNA template

b. Fast cooling to 50-65ºC to hybridize the primers and the polymerases (Taq)

c. Incubation at 72ºC to synthesize the new strands

4 main types of PCR:

1. Standard PCR: to detect small variations in genetic material (e.g. Factor V Leiden

mutation).

2. Multiplex PCR: involves parallel amplification of different pieces of DNA (e.g. Duchene

muscular dystrophy).

3. Long PCR: combines 2 enzymes: Taq polymerase and a proof-reader, which make it

possible to amplify larger DNA fragments (e.g. Friedrich ataxia).

4. Quantitative Real-time PCR: used to quantify the copy number of a specific DNA in the

sample (e.g. translocation 9:22).

Principles of chromatography and its application in diagnostics (Practical topic 25)

Chromatography is used when the substance to be measured needs to be separated from other disturbing components. A small sample volume is injected in a continuous flow of gas or liquid (the mobile phase). The mixture of the sample and the mobile phase flows through the stationary phase, which usually consists of solid particles. On the stationary phase, the

components of the mixture can be separated using different principles, because the different components spend different period of time in the stationary phase due to their different chemical properties. There are 3 main methods:

1. Adsorption chromatography: the particles of the stationary phase bind the components of the sample mixture. Different components remain bound for various amounts of time according to their chemical properties (those with low affinity to the stationary phase spend shorter time).

2. Ion exchange chromatography: the stationary phase consists of ion exchange particles, which bear surface-bound ionic groups with their counter-ions. Components of the sample replace the counter-ions from the surface of the particles. Different components bind to the fixed ions with different affinity.

3. Size exclusion chromatography: molecules are separated based on their physical size.

Column chromatography may utilize 2 methods: gas chromatography and high performance liquid chromatography (HPLC). Gas chromatography: this method utilizes a high purity gas as eluent with constant pressure or flow rate. This technique is suitable for the detection of analytes that can be brought to gas phase and stay stable at the high analysis temperature of 100-350ºC. Gas chromatography can be used both for quantitative and qualitative analysis. HPLC: in the standard HPLC, the mobile phase is a stable liquid, and the stationary phase can be either a liquid or a solid substrate. The eluent circulates in a closed system and the constant flow is provided by a pump. The effluent coming from the column is transferred into a small cuvette of a detector. The detectors can be photometric, fluorimetric, electrochemical and refraction detectors. HPLC is applicable to measure materials which are soluble and stable in the mobile phase. The upgraded methods of HPLC are GC/MS and LC/MS (GS: gas chromatography; LC:

liquid chromatography; MS: mass spectrometry). The different analytes reach the detector (the MS), separated, and analyzed. Clinical uses: PKU, cystinuria, homocystinuria, detection of HbA 1c (on cation exchange column), hemoglobinopathies, vitamin D disorders, and detection of catecholamines and their metabolites (for example, catecholamines are elevated in neuroblastoma and melanoblastoma).

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Clinical Biochemistry

Topic 5

Pathobiochemistry of inflammation

Inflammation can be acute or chronic: the acute one lasts from few minutes to few days, while the chronic one lasts from days to weeks. Acute inflammation is characterized by the presence of exudate and neutrophils, and the chronic one is characterized by the presence of lymphocytes, macrophages and tissue destruction. The chemical mediators released during the inflammatory process are responsible for the vascular response and for the cellular response. The vascular changes: in acute inflammation they manifest in a local increase in blood flow

that results in redness and heat, and in increased vascular permeability that results in transudation. As the intravascular osmotic pressure decreases, the interstitial osmotic pressure increases and exudation replaces transudation. The accumulation of fluids in the extravascular tissue contributes to the edema. As the blood becomes more viscous, stasis ensues. The cellular changes: manifest in emigration of leukocytes from the microcirculation that accumulate in the inflamed site and are activated there. Leukocytes undergo 5 stages:

1. Margination and rolling: this is facilitated by the selectins expressed on the

leukocytes as they are activated. L-selectins bind to E-selectins and P-selectins.

2. Adhesion and transmigration: adhesion is carried out by expression of integrins that bind to ICAM-1 expressed on the endothelium.

3. Chemotaxis and activation

4. Phagocytosis and degranulation

5. Leukocyte-induced tissue injury: injury is caused by lysosomal enzymes, by ROS, and arachidonic acid metabolites. Pain and loss of function result.

Chemical mediators:

1. Chemical mediators of cellular origin:

a. Vasoactive amines stored in secretion granules:

i. Histamine: present in mast cells and basophils. It is released in response to physical injury, immune reactions, C3a and C5a, proteins

released from leukocytes and IL-1 and IL-8 (cytokines). It causes arteriolar dilation, contraction of endothelium in venules and it increases vascular permeability.

ii. Serotonin: present in platelets and is released by platelet-activating agents. Its effects are similar to those of histamine.

b. Enzymes stored in secretion granules:

i. Acid proteases: present in phagolysosomes only.

ii. Neutral proteases: these include elastase, collagenase and cathepsins.

They cause the destruction of the basement membrane and ECM. The control of these proteases is carried out by anti-proteases (e.g. α1-

antitrypsin).

c. Newly synthesized mediators:

i. Prostaglandins: synthesized in leukocytes, platelets and endothelium. PgI 2 causes vasodilation and inhibits platelet activation; PgD 2 , PgE 2 and PgF 2α cause mainly vasodilation and potentiate edema; TXA 2 causes vasoconstriction and activates platelets.

ii. Leukotrients: synthesized in leukocytes. LTB 4 is chemotactic; LTC 4 , LTD 4 and LTE 4 cause vasoconstriction, bronchospasm and increased permeability.

iii. Platelet-activating factor: synthesized in leukocytes and endothelium.

iv. Oxygen radicals: synthesized in neutrophils and macrophages by NADPH oxidase. These include superoxide, hydrogen peroxide, hydroxyl free radical and toxic NO derivatives. They cause endothelial injury and thrombosis, activation of proteases and inactivation of anti-proteases, and injury of other cells. Normally, they are counteracted by catalase, superoxide dismutase and glutathione.

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Clinical Biochemistry

v. NO: synthesized in endothelium and macrophages. It is synthesized from arginine, in the presence of O 2 , NADPH and NOS. It causes vasodilation, inhibits platelet activation and killing of bacteria in activated macrophages.

vi. Cytokines: synthesized in activated lymphocytes, macrophages and endothelium. Cytokines are polypeptides that have local effects on endothelium, leukocytes and fibroblasts, as well as systemic effects.

1. IL-1, IL-6 and TNFα are synthesized in activated macrophages. They are produced in the acute phase reaction and are responsible for the fever, lethargy, neutrophilia, synthesis of acute-phase proteins, corticosteroids and cachexia. They also cause endothelial activation that results in leukocyte adhesion, synthesis of PgI, NO and thrombogenesis.

2. IFN-γ is synthesized in T-cells and activates neutrophils and macrophages, and induces NO synthesis.

3. Chemokines: these are synthesized in activated macrophages, endothelium and fibroblasts. They cause chemotaxis and neutrophil activation (e.g. IL-8).

2. Mediators derived from the plasma:

a. Activation of the kinin system: FXII is activated and cleaves prekallikrein. The formed kallikrein cleaves off bradykinin from kininogen. Bradykinin increases vascular permeability, causes vasodilation, bronchospasm and mediates pain. As FXII is activated, tissue plasminogen activator (t-PA) is also released.

b. Activation of coagulation: induction of tissue factor in macrophages and endothelium.

c. Activation of complement system

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Clinical Biochemistry

Topic 6

Pathobiochemistry of plasma proteins

When measuring the proteins in the plasma it is possible to measure the total protein

concentration, the albumin and globulin fractions (semi-quantitative assessment) or specific proteins. What is the clinical significance in measuring the total serum protein? Total serum protein may decrease in the following settings:

1. Decreased protein synthesis (malnutrition, malabsorption, starvation, hepatic failure)

2. Hemodilution

3. Humoral immunodeficiency

4. Increased loss of proteins

5. Catabolic states

Total serum protein may increase in the following settings:

1. Hemoconcentration (dehydration, stasis, posture)

2. Increased synthesis of plasma proteins (hypergammaglubolinemia, paraproteinemia)

What are the most important serum proteins?

Prealbumin

 

Albumin

 

α1 globulins

α1-antitrypsin

α1-acid glycoprotein

α2 globulins

α2-macroglobins

Haptoglobins

Ceruloplasmin

β globulins

Transferrin

Complement components

γ globulins

IgG, IgA, IgM, IgD, IgE

1. Prealbumin

Prealbumin is a protein status indicator; it has a much shorter half-life and smaller serum pool than albumin. The half-life of prealbumin is approximately 2 days, making prealbumin a more timely and sensitive indicator of protein status. Prealbumin is a tryptophan-rich protein, and like albumin, it is synthesized in the hepatocytes of the liver. Prealbumin's main function is to serve as a binding and transport protein. The term prealbumin is actually a misnomer-the prefix pre implies that it is a precursor for albumin, which it is not. The more accurate name for prealbumin is transthyretin to indicate that it is a serum transport protein for thyroxin and retinol-binding protein. Evaluating prealbumin Like albumin, prealbumin is a negative acute-phase reactant. This limits its use as a screening tool for malnutrition because low levels could result from either inadequate nutrition or inflammatory stress. Rather than a diagnostic tool, prealbumin should be used as an indicator of nutritional improvement and as a measure of how well nutritional interventions are working. The very short half-life and small serum pool allows small changes in nutritional status to be identified in a short time frame. Elevated prealbumin levels may be seen in patients taking corticosteroids and in patients with Hodgkin disease.

2.

Albumin

Low levels of albumin can result from:

a. Overhydration

b. Decreased synthesis (malabsorption, malnutrition, starvation)

c. Increased loss (nephrotic syndrome, Crohn's disease)

The consequences of low levels of albumin may be:

a. Edema

b. Secondary aldosteronism

c. Hypocalcemia (but ionized Ca +2 is not decreased)

d. Increased risk of kernicterus in infants

e. Altered pharmacokinetics of albumin-bound drugs

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Clinical Biochemistry

High levels of albumin may result from:

a. Dehydration

b. Venous stasis during blood collection

c. Overinfusion of albumin

Remember: increased albumin synthesis does not cause hyperalbuminemia! So when do we decide to measure albumin levels in the plasma?

1.

When we have to elucidate the cause of edema;

2.

To follow up nutritional status (also measuring prealbumin);

3.

To evaluate liver function (except for acute hepatitis where albumin is not

informative);

4.

To elucidate the cause of low calcium levels in the plasma;

5.

When we wish to administer a drug that binds to albumin;

6.

When we wish to diagnose analbuminemia.

3.

α1-antitrypsin

AAT is an α1-globulin, a protease inhibitor and an acute phase protein. When it is deficient,

homozygotes are prone to develop emphysema and liver disease. The deficient level is detected by isoelectric focusing, and the genotype (homo- or heterozygote) is determined by PCR techniques.

4. Haptoglobin

This α2-globulin binds free Hb and is an acute phase protein. Its levels decrease in:

a. Intravascular hemolysis (absent) and extravascular hemolysis (low)

b. Chronic liver disease

c. Metastases of carcinoma

d. Severe sepsis

Its levels increase in:

a.

Acute phase reactions

b.

Nephrotic syndrome

5.

α2-macroglobulin

This is a protease inhibitor, whose synthesis is increased in hypoalbuminemia (the decreased

albumin is replaced by the high-molecular-weight macroglobulin)

6. Ceruloplasmin

Ceruloplasmin transports copper. Low levels are associated with Wilson's disease, and high

levels are evident in acute phase reactions, pregnancy and use of oral contraceptives.

Acute phase reactions This is a systemic, non-specific reaction to acute inflammation, infection, burns, tissue necrosis and tumor proliferation. It includes fever, leukocytosis, complex hormonal and metabolic changes (altered levels of acute phase proteins). Acute phase proteins can be positive or negative.

1. Positive acute phase reactants:

Early, sensitive reactants:

a. α1-antitrypsin

b. α1-acid glycoprotein

c. Haptoglobin

d. Fibrinogen

e. C-reactive protein (CRP)

f. Procalcitonin

Late, weak reactants:

a.

Ceruloplasmin

b.

C3, C4

2.

Negative acute phase reactants:

a.

Prealbumin

b.

Albumin

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Clinical Biochemistry

Cytokines, such as IL-1, IL-6 and TNF, increase the mRNA of positive acute phase reactants, and decrease the mRNA of the negative ones. Synergethic hormones, such as glucocorticoids, enhance the reaction. What is the role of the acute phase proteins?

1. They inhibit the destructive proteases released from leukocytes during inflammation.

2. Scavanger proteins (CRP, haptoglobin) help to present cellular debris and breakdown products to the RES in order to process them and retain precious components.

3. Fibrinogen is required for wound healing.

What are the classic parameters used to detect acute phase reactions? Fever, leukocytosis, ESR and serum protein electrophoresis. Of the acute phase reactants,

CRP is the most sensitive and specific. C-reactive protein (CRP) Synthesized in the liver, CRP reacts with the C-polysaccharide of Streptococcus pneumonia.

In the presence of calcium it bnds to phospholipids, polyanions and galactans. Through these molecules it binds to many bacteria, fungi, protozoa and cellular debris.reacts with the C-polysaccharide of Streptococcus pneumonia. In protein electrophoresis, it migrates somewhere between

In protein electrophoresis, it migrates somewhere between the γ band to mid-β region. Its function is to initiate opsonization, phagocytosis and cell lysis. Once it is complexed, it activates the classical complement pathway. CRP is elevated in 4 main scenarios:

1. Inflammation: CRP shows an early rise (4-6 hours), which is more intense than ESR. When the patient recovers, its levels decrease before those of ESR. Mostly, its levels decrease when the inflammatory process is suppressed by steroids or salicylates. Thus, it is an excellent tool to monitor the activity level of rheumatoid arthritis, rheumatic fever, vasculitides, etc. Of particular interest, CRP is significantly higher in Crohn's disease than in ulcerative colitis, and its levels correspond to relapse, remission and response to therapy.

2. Tissue injury and necrosis: in AMI, CRP starts rising within 24-48 hours and reaches its peak on the 3 rd day. It returns to normal level within 1-2 weeks. If there is tissue damage, it manifests in permanent increase of CRP. Its levels are elevated in infarctions of other tissues other than the heart. Other examples of CRP rise are rejection of kidney and bone marrow transplants, burn injuries and following surgery. If its levels fail to decrease after surgery, it is highly indicative of complications.

3. Infections: its levels are the highest in bacterial infections, but it is useful in the diagnosis of both bacterial and viral meningitis. It is useful in monitoring the activity of the disease and the efficacy of therapy. Last but not least, it is useful in diagnosing post-operative and intercurrent infections, mainly in active severe SLE and leukemia.

4. Malignancy: its levels are increased particularly in breast cancer, lung cancer and GI

cancer. It can be thus considered a "general" tumor marker (see Topic 8). Procalcitonin Procalcitonin is a stable precursor of calcitonin coded by CALC-I gene. It is composed of 116 amino acids, and has a half-life of 25-30 hours. It consists of an N-terminal region, calcitonin and katacalcin domains. Procalcitonin is not synthesized in the thyroid, but in the lung, liver and intestines. What is the diagnostic value of procalcitonin? Procalcitonin levels are increased in sepsis and severe bacterial infections. It is practically absent (<0.1µg/L) in the serum of healthy individuals. Its levels correlate well with the activity and extent of systemic bacterial infection. In viral infections, chronic and acute non- bacterial inflammations, however, its level remains unchanged or only slightly increases. Its increase precedes the clinical manifestations of severe generalized sepsis and has a prognostic value to it. Thus, it is useful in monitoring the treatment of such diseases.

generalized sepsis and has a prognostic value to it. Thus, it is useful in monitoring the
generalized sepsis and has a prognostic value to it. Thus, it is useful in monitoring the
generalized sepsis and has a prognostic value to it. Thus, it is useful in monitoring the

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Clinical Biochemistry

Topic 7

Biochemical effects of tumors

The biochemical effects of tumors (malignancies) are indirect: they may secrete biologically active substances even below the threshold of 10 9 cells (a tumor formed after 30 divisions, weighs 1 gram and spans 1 cm in diameter. This is when the tumor is clinically detectable).

A paraneoplastic syndrome (PNS) is a disease or symptom that is the consequence of the

presence of cancer in the body, but is not due to the local presence of cancer cells. These

phenomena are mediated by humoral factors (by hormones or cytokines) excreted by tumor

cells or by an immune response against the tumor. Recognition of a PNS alerts a new diagnosis of cancer. The frequency of PNS is 10-15%. PNS can be endocrine, neurological, hematological, dermatological, etc.

1. Endocrine syndromes:

Cushing syndrome: caused by ectopic secretion of ACTH by lung cancers. SIADH: caused by ectopic secretion of ADH by lung cancers. Gynecomastia: caused by ectopic secretion of hCG by lung and liver cancers. Hypercalcemia: caused by ectopic secretion of PTH-rP by lung cancers. Hypocalcemia: caused by ectopic secretion of calcitonin by breast cancers. Hypoglycemia: caused by ectopic secretion of IGF-II by sarcomas. Carcinoid syndrome: caused by ectopic secretion of serotonin by carcinoid tumors. Carcinoid tumors are discrete, yellow, well-circumscribed tumors that can occur anywhere

along the gastrointestinal tract and in the lung. They most commonly affect the appendix, ileum, and rectum (90%). Carcinoids are tumors of neuroendocrine nature, that originate in the cells of the neuroendocrine system (APUD) and are characterized by production of serotonin (5-hydroxytryptamine; 5-HT). Multiplex Endocrine Neoplasia (MEN): these are familial cancers (AD) characterized by the simultaneous appearance of tumors (benign or malignant) of 2 or more endocrine organs. MEN-I: parathyroid, pancreatic islets, anterior pituitary. MEN-IIa: thyroid (medullary cell carcinoma), adrenal medulla (pheochromocytoma), parathyroid. MENIIb: same as MEN-IIa but with various somatic abnormalities (marfanoid habitus, e.g.).

2. Hematological syndromes:

Anemia: the anemia can be of two types – hypoproliferative or autoimmune hemolytic

anemia. The hypoproliferative anemia is a mild anemia (Hb: 80-120 g/L) due to impaired erythropoiesis (e.g. insufficient EPO production or impaired response of the BM to EPO). The autoimmune hemolytic anemia is due to Ab against RBCs. The Abs can be warm reacting (as

in HL, NHL, CLL, MM, breast and lung cancer) or cold reacting (as in chronic granulocytic

leukemia, carcinoid tumors and adrenocorticocarcinoma). Erythrocytosis: caused by ectopic production of EPO or by hypoxemia (that can be the result of a direct effect of the tumor, as it narrows the O 2 supply highway to the kidney). Erythrocytosis is mainly seen in hypernephromas (30%), cerebellar hemangioblastoma (20%), hepatoma (1%) and Wilm's tumor (1%). Leukocytosis: caused by ectopic production of G-CSF. It is associated with gastric adenocarcinoma, lung cancer, pancreatic cancer, HL, and NHL. Symptoms appear late in the course of the malignant process. The WBC count exceeds 20x10 9 /L without a left shift. Thrombocytosis: caused by thrombopoetin overproduction in 1/3 of cancer patients (HL and NHL). PLT count is mildly high (1000 G/L). Thrombocytopenia: caused by ITP-like or hypoplasia, and is associated with HL, NHL, CLL, lung cancer, breast cancer and testicular cancer. PLT count is less than 30 G/L. Hemostasis disorders: thromboembolism is the second cause of death among cancer patients with promyelocytic leukemia, myeloproliferative disease, primary brain tumor, lung cancer and ovarian cancer. Patients with acute leukemias suffer from hemorrhages due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.

due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.
due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.
due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.
due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.
due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.
due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.
due to consumption coagulopathies. DIC is seen among patients with prostate cancer, adenocarcinoma, and AML-M3.

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Clinical Biochemistry

3. Neurological syndromes

The paraneoplastic neurological syndromes result from autoimmune processes. Certain

tumors produce large amounts of proteins expressed in the PNS or CNS. These molecules stimulate the humoral or the cellular immune system. Lambert-Eaton syndrome: Abs against Ca +2 channels in the presynaptic neuron. Thus, Ach is not released. It is most frequently associated with lung cancer. Cerebellar degeneration: Anti-Yo Abs. Encephalomyelitis: Anti-Hu Abs. Degeneration of peripheral nerves: Anti-Hu Abs. Retinopathy: CAR Abs.

4. Dermatological syndromes

These PNS result from autoimmune processes or from mediators produced locally or

systemically such as in carcinoid tumors.

These PNS result from autoimmune processes or from mediators produced locally or systemically such as in
These PNS result from autoimmune processes or from mediators produced locally or systemically such as in
These PNS result from autoimmune processes or from mediators produced locally or systemically such as in
These PNS result from autoimmune processes or from mediators produced locally or systemically such as in
These PNS result from autoimmune processes or from mediators produced locally or systemically such as in

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Clinical Biochemistry

Topic 8

Tumor markers (TM) in the diagnosis of malignant disease

What is TM? Tumor markers are molecules produced by the tumor or by the host (reacting to the presence of the tumor). These markers can be enzymes (and isoenzymes), hormones, oncofetal antigens, carbohydrate epitopes, cytokeratines, receptors and products of oncogenes. TM can be measured qualitatively and quantitatively by chemical methods, immunochemical methods or molecular biological methods in the serum or in other body fluids (urine, saliva, CSF, nipple discharge). What is the ideal TM? The ideal TM:

Is produced specifically by the malignant tissue or by the premalignant tissue; Is produced in all patients with a specific tumor type; Is produced in an organ-specific manner; Is measurable in an easily accessible body fluid; Its concentration in body fluid correlates with tumor volume or with the biological behavior of the tumor; Has a short half-life for the post-therapy follow-up; Informs of the progression and regression by its rise-and-falls; Is cheap, simple, standardized with an available reproducible assays. What are the "buts" ? But the ideal TM test does not exist yet (if you read this after 2009, double-check if it's still so….) But TM lack specificity: elevated concentrations can be seen in benign diseases too. But TM lack sensitivity for early malignancy. But TM are rarely elevated in all patients of a particular type of cancer. But no marker so far has an absolute organ specificity. What are the types of TM?

an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:
an absolute organ specificity. What are the types of TM? What are TM used for? Screening:

What are TM used for? Screening: serum TM are not ideal for population screening because they have low sensitivity and specificity plus a low positive predictive value of these tests. Aiding in diagnosis: is the tumor benign or malignant? What is the histological type of the tumor (e.g. is it a germ-cell tumor, a seminoma or a non-seminoma tumor?) Is it a metastatic cancer with unknown primary site? Assessing the prognosis: beside the classical prognostic factors (tumor size, grade, stage), TM can serve as independent prognostic factors. Predicting the response to therapy: in breast cancer, for example, the presence or absence of hormone receptors may predict the response to therapy. Monitoring patients with diagnosed disease: TM aid in early detection of recurrent malignancy as well as in monitoring the treatment given to the patient. Those markers that were positive at the time of diagnosis are applied in the monitoring process.

given to the patient. Those markers that were positive at the time of diagnosis are applied
given to the patient. Those markers that were positive at the time of diagnosis are applied
given to the patient. Those markers that were positive at the time of diagnosis are applied
given to the patient. Those markers that were positive at the time of diagnosis are applied
given to the patient. Those markers that were positive at the time of diagnosis are applied

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Clinical Biochemistry

What are the tumor markers used for screening? There are 6 TM that are worth mentioning:

1. VMA (vanillylmandelic acid) and HVA (homovanillic acid) for neuroblastoma (NB):

NB is the most common solid tumor among children younger than 5 years of age. The tumor originates from the primitive nervous tissue, and it synthesizes adrenaline, noradrenaline and dopamine. VMA and HVA are breakdown products of these catecholamines and can be measured in the urine. High levels of these markers can be found in 85-90% of NB patients at 6 months of age. Screening, however, does not reduce mortality.

2. PSA for prostate cancer: prostate cancer is the 4 th most common malignancy in males. PSA and a digital rectal examination are used for screening males older than 50 years of age, and for screening high-risk groups at 40-45 years of age.

3. CA125 for ovarian cancer (OC): OC is the most frequent cancer in females in the western world. CA125 combined with recto-vaginal pelvic examination and trans- vaginal ultrasound (TVUS)/transabdominal US (TAUS) and Doppler test are used for screening. Screening, however, is recommended for high-risk population.

4. AFP for hepatocellular cancer: HC cancer is the 5 th most common cancer in males and the 8 th most common in females. It is associated with HBV and HCV infections, hemachromatosis and liver cirrhosis. AFP combined with TAUS are used for screening. Screening is recommended for high-risk groups (those with chronic active hepatitis, cirrhosis, etc.). When performed, AFP is screened for every 3-4 months, and TAUS is performed every 4-6 months.

5. hCG for choriocarcinoma: this tumor is composed of villous trophoblasts, and may follow normal pregnancy, non-molar abortion, ectopic pregnancy or hydatidiform

mole. hCG is elevated in almost all patients with choriocarcinoma. It is very sensitive and is considered a nearly-ideal TM. Even though a good therapy is available, screening is recommended for high-risk groups only (e.g. hydatidiform mole).

6. Calcitonin for medullary thyroid cancer: this is a rare malignancy that can be hereditary or acquired, solitary or in combination with other tumors (MEN2A, MEN2B). The parafollicular cells are malignant. As they produce calcitonin in basal values or in stimulated values (following the administration of pentagastrin or calcium gluconate), screening is recommended for high-risk groups once a year.

What are the TM for germ-cell tumors? Seminoma: AFP Non-seminoma: hCG Mixed germ-cell tumor: LDH, P-ALP What are the gynecological tumor markers? Ovarian cancer: CA125 (to a lesser degree: CA15-3, CA72-4, TPA) Cervical cancer: SCC Endometrial cancer: CA125 What are the lung cancer tumor markers? Adenocarcinoma: cyfra21-1, CEA (to a lesser extent: CA125) Squamous cell carcinoma: cyfra21-1, SCC Small cell carcinoma: NSE, cyfra 21-1 Large cell carcinoma: cyfra21-1, CEA (to a lesser extent: CA125). What are the breast cancer TM? CA15-3, CEA (to a lesser extent: TPA, Cyfra21-1) What are the prostate cancer TM? PSA, fPSA (to a lesser extent: P-AcP (prostatic acid phosphatase)) What are the GI cancer TM? Colorectal cancer: CEA Gastric cancer: CEA<CA19-9<CA72-4 Esophageal cancer: SCC, TPA, cyfra21-1 (to a lesser extent: CA19-9) Pancreatic cancer: CA19-9 Hepatocellular cancer: AFP

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Clinical Biochemistry

What do CA, TPA, SCC, Cyfra, hCG, AFP, CEA, PSA abbreviate? CA – carbohydrate antigen; TPA – tissue polypeptide antigen SCC – squamous cell carcinoma antigen Cyfra – cytokeratine 19 fragment hCG – human chorionic gonadotropin AFP – alpha-feto-protein CEA – carcino-embryonic antigen PSA – prostatic specific antigen What is the frequency of TM determination? Before first therapy 2-10 days after therapy (according to the half-life of the TM) In intervals of 3 months during the 1 st and 2 nd year In intervals of 6 months in the 3 rd , 4 th and 5 th year after the first therapy Before any change of therapy If a relapse or metastasis are suspected If restaging is to be carried out 2-4 weeks after the first occurrence of a significant increase of the TM How do we evaluate the results? During monitoring of therapy, a decrease of at least 50% in the concentration of the TM indicate partial remission. TM cannot be used to determine complete remission. If an increase of the TM concentration is detected, repeat the measurement in 2-4 weeks (twice; all-in-all: 3 measurements). An increase of at least 25% in the TM concentration is significant enough to determine progression of the tumor. What are the factors that alter the serum concentration of TM? The production of the TM: how much TM is produced. The release of the TM: the rate of release, and whether it is released or not. The mass of the tumor: the larger the tumor is, the more TM might be produced. Blood supply of the tumor: the presence of the TM in the serum depends on that. Diurnal variation Position of the body during blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.

blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.
blood drawing Iatrogenic influences Catabolism of the tumor marker Life-style (habits): smoking, alcohol consumption.

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Clinical Biochemistry

Topic 9

Iron metabolism, hemochromatosis, iron deficiency anemia (note: this topic includes practical topics 2, 3 and 6)

The daily iron intake is about 15 mg, but less than 10% is absorbed (0.6-1.5 mg). The total body content of iron is ~4 g: 3g in hemoglobin, ~0.5g in myoglobin and enzymes and ~4mg in the plasma. The body stores up to 1.5g of iron. The average male loses ~1mg per day and a female loses ~2.5mg per day. The regulation of iron absorption is complex. There are three major influences:

The state of the body's iron stores (absorption is increased when they are depleted and decreased when they are replete) Erythropoiesis (absorption is increased when erythropoiesis is increased, irrespective of the state of the iron stores) Recent iron intake (a dietary iron bolus decreases iron absorption for several days). The main site of iron absorption is the proximal small bowel. Iron is more readily absorbed in the Fe 2+ form but dietary iron is mainly in the Fe 3+ form. Gastric secretions are important in iron absorption in that they liberate iron from food (although heme can be absorbed intact) and promote the conversion of Fe 3+ to Fe 2+ . Ascorbic acid and other reducing substances facilitate iron absorption while phytic acid (in cereals), phosphates and oxalates form insoluble complexes with iron and decrease its absorption. Iron is continuously recycled in an almost closed system – there is no significant excretion. Body stores are determined by the control of intestinal absorption. Free iron is toxic, and thus, iron is protein-bound (i.e. it is transported bound to transferrin and stored in ferritin and hemosiderin).

to transferrin and stored in ferritin and hemosiderin). Once absorbed into the intestinal mucosal cells, iron
to transferrin and stored in ferritin and hemosiderin). Once absorbed into the intestinal mucosal cells, iron
to transferrin and stored in ferritin and hemosiderin). Once absorbed into the intestinal mucosal cells, iron
to transferrin and stored in ferritin and hemosiderin). Once absorbed into the intestinal mucosal cells, iron

Once absorbed into the intestinal mucosal cells, iron is either transported directly into the bloodstream, or else combines with apoferritin, a complex iron-binding protein, to form ferritin. This iron is lost into the lumen of the gut when mucosal cells are shed. In iron deficiency, the apoferritin content of mucosal cells decreases and a greater proportion of absorbed iron reaches the bloodstream. Transport: In the blood, iron is transported mainly bound to transferrin, each molecule of which binds two Fe 3+ ions. Transferrin concentration is increased in pregnancy, among women who take oral contraceptive, estrogen, and in iron deficiency. Its concentration is decreased in chronic inflammation, malignancies, nephrotic syndrome and iron overload. The transferrin saturation is normally 15-45%. In iron deficiency, saturation is < 15% and in iron overload it is > 60%. Iron can be taken from the plasma by cells other than erythrocytes,

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Clinical Biochemistry

as these cells express transferrin receptors. Transferrin receptor (TfR) is present on the surface of all cell types (80% on erythroid precursors). It is a transmembrane protein that consists of 2 identical subunits. Its extracellular part is cleaved off by a serine protease and the receptor is liberated (mainly from late erythroid precursors, i.e. normoblasts and reticulocytes). Once in the circulation, it is complexed with transferrin. Iron uptake is regulated by the number of transferrin receptors. Storage: ferritin and hemosiderin store iron. Ferritin is found in all cell types. It has a Fe +3 hydroxide-phosphate core covered by 24 subunits of protein shell (apoferritin). On the surface of this "ball" there are channels through which Fe +3 enters and leaves. There are 2 types of subunits: a heavy chain, which has a ferroxidase activity, and a light chain that is responsible for the building up of the iron core. Each subunit (of the 24 subunits) is called an apoferritin. Each apoferritin binds 3000-4500 iron ions. Small amount circulates in the serum and is in equilibrium with that in stores. Serum ferritin reflects the size of iron stores. Ferritin is an acute phase reactant. It is increased in iron overload, liver disease, cancer, and in acute and chronic inflammation. It is decreased in iron deficiency.

chronic inflammation. It is decreased in iron deficiency. Iron regulation of gene expression: Cells in rest

Iron regulation of gene expression:

Cells in rest have very low transferrin receptors. When the iron level is low, cells need more transferrin receptors in order to get enough iron from the circulation. However. They do not require much storing proteins (ferritin). The opposite is true when the iron concentration in the blood is high. Both transferrin receptors and ferritin are regulated at the mRNA level, according to the cytosolic iron level. Both mRNAs have a iron-response element sequence, to which binding proteins (IRE-BP) can bind. When the binding proteins bind to ferritin mRNA, translation is blocked, but when they bind to transferrin receptor IRE, translation takes place.

bind to ferritin mRNA, translation is blocked, but when they bind to transferrin receptor IRE, translation

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Clinical Biochemistry

Hepcidin: this is a small peptide hormone synthesized in the liver. It has a regulatory role in iron homeostasis and an antimicrobial effect. In case of inflammation (IL-6), its levels

increase. It then binds to ferroportin and leads to its degradation. Thus, iron is trapped in the enterocyte. Some 48 hours following ferroportin degradation, the enterocyte is desquamated and the iron content spills. Iron is trapped in macrophages as well leading to anemia of chronic disease. Iron deficiency anemia:

This anemia is microcytic (MCV<80fL) and hypochromic (MCH < 27 pg). Iron deficiency can be caused due to increased demand for iron or due to a decreased supply.

Increased demand for iron can be due to:to increased demand for iron or due to a decreased supply. o Chronic blood loss (GI,

o

Chronic blood loss (GI, urogenital, respiratory system)

o

Growth

o

Pregnancy and lactation

Decreased supply can be due to:system) o Growth o Pregnancy and lactation o Diet with low amount of heme iron o

o Diet with low amount of heme iron

o Impaired iron absorption (intestinal disease, inhibitors of absorption such as phosphates, phytates that inhibit reduction of Fe +3 , HCl deficiency and accelerated passage). Development of iron deficiency anemia:

accelerated passage). Development of iron deficiency anemia: Lab diagnosis of iron deficiency anemia: 1. hematological

Lab diagnosis of iron deficiency anemia:

1. hematological tests: Hb, hematocrit, MCV, MCH, MCHC, RDW, blood smear and bone marrow smear.

2. Biochemical tests: serum iron, serum transferrin, transferrin saturation, serum ferritin, soluble transferrin receptor.

Procedure of blood drawing, vacutainer tubes (practical topic 3)

1.

Vacutainer tubes:

 
 

Anticoagulant

Common tests

Color of stopper

None

Chemistry

Red

EDTA

(ethylene

diamine

Hematology,

molecular

Lavender

tetra-acetic acid)

genetic tests

Sodium citrate

 

Hemostasis,

molecular

Blue

 

genetic tests

Sodium/lithium-heparin

Flow

cytometry,

Green

chromosome analysis

Sodium fluoride

 

Glucose determination from stored samples

Gray

ACD (acid citrate dextrose)

Blood culture

Yellow

2. Needle size: the gauge is a measurement of the needle diameter – the larger the number is, the smaller the diameter is (gauge 21 or 23 are usually used).

3. Needle type: multiple draw needle, butterfly needle, blood lancet.

4. Tube holder: barrel (a device for a safe and secure blood drawing)

5. Alcohol swabs: isopropyl alcohol (does not induce hemolysis)

6. Tourniquets

7. Gloves

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Clinical Biochemistry

9. Adhesive bandages/tapes

10. Needle disposal container

Procedure:

1. Wear gloves

2. Positioning of the patient

3. Apply tourniquet in the upper arm

4. Locate the vein (middle cubital for example)

5. Assemble the equipment

6. Clean the site

7. Perform venipuncture

8. Release tourniquet

9. Remove the needle

10. Apply the gauze

11. Dispose the needle

12. Label the specimen

Microcapillary blood collection: used in case of newborns and infants. The site chosen is

usually the finger or the heel in case of an infant (for PKU testing for example). The skin can

be

perpendicular to the lines of the fingerprint. Then, collect the blood by first wiping off the

first drop and proceed as above-mentioned.

Determination of hemoglobin and hematocrit (practical topic 2)

The Hb-cyanide method:

Advantages:

necessary. After cleaning the site, puncture the skin off-center and

preheated

if

1. Beside Hb, methemoglobin and carboxyhemoglobin (HbCO) can be measured.

2. Stable cyanmethemoglobin standards are available for calibration.

Disadvantages:

1. It does not measure sulphhemoglobin, which is rarely present.

2. The total conversion of HbCO is slow – it takes 30 minutes. If during determination the extinction of the solution is measured exactly at the 3 rd minute in the presence of 20% HbCO (which may be found in heavy smokers) the Hb concentration is overestimated by 6% (HbCO absorbs more light at 540nm than does hemoglobin cyanide).

3. Considering that the reagents contain cyanide, special fluid waste disposal is required.

Procedure:

1. Collection of whole blood anticoagulated with EDTA

2. Lysis of RBCs by SLS (Sodium-Lauryl-Sulphate)

3. Hb ----------> hemiglobin -------------> HiCN.

The reagent contains K 3 Fe(CN) 6 that carries out the first reaction, and KCN (CN - ) that carries out the 2 nd reaction.

4. A non-ionic detergent (Nonidet P40) is added to assure total and rapid hemolysis and for the prevention of precipitation of proteins and lipoproteins. 5. The extinction of the color product is measured at 540 nm. The transformation of Hb, HbO 2 and Hi occurs in 5 minutes. If there is a high WBC count, PLT count or high concentration of lipoproteins, turbidity is visualized and the specimen should be centrifuged before measurement. Reference interval:

Male: 140-180 g/L Female: 120-160 g/L Hematocrit:

Htc can be measured using centrifugation, measuring impedance and by calculation. Centrifugation:

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Clinical Biochemistry

2. Fill the blood into the capillary and close with wax. Make sure the closing is perfect and the surface of the wax is horizontal.

3. Place the capillary in the centrifuge, with the closed end facing outwards.

4. Centrifuge for 5 minutes with RPM corresponding to 12000g.

5. Transfer the capillary to the reading device. The bottom of the sample should fall exactly on the zero level. The capillary holder should be moved horizontally till the top of the plasma reaches the line corresponding to the 1.00 value.

6. The line marking the top of the RBCs cylinder gives the Htc value.

7. Multiple the value by 0.97: sedimented RBCs enclose about 3% plasma (trapped plasma). This value can be higher when the form of the RBC is altered (like in sickle cell anemia, when the trapped plasma can reach 20%).

Reference interval:

Male: 0.38-0.52 Female: 0.37-0.46 Errors may occur:

1. If the capillary is filled with incorrectly suspended blood.

2. If the capillary is not well closed – some RBC may leak.

3. If the closing wax at the bottom of the capillary is not horizontal (the setting of the zero line is not correct).

4. If the WBC count is high, and not taken into consideration, it may lead to falsely high Htc value. In this case, the Htc should be read at the top of RBC layer.

5. Unequal diameter of the capillary, inadequate RPM, short centrifugation time…

Calculation:

Automated hematology analyzers obtain Htc value as a calculated parameter derived from

RBC count and MCV:

RBC

×

MCV

Htc =

. (RBC in T/L (10 12 ) and MCV in fL (10 -15 ) ).

1000

Avi Sayag

Clinical Biochemistry

Topic 10

Laboratory diagnostics of hemoglobinopathies

Globin chains are synthesized in the liver of the fetus and in the bone marrow of adults. Adults Hb (HbA) can be of 2 type:

HbA 1 – composed of 2α2β chains (96-98%) HbA 2 – composed of 2α2δ (2%) Fetal Hb (HbF) is composed of 2α2γ (0.5-0.8%) Gene defects in the Hb molecule are the most common genetic disorders in the world. There are about 400 Hb variants registered. Abnormal Hb can be detected in electrophoresis. The most common variants are: HbS, HbC, HbS-C, HbE. Sickle cell anemia (HbS) The cause of the disease is a point mutation in the β chain of the globin: the β-globin gene is found on the short arm of chromosome 11. The association of two α-globin subunits with two mutant β-globin subunits forms hemoglobin S (HbS). Valine replaces glutamate residue at position 6 of the β chain located at the surface of the Hb, which is exposed to water. Glu is a polar amino acid while Val is non-polar. Thus, the replacement causes the formation of a sticky patch on the surface of the β chains. On the surface of the deoxyHb there is another sticky patch that causes polymerization of Hb into a fibrous structure.

that causes polymerization of Hb into a fibrous structure. The loss of red blood cell elasticity

The loss of red blood cell elasticity is central to the pathophysiology of sickle-cell disease. In sickle-cell disease, low oxygen tension promotes red blood cell sickling and repeated episodes of sickling damage the cell membrane and decrease the cell's elasticity. These cells fail to return to normal shape when normal oxygen tension is restored. Consequently, these rigid blood cells are unable to deform as they pass through narrow capillaries, leading to vessel occlusion and ischemia. Thus, the hemolytic anemia dominates the clinical features with characteristic crises:

1. Infarcts in bones, lungs, spleen (vaso-occlusive crisis)

2. Sequestration in visceral organs

3. Aplastic crises (infections; decreased Hb and reticulocytes)

4. Hemolytic crises (decreased Hb but increased reticulocytes)

25% of Africans are heterozygote for the disease and are therefore resistant to malaria, since

the sickle red blood cells are not conducive to the parasites. In areas where malaria is common there is a survival value in carrying the sickle-cell genes. Apart from middle Africa, sickle cell anemia is also common in south Europe and in some parts of the Saudi Arabia. Diagnosis

1. In HbSS, Hb levels are in the range of 6–8 g/dL with a high reticulocyte count.

2. A blood film may show features of hyposplenism (target cells and Howell-Jolly bodies).

3. Sickling of the red blood cells, on a blood film, can be induced by the addition of sodium

metabisulfite. The presence of sickle Hb can also be demonstrated with the "sickle solubility test". A mixture of Hb S in a reducing solution (such as sodium dithionite) gives a turbid appearance, whereas normal Hb gives a clear solution.

4. Abnormal Hb forms can be detected on electrophoresis

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Clinical Biochemistry

Avi Sayag Clinical Biochemistry 5. The diagnosis can be confirmed with high-performance liquid chromatography (HPLC). 6.

5. The diagnosis can be confirmed with high-performance liquid chromatography (HPLC).

6. An acute sickle-cell crisis is often precipitated by infection. Therefore, a urinalysis to

detect an occult urinary tract infection, and chest X-ray to look for occult pneumonia should be routinely performed. It should be noted that the sickle cell trait is a benign condition with no anemia. The RBCs appear to be normal, and crisis can be caused by extreme stress (anoxia, infection).

crisis can be caused by extreme stress (anoxia, infection). Note the presence of the A band

Note the presence of the A band as opposed to the previous one where no A band is present (this is because in homozygotes only HbS is present, while in heterozygotes 40% is HbS and the remainder is HbA). Hb C disease Hb C is an abnormal Hb with substitution of a lysine residue for a glutamic acid residue at the 6th position of the β-globin chain. This mutated form reduces the normal plasticity of host erythrocytes causing a hemoglobinopathy. In those who are heterozygous for the mutation, about 28–44% of total hemoglobin (Hb) is HbC, and no anemia develops. In homozygotes, nearly all Hb is in the HbC form, resulting in mild hemolytic anemia. Target cells (codocytes), microspherocytes and HbC crystals are found in a blood smear from a homozygous patient. The reticulocyte count is high, as well as serum bilirubin. Hb E disease Lysine substitutes glutamate on position 26 in the β chain. Most common in south east Asia. Homozygotes present with mild hypochromic, microcytic anemia with target cells.

Thalassemias Thalassemia is an inherited autosomal recessive blood disease. In thalassemia, the genetic defect results in reduced rate of synthesis of one of the globin chains that make up hemoglobin. Reduced synthesis of one of the globin chains causes the formation of abnormal Hb molecules, and this in turn causes the anemia which is the characteristic presenting symptom of the thalassemias. The cause of the disease might be a missing gene, improper processing of mRNA, premature termination of protein synthesis, frameshift mutation, etc. The thalassemias are classified according to which chain of the hemoglobin molecule is affected. In α thalassemias, production of the α globin chain is affected, while in β thalassemia production of the β globin chain is affected. β globin chains are encoded by a single gene on chromosome 11; α globin chains are encoded by two closely linked genes on chromosome 16. Thus in a normal person with two copies of each chromosome, there are two loci encoding the β chain, and four loci encoding the α chain. α-thalassemia: There are four genetic loci for α globin, two of which are maternal in origin and two of which are paternal in origin. The severity of the α thalassemias is correlated with the number of affected α globin loci: the greater the number of affected loci, the more severe the manifestations of the disease will be. If 1 copy is defective- silent α-thalassemia carrier; no significant signs or symptoms (except maybe low MCV and low MCH)

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Clinical Biochemistry

If 2 copies are defective – α thalassemia triat. Two α loci permit nearly normal erythropoiesis, but there is a mild microcytic, hypochromic anemia. If 3 copies are defective – mild-to-moderate hemolytic anemia (HbH disease), resulting in poor O 2 delivery (too high affinity to O 2 ). If 4 copies are defective - the fetus cannot live once outside the uterus and may not survive gestation: most such infants are dead at birth with hydrops fetalis, and those who are born alive die shortly after birth. They are edematous and have little circulating hemoglobin, and the hemoglobin that is present is all tetrameric γ chains. β-thalassemia: as mentioned, there are 2 copies of the β-chain gene:

If 1 copy is defective – minor β-thalassemia – mostly asymptomatic (perhaps mild microcytic, hypochromic anemia may develop). HbF is increased and HbA 2 > 3.5%; target cells are present as well as ovalocytes, poikilocytosis and basophil stippling. If 2 copies are defective – major β-thalassemia – appears after birth. Severe microcytic anemia develops (Cooley's anemia), and the patient depends on constant blood transfusion. This offered treatment is life-saving, but results in iron overload. The patient may die at 20-25 years of age. The ultimate cure is bone marrow transplantation. The Hb levels are 30-40 g/L, MCV – 51-61 fL, reticulocytes – 1-8%, and both HbF and HbA 2 are elevated. The bone marrow shows erythroid hyperplasia (E:M is 20:1) seFe is elevated, Tfsat is > 80%. There is also thalassemia intermedia in which the morphology is the same as for the major form, but the HbF is 2-100%, the HbA 2 does not exceed 7%. Diagnosis

HbF is 2-100%, the HbA 2 does not exceed 7%. Diagnosis Sideroblastic anemia Sideroblastic anemia is

Sideroblastic anemia Sideroblastic anemia is caused by abnormal production of red blood cells usually as part of myelodysplastic syndrome, which can evolve into hematological malignancies (especially acute myelogenous leukemia). The body has iron available but cannot incorporate it into hemoglobin. Sideroblasts are seen, which are nucleated erythrocytes with granules of iron in their cytoplasm The problem lies in a failure to completely form heme molecules, whose biosynthesis takes place partly in the mitochondrion. This leads to deposits of iron in the mitochondria that form a ring around the nucleus of the developing red blood cell. It can be inherited (X-linked) or acquired. If acquired, it can be primary, as part of a myelodysplastic syndrome, or secondary due to:

Toxins: lead or zinc poisoning

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Clinical Biochemistry

Drug-induced: ethanol, isoniazid, chloramphenicol, cycloserine Nutritional: pyridoxine or copper deficiency Genetic: ALA synthase deficiency Diagnosis Specific test: Prussian Blue stain of RBC in marrow. Shows ringed sideroblasts. Increased ferritin levels Decreased total iron-binding capacity Hematocrit of about 20-30% Serum Iron: High High transferrin saturation MCV is usually normal or slightly increased; although it may occasionally be low, leading to confusion with iron deficiency. With lead poisoning, there is coarse basophilic stippling of red blood cells on peripheral blood smear.

Summary and presentation of the topic:

General features of Hb – types, %, structure Disorders: HbS, HbC, HbC-S, HbE, thalassemia, sideroblastic anemia Diagnosis of HbS: Hb, ret, blood film, Na-metabisulfite, sickle solubility test, electrophoresis, HPLC (sickle cell trait) Diagnosis of thalassemia: history, clinical signs, blood (Hb, MCV, MCH, RDW, ret), electrophoresis, HPLC, β-chain sequencing Diagnosis of sideroblastic anemia: Prussian blue stain, ferritin, iron binding capacity, HTC, Fe, Tfsat, MCV, lead poisoning: basophilic stippling on peripheral smear.

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Clinical Biochemistry

Topic 11

Laboratory diagnostics of hemolytic anemias

An increased rate of red cell destruction;

An increased rate of red cell destruction;

A compensatory increase in erythropoesis that results in reticulocytosis;

A

compensatory increase in erythropoesis that results in reticulocytosis;

The retention by the body of the products of red cell destruction (including iron). Because

The retention by the body of the products of red cell destruction (including iron). Because

the iron is conserved and recycled readily, red cell regeneration can keep pace with the hemolysis. Consequently, these anemias are almost invariably associated with a marked erythroid hyperplasia within the marrow and an increased reticulocyte count in peripheral blood. In severe hemolytic anemias, extramedullary hematopoiesis often develops in the spleen, liver, and lymph nodes.

Destruction of red cells can occur within the vascular compartment (intravascular hemolysis) or within the cells of the mononuclear phagocyte (reticuloendothelial) system (extravascular hemolysis).

Intravascular hemolysis can result from mechanical trauma (e.g., a defective heart valve) or biochemical or physical agents that damage the red cell membrane (e.g., fixation of complement, exposure to clostridial toxins, or heat). Regardless of the cause, hemolysis leads to hemoglobinemia, hemoglobinuria, and hemosiderinuria. The conversion of the heme pigment to bilirubin can result in unconjugated hyperbilirubinemia and jaundice. Haptoglobin, a circulating protein that binds and clears free hemoglobin, is often absent from the plasma.(reticuloendothelial) system (extravascular hemolysis). Extravascular hemolysis (more common) takes place largely

Extravascular hemolysis (more common) takes place largely within the phagocytic cellsand clears free hemoglobin, is often absent from the plasma. of the spleen and liver. The

of the spleen and liver. The RES removes damaged red cells from the circulation. The red

cells need to be highly deformable to travel in the splenic sinusoid. Therefore, any change

in that feature leads to red cells being stuck in the spleen (and phagocytosed there). Extravascular hemolysis is not associated with hemoglobinemia and hemoglobinuria, but

it often produces jaundice and, if long-standing, can lead to the formation of bilirubin-rich

gallstones. Haptoglobin amounts are always decreased, because some hemoglobin invariably escapes into the plasma. In most forms of hemolytic anemia there is a reactive hyperplasia of the RES (splenomegaly). In chronic hemolytic anemias, changes in iron metabolism lead to increases in iron absorption from the gut. Because the pathways for the excretion of excess iron are limited, this often causes iron to accumulate, giving rise to systemic hemosiderosis.

The intracorpuscular abnormalities can be either hereditary or acquired. The hereditary causes might be:

Membrane defects:hereditary or acquired. The hereditary causes might be : o In the setting of spherocytosis (spectrin

o In the setting of spherocytosis (spectrin deficiency), jaundice may follow, splenomegaly, gallstones and most probably anemia. Spherocytosis is AD. Microspherocytes and elevation of reticulocytes (5-20%) are evident in blood film. The mechanism underlying spherocytosis starts with spectrin deficiency. This leads to decreased protein density of the RBC's cytoskeleton and as a result parts of the erythrocyte's bilayer membrane are released as microvesicles. Thus, the surface area of the RBC is decreased (spherocytosis). This condition impairs the deformability of the red blood cell, and they are therefore entrapped in the spleen. Trapped cells cause splenic conditions leading to further loss of surface area, which then again lead to the release of RBCs' membrane as microvesicles. Other forms of hereditary spherocytiosis result from mutations that involve ankyrin, band 4.2 and band 3.

microvesicles. Other forms of hereditary spherocytiosis result from mutations that involve ankyrin, band 4.2 and band

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Clinical Biochemistry

o

Diagnosis: the classic laboratory features of HS include minimal or no anemia, reticulocytosis, an increased mean corpuscular hemoglobin concentration (MCHC), spherocytes on the peripheral blood smear, hyperbilirubinemia, and abnormal results on the osmotic fragility test (the most sensitive test to help detect HS is the incubated osmotic fragility test performed after incubating RBCs for 18-24 hours under sterile conditions at 37°C - hemolysis of HS cells may be complete at a solute concentration that causes little or no lysis of normal cells).

o

RBC morphology is distinctive yet not diagnostic.

o

Other biochemical changes of hemolysis also are present, including increased LDH, increased unconjugated bilirubin, and decreased serum haptoglobin.

o

An increased MCHC obtained from an electronic cell counter is a characteristic feature of red cells in HS. MCHC values greater than the upper limit of normal (35-36%) are common. This increased MCHC is a result of mild cellular dehydration. The MCV in patients with HS is low. This relatively low MCV may reflect membrane loss and cell dehydration.

o

Further characterizing the specific membrane lesion by looking for abnormalities in spectrin, ankyrin, pallidin, or band 3 is possible. However, these studies are not routine and are available only in select research laboratories.

o

The initial workup if hemolysis is suggested should include the following:

Hb, MCHC, MCV, Reticulocyte count

Lactate dehydrogenase

Fractionated bilirubin

Haptoglobin

Peripheral smear: Howell-Jolly bodies may be present, indicating remnant splenic

tissue if the patient has had their spleen removed. Findings also may include megalocytosis. Vitamin B-12 and folate: This should be measured to determine the nutritional stores during recovery from an aplastic crisis. Herpes simplex virus, HPV type 19, and infectious mononucleosis: Testing for these may help identify an infectious etiology for the aplastic crisis.

o

Elliptocytosis – due to a mutated band 4.1

o

Membrane lipids: a-β-lipoproteinemia

Enzymatic causes: G6PD (x-linked), glutathione synthetase, pyruvate kinase, hexokinaseband 4.1 o Membrane lipids: a- β -lipoproteinemia Disorders of hemoglobin synthesis (hemoglobinopathies):

Disorders of hemoglobin synthesis (hemoglobinopathies): thalassemias, sickle cell anemia The acquired cause is due to Paroxysmal Nocturnal Hemoglobinuria (PNH): The acquired cause is due to Paroxysmal Nocturnal Hemoglobinuria (PNH):

o The problem lies in the lack of anchoring proteins for DAF and MIC, leading to non- inhibition of the complement membrane destruction complex. Extracorpuscular The acquired causes can be:

Immune causes: autoimmune (as in transfusion reactions and SLE), alloimmune (as in the Rh disease of the newborn) or drug-associated (penicillin or methyl-DOPA).complex. Extracorpuscular The acquired causes can be : RBC fragmentation (physical damage on abnormal surfaces

RBC fragmentation (physical damage on abnormal surfaces (artificial heart valves) or microangiopathic damage: disease of small blood vessels, in DM for example, which may lead to DIC, HUS or TTP (thrombic thrombocytopenia purpura).the newborn) or drug-associated (penicillin or methyl-DOPA). March hemolytic anemia Infections with meningococci, malaria

March hemolytic anemialead to DIC, HUS or TTP (thrombic thrombocytopenia purpura). Infections with meningococci, malaria or Clostridium

Infections with meningococci, malaria or Clostridium perfringens(thrombic thrombocytopenia purpura). March hemolytic anemia Secondary to liver disease or renal disease. Due to chemical

Secondary to liver disease or renal disease.with meningococci, malaria or Clostridium perfringens Due to chemical or physical agents such as snake venom,

Due to chemical or physical agents such as snake venom, insect bite and burns.anemia Infections with meningococci, malaria or Clostridium perfringens Secondary to liver disease or renal disease.

Avi Sayag

Clinical Biochemistry

Topic 12

Laboratory diagnostics of megaloblastic anemias

Megaloblastic anemias are macrocytic (MCV>100 fL) and hyperchromic (MCH>31pg/RBC). The 2 main causes are vitamin B12 deficiency and folic acid deficiency. Folic acid deficiency Diet is not a common cause for folic acid deficiency. Although it is present in almost all foods, it is destroyed in 10-15 minute cooking. It is distributed widely in green leafy vegetables, citrus fruits, and animal products. Humans do not generate folate endogenously because they cannot synthesize PABA (p-aminobenzoic acid), nor can they conjugate the first glutamate.

Folates are present in natural foods and tissues as polyglutamates because these forms serve to keep the folates within cells. In plasma and urine, they are found as monoglutamates because this is the only form that can be transported across membranes. Enzymes in the lumen of the small intestine convert the polyglutamate form to the monoglutamate form of the folate, which is absorbed in the proximal jejunum via both active and passive transport. Within the plasma, folate is present, mostly in the 5-methyltetrahydrofolate (5-methyl THF) form, and is loosely associated with plasma albumin in circulation. The 5-methyl THF enters the cell via a diverse range of folate transporters with differing affinities and mechanisms (i.e., ATP–dependent H+ co-transporter or anion exchanger). Once inside, 5-methyl THF may be demethylated to THF, the active form participating in folate-dependent enzymatic reactions. Cobalamin (B12) is required in this conversion, and in its absence, folate is "trapped" as 5-methyl THF. From then on, folate is no longer able to participate in its metabolic pathways, and megaloblastic anemia results. Large doses of supplemental folate can bypass the folate trap, and megaloblastic anemia will not occur. The biologically active form of folic acid is tetrahydrofolic acid (THF), which is derived by the 2-step reduction of folate involving dihydrofolate reductase. THF plays a key role in the transfer of 1-carbon units (such as methyl, methylene, and formyl groups) to the essential substrates involved in the synthesis of DNA, RNA, and proteins. More specifically, THF is involved with the enzymatic reactions necessary to synthesis of purine, thymidine, and amino acid. Manifestations of folate deficiency thereafter, not surprisingly, would involve impairment of cell division, accumulation of possibly toxic metabolites such as homocysteine, and impairment of methylation reactions involved in the regulation of gene expression, thus increasing neoplastic risks.

A healthy individual has about 500-20,000 mcg of folate in body stores. Humans need to

absorb approximately 50-100 mcg of folate per day in order to replenish the daily degradation and loss through urine and bile. Otherwise, signs and symptoms of deficiency can manifest after 4 months. Signs and symptoms:

Anemia: weakness, vertigo, tinnitus, palpitations, angina, pallor with slightly icteric skin and eyes.deficiency can manifest after 4 months. Signs and symptoms: GI: glossitis (sore tongue), cheilosis angularis, diarrhea,

GI: glossitis (sore tongue), cheilosis angularis, diarrhea, weight lossangina, pallor with slightly icteric skin and eyes. There are no neurological signs. B12 deficiency Vitamin

There are no neurological signs.(sore tongue), cheilosis angularis, diarrhea, weight loss B12 deficiency Vitamin B12 is absorbed in the ileum

B12 deficiency Vitamin B12 is absorbed in the ileum complexed to intrinsic factor secreted from the parietal cells of the stomach. It is transported in the blood bound to transcobalamin II. In its methylated form it facilitates the conversion of homocysteine to methionine – a reaction which is carried out by the conversion of methyl-THF to THF. As mentioned, THF is required

to synthesize purines (thymidine).

Pathophysiology: vitamin B12 deficiency is caused by failure to absorb the vitamin due to autoantibodies directed against the gastric parietal cells, the intrinsic factor, the IF-B12 complex or against the receptors in the ileum.

Avi Sayag

Signs and symptoms:

Clinical Biochemistry

 
  Anemia: weakness, vertigo, tinnitus, palpitations, angina, pallor with slightly ichteric skin and eyes.

Anemia: weakness, vertigo, tinnitus, palpitations, angina, pallor with slightly ichteric skin and eyes.

GI: glossitis (sore tongue), cheilosis angularis, diarrhea, weight loss

GI: glossitis (sore tongue), cheilosis angularis, diarrhea, weight loss

Neurological signs: numbness and parasthesias in the extremities, ataxia,

Neurological signs: numbness and parasthesias in the extremities, ataxia,

 

poor

finger coordination, sphincter disturbances, forgetfulness, severe

dementia, and psychosis.

Lab:

 
  MCV > 100 fL (macrocytic anemia)

MCV

> 100 fL (macrocytic anemia)

In the peripheral smear macro-ovalocytes can be found as well as hypersegmented granulocytes, and the

In the peripheral smear macro-ovalocytes can be found as well as hypersegmented granulocytes, and the reticulocyte count is low.

Unconjugated serum bilirubin is elevated as well as LDH1. Autoantibodies can also be found in

Unconjugated serum bilirubin is elevated as well as LDH1. Autoantibodies can also be found in the serum or in the gastric juice.

In the bone marrow there are megaloblasts and ineffective erythropoesis.

In the bone marrow there are megaloblasts and ineffective erythropoesis.

Endoscopy may reveal atrophy of the gastric mucosa that may lead to achlorhydria. This poses

Endoscopy may reveal atrophy of the gastric mucosa that may lead to achlorhydria. This poses an increased risk for gastric carcinoma.

Serum B12 can be directly measured (plasma immunoassay)

Serum B12 can be directly measured (plasma immunoassay)

Schilling test is positive and helps to differentially diagnose GI disease from pernicious anemia:

Schilling test is positive and helps to differentially diagnose GI disease from pernicious anemia:

i. First, IM injection of B12 is given to saturate the transcobalamin II stores. At the same time, the patient is given labeled B12 per os (the

most commonly used radiolabels are 57 Co and 58 Co).

ii. Urine is then collected over 24 hours. A normal result is documented when more than 10% of the given labeled B12 is excreted in the urine.

iii. If less than 10% is collected, the test is repeated with a modification:

labeled B12 and IF are given per os, and urine is collected over 24 hours. If more than 10% is excreted this time, then pernicious anemia is diagnosed. However, if the result is still not normal, then a GI disease is suspected and should be further investigated.

Summary and presentation of the topic:

Classify megaloblastic anemia (hyperchromic, macrocytic) and give values of MCV and MCHfurther investigated. Summary and presentation of the topic: Mention the 2 main causes: folate and B12

Mention the 2 main causes: folate and B12 deficiency(hyperchromic, macrocytic) and give values of MCV and MCH Speak about folate: o Forms and evolution:

Speak about folate:and MCH Mention the 2 main causes: folate and B12 deficiency o Forms and evolution: poly

o

Forms and evolution: poly mono (methyl-FH4) FH4 (active form)

o

Absorbance (prox. jejunum, active + passive)

o

Participation in metabolism and the role of B12

o

Reference range: 3-20 µg/L

o

Signs and symptoms of folic acid deficiency

o

Diagnosis: plasma immunoassay (RBC is better, as the concentration in RBCs reflects the body's folate reserves, range >140µg/L , while plasma concentration reflects recent dietary intake).

Speak about B12:while plasma concentration reflects recent dietary intake). o Absorbance in the ileum + IF o Transport

o

Absorbance in the ileum + IF

o

Transport in the blood with transcobalamine II

o

Functions of B12

o

Normal range: 130-700 ng/L

o

Recommended daily intake: 3-5µg (the liver stores ~3mg – enough for years)

o

Sources of deficiency (autoimmune, gastrectomy, Crohn's, low intake)

o

Signs and symptoms of deficiency

o

Diagnosis: plasma immunoassay, Schilling test

Lab: Hb, MCV, MCH, B12, folic acid + normal ranges + lab proceduresdeficiency o Diagnosis: plasma immunoassay, Schilling test Additional tests: endoscopy, BM examination, blood smear

Additional tests: endoscopy, BM examination, blood smearo Diagnosis: plasma immunoassay, Schilling test Lab: Hb, MCV, MCH, B12, folic acid + normal ranges

Avi Sayag

Clinical Biochemistry

Topic 13

Major laboratory characteristics of acute and chronic lymphoid leukemias

ALL

Most common between 2-10 years of age.characteristics of acute and chronic lymphoid leukemias ALL 85% B-cells, 15% T-cells. More then 20% blasts

85% B-cells, 15% T-cells.leukemias ALL Most common between 2-10 years of age. More then 20% blasts in peripheral blood

More then 20% blasts in peripheral blood and BM.common between 2-10 years of age. 85% B-cells, 15% T-cells. Risk factors: Down syndrome, Fanconi's anemia,

Risk factors: Down syndrome, Fanconi's anemia, AT, chemical drugs, in utero radiation.T-cells. More then 20% blasts in peripheral blood and BM. Involves the enlargement of lymph nodes,

Involves the enlargement of lymph nodes, spleen and liver.anemia, AT, chemical drugs, in utero radiation. Complete remission in 90% of cases and while 66%

Complete remission in 90% of cases and while 66% undergo full recovery.Involves the enlargement of lymph nodes, spleen and liver. FAB classification: o L1: small blasts, homogenous

FAB classification:in 90% of cases and while 66% undergo full recovery. o L1: small blasts, homogenous population,

o

L1: small blasts, homogenous population, narrow cytoplasm.

o

L2: larger blasts, heterogeneous population, prominent nucleolus, wider cytoplasm.

o

L3: homogenous population, middle-large blasts, basophilic cytoplasm with vacuoles (Burkitt lymphoma).

Another classification according to the immunophenotyping:basophilic cytoplasm with vacuoles (Burkitt lymphoma). o Pre-B-cell ALL: CD19, CD79a o Common ALL: CD19,

o

Pre-B-cell ALL: CD19, CD79a

o

Common ALL: CD19, CD79a, CD10

o

Late pro-B-cell ALL: CD19, CD79a, CD10, µ-chain

o

B-cell ALL: with surface Ig.

B-cell markers: CD 19, CD 20, CD 22, CD 79a, HLA-DR (young: CD 10)CD79a, CD10, µ-chain o B-cell ALL: with surface Ig. Prognosis: o L3 - unfavorable. o CD

Prognosis:markers: CD 19, CD 20, CD 22, CD 79a, HLA-DR (young: CD 10) o L3 -

o

L3 - unfavorable.

o

CD 10, T-cell ALL - unfavorable.

o

Adult > 30 G/L and children > 50 G/L - unfavorable.

o

Age: less then 2 years, older then 10 years, older then 35 (adult) - unfavorable.

o

Less then 45 chromosomes – unfavorable.

o

More then 50 chromosomes – favorable.

o

Translocations t(8:14), t(9:22), t(1:19), t(4:11) – unfavorable.

o

Translocations t(12:21) – favorable.

CLL

In the spleen the germinal center is surrounded by two separate zones: the mantle zone (close to the germinal center and IgD positive) and the marginal zone. Both zones are populated by B-lymphocytes (the marginal zone lymphocytes have irregular nuclei and the cytoplasm appears more empty).o Translocations t(12:21) – favorable. CLL In the lymph node, there is a mantle zone, and

In the lymph node, there is a mantle zone, and B-cells surround it. However, they do not form a special zone, but are rather mixed with the lymphocytes in the mental zone.have irregular nuclei and the cytoplasm appears more empty). CLL is the leukemic counterpart of SLL

CLL is the leukemic counterpart of SLL (small lymphocytic lymphoma)are rather mixed with the lymphocytes in the mental zone. Usually affects people over 50. Not

Usually affects people over 50.the leukemic counterpart of SLL (small lymphocytic lymphoma) Not aggressive. Often asymptomatic, those who are

Not aggressive.(small lymphocytic lymphoma) Usually affects people over 50. Often asymptomatic, those who are symptomatic have general

Often asymptomatic, those who are symptomatic have general symptoms: general symptoms of malignancy, lymphadenopathy, hepatosplenomegaly, leukocytosis (around 200000/µL), hypogammaglobulinemia (prone to infections), anemia, autoimmune hemolytic anemia, and thrombocytopenia. The organs most commonly involved are the bone marrow and the blood (these are the primary sites), the lymph nodes, spleen, liver, skin and tonsils.lymphoma) Usually affects people over 50. Not aggressive. Does not transform into ALL Clonal expansion of

Does not transform into ALLsites), the lymph nodes, spleen, liver, skin and tonsils. Clonal expansion of mature lymphocytes (98% B-cell,

Clonal expansion of mature lymphocytes (98% B-cell, 2% T-cell). mature lymphocytes (98% B-cell, 2% T-cell).

Gumprecht shadow.expansion of mature lymphocytes (98% B-cell, 2% T-cell). More than 30% lymphocytic infiltration of the bone

More than 30% lymphocytic infiltration of the bone marrow.tonsils. Does not transform into ALL Clonal expansion of mature lymphocytes (98% B-cell, 2% T-cell). Gumprecht

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Clinical Biochemistry

In the serum of these patients there are low levels of Igs, paraproteins are rare and uric acid level is elevated.Avi Sayag Clinical Biochemistry Immunophenotyping: B-cell markers (CD19, CD20, CD22), T-cell markers (CD5), CD23.

Immunophenotyping: B-cell markers (CD19, CD20, CD22), T-cell markers (CD5),Igs, paraproteins are rare and uric acid level is elevated. CD23. Cytogenetics: 12 trisomy; deletions 11,13,17;

CD23.

Cytogenetics: 12 trisomy; deletions 11,13,17; no translocation involving CycD (no t(4:11) ), as it must be differentially diagnosed from mantle zone lymphoma (centrocytic lymphoma) which is characterized by t(4:11)(cycD+), CD5+, no +12, and CD43.markers (CD19, CD20, CD22), T-cell markers (CD5), CD23. CLL/SLL ALL 98% B cells 85% B cells

CLL/SLL

ALL

98% B cells

85% B cells

No translocations associated

 

t(4:11), t(8:14), t(1:19), t(9:22), t(12:21)

+12, deletions 11, 13, 17

 

Chromosomes < 45, >50

CD19, CD20, CD22, CD23, CD5

 

CD19, CD20, CD22, CD10

Associated

with

hypo-γ-glubolinemia

and

 

hyperuricemia

 

Stem cell early pro-B late pro-B large pre-B small pre-B immature B mature B In general, ALL is characterized by arrested maturation somewhere between late pro-B and small pre-B/immature B, while CLL is characterized by arrested maturation and proliferation of activated B cell or memory B cell.

Characterization of leukemic cells by morphology (practical topic 7)

Myelopoiesis:

PMNs)

Myeloblast promyelocyte myelocyte metamyelocyte band neutrophils/eosinophils/basophils

1. Myloblasts: > 10µm, basophilic plasma, no granulations, loose chromatin in the nucleus, multiple nucleoli, a halo around the nucleus.

2. Promyelocyte: the largest cell in the lineage, many azurophilic granules, decreased nucleus:plasma ratio.

form

(for

3. Myelocytes: less basophilic plasma, lower number of granules, no nucleolus.

4. Metamyelocytes: secondary/specific granules appear; the nucleus is indented and bean-shaped.

5. Band form: stick-shaped nucleus; grayish-blue granulation

6. Neutrophils: the nucleus has several lobes; bluish granulation.

7. Eosinophils: bi-lobed nucleus, pink plasma with reddish-brown large granules. 12-17µm.

8. Basophils: rough, purple-black granulation that covers even the nucleus. 10-

14µm.

9. Monocytes: grayish-blue plasma with azurophilic granules. The nucleus is bean-shaped. 12-20µm.

10. Lymphoblasts: large nucleus:plasma ratio; purplsh plasma; nucleoli are present; it takes an expert to distinguish them from myeloblasts.

11. Small lymphocytes: small cells (10-12µm); basophilic cytoplasm; the nucleus is condensed and purple.

12. Large lymphocytes (activated): loose chromatin and less basophilic cytoplasm.

13. Large granular lymphocytes: large cells, rough azurophilic granules.

14. Plasma cells: blue cytoplasm; dark-purple nucleus in the periphery.

15. Megakaryocyte: large cell; cloudy cytoplasm with azurophilic granulation;

sometimes detached platelets can be seen in the surrounding; the nucleus is multilobed. 50-70µm. As a general rule, when cells mature, their size decreases as well as the nucleus:plasma ratio. Nucleoli disappear from the nucleus, and the nuclear material becomes more condensed.

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Clinical Biochemistry

Topic 14

Major laboratory characteristics of acute and chronic myeloid leukemias

AML

AML primarily affects older adults, with the median age being 50 years (nonetheless, it can affect all age groups).characteristics of acute and chronic myeloid leukemias AML The clinical signs and symptoms are usually related

The clinical signs and symptoms are usually related to marrow failure caused by the replacement of normal marrow elements by leukemic blasts.being 50 years (nonetheless, it can affect all age groups). Fatigue and pallor, abnormal bleeding, and

Fatigue and pallor, abnormal bleeding, and infections are commonreplacement of normal marrow elements by leukemic blasts. Splenomegaly and lymphadenopathy are in general less

Splenomegaly and lymphadenopathy are in general less prominent than in ALL, but, rarely, AML presents as a discrete tissue mass (a so-called granulocytic sarcoma). Ideally, the diagnosis and classification of AML are based on the results of morphologic, histochemical, immunophenotypic, and karyotypic studies. Of these tests, karyotyping is most predictive of outcome:and pallor, abnormal bleeding, and infections are common Most AMLs are associated with acquired mutations in

Most AMLs are associated with acquired mutations in transcription factors that inhibit normal myeloid differentiation, leading to the accumulation of cells at earlier stages of development.Of these tests, karyotyping is most predictive of outcome: t(15;17) translocation occurs in acute promyelocytic

t(15;17) translocation occurs in acute promyelocytic leukemia. This translocation results in the fusion of the retinoic acid receptor α (RARA) gene on chromosome 17 with the PML gene on chromosome 15. The chimeric α (RARA) gene on chromosome 17 with the PML gene on chromosome 15. The chimeric gene(s) produce abnormal PML/RARA fusion proteins that block myeloid differentiation at the promyelocytic

stage. Pharmacologic doses of retinoic acid, a vitamin A analogue, overcome this block and cause the neoplastic promyelocytes to terminally differentiate into neutrophils and die. Because neutrophils live, on average, for 6 hours, the result is the rapid clearance of tumor cells and remission in a high fraction of patients. The effect is very specific; AMLs without translocations involving RARA do not respond to retinoic acid.

Classification:involving RARA do not respond to retinoic acid. o o o o o M0 – AML

o

o

o

o

o

M0 – AML without maturation:

2% of AML cases; Diagnostic criteria: in the bone marrow – there are no azurophilic granulation, no Auer rods in the blast cells; MPO is positive in less than 3% of cells, and immunophenotyping is needed.

M1 – AML with minimal maturation:

10-20% of AML cases. Diagnostic criteria (in the bone marrow): azurophilic granules and/or Auer rods in less than 10% of cells; 90% of non-erythroid cells are myeloblasts; MPO (or Sudan black) is positive in more than 3% of

blasts.

M2 – AML with maturation:

30-45% of AML cases. Diagnostic criteria (in the bone marrow): azurophilic granules and/or Auer rods in more than 50% of cells; 20-90% of non-erythroid cells are myeloblasts; less than 20% of cells are monocyte precursors; MPO (or Sudan black) is positive

M3 – AML promyelocytic (hypergranular)

10-15% of AML cases Diagnostic criteria (in the bone marrow): strong granulation and lots of Auer rods; more than 50% of cells are abnormal promyelocytes; DIC is characteristic of this class.

M4 – AML myelomonocytic

15-20% of AML cases Diagnostic criteria (in the bone marrow): more than 20% of nucleated cells in the bone marrow should be myeloblasts and promyelocytes;

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Clinical Biochemistry

o

o

o

o

more than 20% of bone marrow cells should be promonoblasts and monoblasts (stain with esterases such as NSE). Gum hypertrophy and tissue infiltration are characteristic of this class.

M5a – AML monoblastic Diagnostic criteria (in the bone marrow): more than 80% of non- erythroid cells in the bone marrow belong to the monocytic lineage; more than 80% are monoblasts. Gum hypertrophy and tissue infiltration are characteristic of this class.

M5b – AML monocytic Diagnostic criteria (in the bone marrow): more than 80% of non- erythroid cells in the bone marrow belong to the monocytic lineage; less than 80% are monoblasts; monocytes and promonocytes comprise more than 20%. Gum hypertrophy and tissue infiltration are characteristic of this class.

M6 – AML erythroleukemia Diagnostic criteria (in the bone marrow): more than 50% of nucleated cells in the bone marrow are early erythroid precursors; more than 20% of non-erythroid cells in the bone marrow are myeloblasts; PAS+ erythroids.

M7 – AML megakaryoblastic Diagnostic criteria (in the bone marrow): more than 20% of non- erythroid cells in the bone marrow are blasts showing megakaryocytic differentiation ("budding" cytoplasm).

Prognosis in AML:differentiation ("budding" cytoplasm). o o Age of onset below 2 years and above 60 years is

o

o

Age of onset below 2 years and above 60 years is not favorable;

Pathomechanisms involving deletions in 5, 7 chromosomes, t(9:22) and t(11q23) are nor favorable.

However, t(8:21) in M2 and inversion in chromosome 16 in M4 are favorable. t(15:17) in M3 is intermediately unfavorable.

CML

It is most common between 40-60 years of age, and more males are affected.favorable. t(15:17) in M3 is intermediately unfavorable. CML are 3 phases to the disease: The chronic

are 3 phases to the disease:between 40-60 years of age, and more males are affected. The chronic phase: 1/3 of cases

The chronic phase: 1/3 of cases are asymptomatic, while those who are have general symptoms such as anemia, fatigue, dyspnea, tachycardia, hepatosplenomegaly, abdominal discomfort, weight loss, night sweats and bleedings.

The accelerated phase: in this phase, patients exhibit enhanced splenomegaly, subfever (or fever), there are 5-20% blasts in the bone marrow and in the peripheral blood, thrombocytopenia or thrombocytosis that is unresponsive to treatment, an increase in the WBC count, and other genetic alterations that were not present at time of diagnosis.

The blastic phase (the crisis phase): in this phase there are more than 20% blasts in the bone marrow and in the peripheral blood. 1/3 of cases will transform to ALL and 2/3 to AML.

should be differentially diagnosed from leukemoid reaction: in leukemoidblood. 1/3 of cases will transform to ALL and 2/3 to AML. reactions there is a

reactions there is a dramatic increase in granulocyte count due to inflammatory

processes going on. Therefore, GAPA score is positive in these reactions (Granulocyte Alkaline Phosphatase Activity on a 0-4 scale).

Pathogenesis: t(9:22) BCR-ABL forming mutated tyrosine kinase; 95% of cases show this Philadelphia chromosome. BCR-ABL forming mutated tyrosine kinase; 95% of cases show this Philadelphia chromosome.

There

o

o

o

CML

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Lab:Avi Sayag Clinical Biochemistry o Blood count: anemia; thrombocytosis; leukocytosis o Bone marrow: hypercellular

Clinical Biochemistry

o

Blood count: anemia; thrombocytosis; leukocytosis

o

Bone marrow: hypercellular (myeloids > erythroids); eosinophils and basophils; megakaryocytes are smaller

o

Cytochemistry: decreases GAPA

o

Cytogenetics: Ph. Chromosome

Therapy:decreases GAPA o Cytogenetics: Ph. Chromosome o Drugs (cytostatics) o IFN- α (arrests cell cycle)

o

Drugs (cytostatics)

o

IFN-α (arrests cell cycle)

o

TK inhibitors (imatinib)

o

BM transplantation (allo).

Characterization

of

leukemic

cells

by

cytochemistry

and

immunophenotyping (practical topic 8)

Cytochemical reactions For the identification of leukemic cells and subtype the evaluation of the intracellular material is needed (enzymes, stored substances), since undifferentiated cells display a lot of similarities.

1. Myeloperoxidase (MPO): used to differentiate AML from ALL. Positive reaction:

grayish-black.

2. Sudan black: detects the phospholipids in leukocyte granules. Positive reaction: black.

3. Non-specific esterase: the most abundant activity is detected in monocytes and promyelocytes. Positive reaction: light brown.

4. PAS: detects the glycogen stored in the cytoplasm. Positive reaction: red.

5. Lysozyme: the enzyme dissolves the bacterial cell wall. Monoblasts and immature monocytes are positive.

6. Prussian blue: detects iron depletion. Positive in ring sideroblasts. Positive reaction:

blue.

7. GAPA: granulocyte alkaline phosphatase converts α-naphtyl-phosphate into a brownish precipitate in alkaline pH. The phosphatase reaction is low or absent in CGL, but its activity is enhanced in myeloproliferative disorders and in leukemoid reactions.

8. Acid phosphatase: positive reaction: red. Positive in MM and HCL.

Sudan black: positive in – M1, M2, M3, M4. M5: 0/+, all the rest: 0. MPO: positive in M3, M4. All the rest: 0/+ NSE: positive in M3, M4, M5. ALL: 0, all the rest: 0/+ PAS: positive in M6, ALL, all the rest: 0/+. Acid phosphatase: positive in MM and HCL (TRAP+).

Type

MPO

Sudan

PAS

NSE

Lysozyme

AP

AML- M1,

0/+

++

0/+

0/+

0

 

M2

M3

++/+++

++

0/+

+++

0

 

M4

+

++

0/+

++

0/+

 

M5

0/+

0/+

0/+

+++

++

 

M6

0/+

0

+

0/+

0

 

M7

0/+

0

0/+

0/+

0

 

ALL

0

0

+

0

0

 

MM

         

+++

HCL

         

++

(TRAP)

Immunophenotyping

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Clinical Biochemistry

This method is based on the detection of cell surface markers and intracytoplasmic markers. It is based on the identification of specific CD markers on pathological cells by fluorophor labeled monoclonal Abs. Since these antigens are already present on immature cells, the method allows the analysis of undifferentiated cells and the determination of subgroups of leukemias. The primary diagnostic goal in acute leukemias is the characterization of the blast cells and the detection of abnormal marker expressions. The most important CDs characteristic for cell lines are as follows:

B-cells:

CD19, CD20, CD22, HLA-DR CD10: cALLA (common acute lymphoid leukemia antigen) can be found on B cell precursors, it is absent on B cells in the peripheral blood of healthy individuals. T-cells:

CD3, CD5, CD7, CD4, CD8 Myeloid cells:

CD13, CD33, CD15, MPO Monocytes: CD14, HLA-DR Megakaryocytes: CD41, CD42, CD61 Stem cell marker:

CD34: not detectable in the peripheral blood and less than 5% in the bone marrow of healthy individuals.

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Clinical Biochemistry

Topic 15

Laboratory diagnostics of quantitative platelet disorders

 

Platelet disorders

Qualitative

 

Quantitative

Platelet

function

disorders

Thrombocytopenia :

Thrombocytosis

(topic 19)

Reduced production Increased destruction Loss from the body Abnormal distribution/increased trapping in the spleen

Thrombocytopenia Thrombocytopenia is characterized by spontaneous bleeding, a prolonged bleeding time, and a normal PT and APTT. Normal platelet count – 150-400 G/L Thrombocytopenia – less than 100 G/L Thrombocytopenia with tendency to bleed – less than 50 G/L Thrombocytopenia with spontaneous bleeding – less than 10 G/L (in some sources – less than 20 G/L). Larger hemorrhages into the central nervous system are a major hazard in patients with markedly depressed platelet count. In most cases in which the cause is accelerated destruction, the bone marrow reveals a compensatory increase in the number of megakaryocytes. Hence, bone marrow examination can help to distinguish the two major categories of thrombocytopenia. It is also worth emphasizing that thrombocytopenia is one of the most common hematologic manifestations of AIDS.

1. Decreased production

Congenital

Neonatal

Acquired

 

May-Hegglin anomally: a genetic disorder of platelets that causes them to be abnormally large. The cause is a mutation in the gene of non-muscle myosin heavy chain IIA. The pathogenesis

Typical in newborns infected with rubella, and in babies of mothers who take thiazides or tolbutamine.

As

part

of a general bone depression with megakaryocyte

marrow

selective

depression. Causes include:

   

1. Chemotherapy/radiation

2. Alcohol (suppresses production of megak.)

is unknown. It

is

3. Cytotoxic drugs

characterized by hypoplasia of megakeryocytes, Dohle bodies (small inclusions in PMNs) and giant platelets.

4. Aplastic anemia

5. Pernicious anemia

6. Infection with CMV, EBV, VZV, rubella, measles vaccine

 

7. Infiltration of BM with tumor cells.

8. Myeloid dysplasia with primary myelosclerosis.

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2. Increased destruction This can be due to 2 reasons:

Clinical Biochemistry

1. Destruction by immune mechanism:

a. Chronic idiopathic thrombocytopenia purpura (ITP)

i. The most common form in women between 15-50 years old that suffer from petechial hemorrhages and are easily bruised.

ii. Can be secondary to SLE, HIV, CLL, etc (but then it is not idiopathic).

iii. Platelets can be sensitized by Ab directed against GpIIb-IIIa

iv. Premature removal by the RES life-span is reduced to few hours.

v. The platelet count is 10-50 G/L.

vi. Platelets are large.

vii. Lab detection of Ab in the serum or on platelets.

viii. There is normal to increased number of megakaryocytes.

b. Acute ITP

i. Most common in children

ii. 75 % of them get it after vaccination or infection.

iii. There is a spountandeous remission.

iv. 5-10% become chronic.

c. Drug induced thrombocytopenia

i. Abs against the drug and the carrier protein are produced the circulating immune complexes are adsorbed into the platelets platelets are removed by the RES or lysed by complement.

ii. Quinine, quinidine, heparin

iii. Count: less then 10 G/L.

iv. Normal to increased number of megakaryocytes.

d. Neonatal alloimmune: the mother becomes sensitized against platelet-specific antigen of the fetus.

e. Neonatal autoimmune: ITP develops in pregnant women; Ab crosses the placenta.

f. Post-transfusion thrombocytopenia:

following

Anti-Pl

Abs

develop

transfusion.

g. Secondary autoimmune: in CLL, SLE, etc.

2. Destruction by non-immune mechanism:

a. In pregnancy of pre-eclampsia (ischemia vascular injury activation of DIC)

b. Thrombotic thrombocytopenia purpura (TTP)

i. Thrombi in capillaries and arterioles RBCs and PLTs are mechanically destroyed by the thrombi intravascular hemolysis reticulocytosis

ii. Women between 20-50 are usually affected.

iii. Severe organ damage.

iv. Count less then 20 G/L.

v. Pathomechanism:

1. Large vWF – deficiency in a MMP called ADAMTS13 which degrades very-high-molecular-weight multimer of

vWF allows multimers of vWF to accumulate in plasma.

2. Endothelial damage.

3. Defective prostacyclin production.

c. In hemolytic uremic syndrome (HUS)

i. Hemolytic anemia (decreased Hb, increased ret., schistocytes)

ii. Renal failure (elevated urea, creatinine, RBCs, proteins)

iii. Thrombocytopenia

iv. Disease of childhood (6 months – 4 years).

Avi Sayag

Clinical Biochemistry

v. Often follows acute viral infection or E. coli (verotoxin damages the endothelium bleeding, activation and consumption of PLTs)

vi. Resembles TTP with no neurological symptoms.

vii. High mortality rate.

d.

DIC

3. Loss from the body

4. Disorders related to distribution/dilution

i. Increased pooling in splenomegaly.

ii. Hypothermia

iii. Extracorporal circulation

iv. Massive blood transfusion

Thrombocytosis

1. Reactive thrombocytosis: blood loss, surgery, post-splenectomy, iron deficiency

due to blood loss, inflammation, stress, exercise.

2. Myeloproliferative disorders: polycytemia vera, myelofibrosis with myeloid metaplasia.

3. Essential thrombocytemia: megakaryocyte proliferation with high PLT count (500-2000G/L); recurrent hemorrhage and thrombosis; abnormally large PLT and megakaryocyte fragments in blood film; may transform to polycytemia vera, myelofibrosis or acute leukemia, but may remain stationary for many years.

Avi Sayag

Clinical Biochemistry

Topic 16

Inheritance of ABO blood group system and its clinical significance

The ABO antigens are added stepwise to proteins or to lipids on the erythrocytes and they appear on day 40 of gestation. The substrate molecule is L-fucose (the H antigen).

The A antigen is N-acetyl-galactosamine (GalNAc);The substrate molecule is L-fucose (the H antigen). The B antigen is galactose (Gal); A and

The B antigen is galactose (Gal);antigen). The A antigen is N-acetyl-galactosamine (GalNAc); A and B genes code for transferase enzymes, such

(GalNAc); The B antigen is galactose (Gal); A and B genes code for transferase enzymes, such

A and B genes code for transferase enzymes, such that transferase A is alpha 1-3-N- acetylgalactosaminyltransferase and transferase B is alpha 1-3-galactosyltransferase. The antigens are found not only on RBCs, but also on most body cells (leukocytes, platelets, etc.) The gene coding for blood type lies on chromosome 9q34. However, other separate genes on chromosome 11 and 19 actually interact with the blood type gene, determining our ability to secrete our ABO blood type antigens into our body fluids and secretions. This is called the secretor gene, and by testing for this gene we can determine whether we are secretors or non-secretors. In the genetics of the secretor system two options exist. A person can be either a secretor (Se) or a non-secretor (se). This is completely independent of whether one's blood type is A, B, AB, or O. Thus a person could be an A secretor or an A non- secretor, a B secretor or a B non-secretor, etc. Secretors: in a simplified sense, a secretor is defined as a person who secretes one's blood type antigens into body fluids and secretions like the saliva, the mucus in the digestive tract and respiratory cavities, etc. Non-Secretors: non-secretors on the other hand put little to none of their blood type into these same fluids. As a general rule, in the US about 15-20% of the population are non-secretors with the remaining 80-85% being secretors. Aside from the physical implications centering around whether you have blood type antigens in your body fluids or not, the secretor genetics have additional significance through the effects of gene linkage: in other words, the outcome of your secretor genetics ‘links’ to other seemingly unrelated genes and influences their function. Your secretor status drastically alters the carbohydrates present in your body fluids and secretions in addition to several important aspects of your metabolism and resistance. These factors include the activity of an enzyme called intestinal alkaline phosphatase, the overall composition of bacteria in your intestinal ecosystem, your propensities toward blood clotting, your level of carbohydrate tolerance, and your resistance to certain parasites and yeast (based on these features, new diet regimes have been proposed tailored to the blood type of those who are desperately willing to try anything to lose weight). The natural ABO antibodies are IgM, which are produced after the first few months of life (after the 4 th month). These antibodies work in "cold" temperatures (room temperature) and may fade in old age. Other irregular Ab are produced after immunization (incompatible

Avi Sayag

Clinical Biochemistry

transfusion or pregnancy). Most complications after such incompatible transfusions are caused by these Ab (mostly IgG – warm Ab that cross the placenta). Antigens and antibodies:

Blood group – Ag on RBC – Ab in serum – Genotype

A – A – anti B – AA or Ao

B – B – anti A – BB or BO

AB – A and B – none – AB

O – none (H) – anti A and anti B - OO

Subtype A The A blood type contains about 20 subgroups, of which A1 and A2 are the most common. 80% of which are A1. These cells carry about 1 million antigens on a single RBC. The A2 cells, however, carry only 200,000 antigens on a single RBC. A1 and A2 differ in such a way that type A2 people can produce anti-A1 antibodies. As mentioned, there are weaker subgroups in the A subtype (about 18) due to mutations in the A gene that may lead to dysfunctional transferase (3-α-N-acetylgalactosaminyl-transferase).

The clinical significance of the ABO group manifest in mismatched transfusions, although rare. However, should they occur, they may be life-threatening, as they may lead to intravascular hemolysis. It is more severe in group O patients, as they have very reactive anti-

A and anti-B antibodies.

Universal donors and recipients:

O group carries no A or B antigens.

The packed and processed units have little or no antibody content. The AB group has no antibodies whatsoever, and therefore cannot lyse

any transfused cells. Other antibodies, however, may be present!

Prevalence:

Bororo (brazil) and Peru (Indian) – 100% type O North American Indians – 82% type A Type O is the most prevalent in all parts of the world, except N. American

Indians. The Bombay phenotype Individuals with the rare Bombay phenotype (hh) do not express antigen H on their red blood cells. As H antigen serves as precursor for producing A and B antigens, the absence of H antigen means the individuals do not have A or B antigens as well (similar to O blood group). However, unlike O group H antigen is absent, hence the individuals produce isoantibodies to antigen H as well as to both A and B antigens. In case they receive blood from O blood group, the anti-H antibodies will bind to H antigen on RBC of donor blood and destroy the RBCs by complement-mediated lysis. Therefore, Bombay phenotype can receive blood only from other hh donors (although they can donate as though they were type O). It should be mentioned that the transferase enzyme IS produced in this phenotype.

O

A B AB
A
B
AB

Summary and presentation of the topic:

Present the blood types and the biochemistry of the various types + prevalence Explain the difference between them in terms of chemistry, Ag, Ab and transferases Chromosome 9q34 and interactions with chromosomes 11 and 19 – Se and se (secretor and non-secretor). The significance of being an secretor or a non-secretor. Subtype A – distribution of A1 and A2 Clinical significance: blood transfusion – universal donors and recipients Bombay phenotype

Avi Sayag

Clinical Biochemistry

Topic 17

Inheritance and clinical significance of Rh blood group system

The Rh blood group system is the second most important system and is the most complex. It is important because it is associated with hemolytic transfusion reactions and with development of severe hemolytic disease of the newborn (HDN). Rh antigens are proteolipids and lack carbohydrate. Rh antigens are present on RBCs only, and there are 20,000 antigen

sites on each erythrocyte. They are relatively small (32KDa). The inheritance of Rh antigens

is determined by a complex of 2 closely linked genes on chromosome 1. One gene codes for

the protein carrying D expression; the other codes for the proteins carrying C or c and E or e expression. Rh-positive individuals have both a D and a CE gene while Rh-negative individuals have only a CE gene. Depending on which genes are present on a chromosome, 8 common antigen combinations or haplotypes are possible: Dce, DCe, DcE, DCE, dce, dCe, dcE, dCE. Common phenotypes and genotypes are given below.

D antigen is the most important Rh antigen. Presence of a single D antigen confers upon an

individual the designation Rh-positive; its absence means that the person is Rh-negative. Eighty five percent of Caucasians, 92% of African Americans and 99% of Asian Americans are Rh positive. The letter d is commonly used to indicate the lack of D in Rh-negative individuals, but neither d antigen nor anti-d has been detected. The Fisher-Race nomenclature has been more widely adopted over the more complex Wiener nomenclature for Rh antigens. However, an abbreviated version of the Wiener system is useful to describe Rh genotypes.

Wiener is covenient because it uses a single letter, R or r, with superscripts to name a 3 locus haplotype. It is possible to translate from one nomenclature to the other by remembering a few rules:

In the Wiener system, D is indicated by an uppercase R and the absence of D is indicated by

lower case r.

In the Wiener system, superscripts or numbers are used to indicate which Cc or Ee genes are

present. Numbers are used with R and primes are used with r.

In

the Fisher Race system, loci are lined up in the order Dd, Cc, Ee (e.g. DCE).

In

the Wiener system, the Dd position is numbered 0, Cc position is 1 and Ee position is 2.

The Wiener superscript of 0, 1, 2 indicates which of the Fisher Race loci is in its uppercase form (D, C, or E). For example, 1 or prime indicates that C is capitalized, while a 2 or double prime indicates that E is capitalized.

Rh Nomenclature and Haplotype Frequencies

Fisher Race

Wiener

Dce

Ro

DCe

R1

DcE

R2

DCE

Rz

dce

r

dCe

r'

dcE

r"

dCE

ry

The most common haplotye in Caucasians and Asian Americans is DCe (R1), while the most common phenotype in African Americans is Dce (Ro). Red blood cells that fail to react with all Rh antibodies are called Rh null. An individual's genotype can only be determined with certainty by performing DNA analysis and family studies. Unlike the ABO naturally occurring antibodies, Rh antibodies are produced in response to an incompatible transfusion or pregnancy. The D antigen is the most immunogenic of the Rh antigens, causing immunization at least 80% of the time when a D-negative person receives a single unit of D-positive blood. Anti-c is the second most important Rh antibody. Although anti-E is more common than anti-c, anti-E is frequently a naturally occurring antibody. Anti-c and anti-e only occur after an antigenic stimulus.

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Clinical Biochemistry

Weak D (D u ): some individuals have a weak expression of the D antigen for either which of 3 reasons:

Individuals who lack part of the D antigen (partial D) have a weak expression on their RBCs. If they are immunized, they produce antibodies to the portion they lack. The D gene encodes all epitopes of the D antigen, but the antigen number on RBCs is less than normal. In some cases a C transposition to a D gene occurs (Dce/Ce or DCe/Ce) which weakens the expression of D. Significance:

80% of Rh(D)- persons exposed to Rh(D)+ blood will develop anti-D antibodies. Anti-D antibodies can also be stimulated by pregnancy with an Rh(D)+ baby. Sensitization can be prevented by the use of anti-D Ig antenatally and postnatally. Rh(D)- females of childbearing potential should never be given Rh(D)+ blood. Antibodies to Rh antigens are primarily IgG antibodies which can cross the placenta. IgG is too small to make bridges between RBCs, so agglutination doesn't occur in saline. These antibodies work best in "warm" temperatures. The D antigen is the most important cause of Hemolytic Disease of the Newborn (HDN). Other antigens can also cause it (C, c, E, e) as well as other blood groups (rare). Finally, it should be noted that the inheritance of ABO and Rh are not linked and are inherited independently.

ABO and Rh are not linked and are inherited independently. Summary and presentation of the topic:
ABO and Rh are not linked and are inherited independently. Summary and presentation of the topic:
ABO and Rh are not linked and are inherited independently. Summary and presentation of the topic:
ABO and Rh are not linked and are inherited independently. Summary and presentation of the topic:
ABO and Rh are not linked and are inherited independently. Summary and presentation of the topic:
ABO and Rh are not linked and are inherited independently. Summary and presentation of the topic:

Summary and presentation of the topic:

Rh Ags are proteolipids found only on RBCs, with 20000 Ags on each RBC Inheritance – 2 genes on chromosome 1 (D and CE) 8 combinations Fisher-Race vs. Wiener Most common DCe (R1) – Caucasians and Asian Americans Most common Dce (R0) – African Americans Anti-Rh Abs are produced in response to transfusion, pregnancy, etc. D Ag is the most immunogenic c Ag is the second most immunogenic, and NOT naturally occurring Anti-e is not naturally occurring Anti-E is frequently naturally occurring and more common than anti-c. 3 causes of weak D (D u ) Significance of Rh: transfusion, pregnancy, transfusion to women in childbearing age Abs are IgG Rh and ABO are not genetically linked

Avi Sayag

Clinical Biochemistry

Determination of ABO and Rh blood group (practical topic 9)

ABO determination

Abs in the ABO group are naturally occurring ones: cross-reacting carbohydrate structures on

environmental agents stimulate the thymus-independent production of IgM anti-A and/or anti-

B in individuals who are not tolerant to these antigens. The IgM Abs then directly agglutinate

the appropriate antigen-positive RBCs, preferentially at room temperature.

The Landsteiner rule: sera of healthy adults contain ABO Ab that reacts with the ABO Ags

missing from the person's RBCs, but it must not contain any Ab that reacts with the Ags on the person's RBCs.

Blood group

Ag on RBC

Ab in serum

Genotype

A

A

Anti-B

AA or AO

B

B

Anti-A

BB or BO

AB

A and B

None

AB

O

None

Anti-A and anti-B

OO

Bedside blood group determination can be one-sided or two-sided.

1. One-sided

This examination is based on agglutination by addition of anti-A, anti-B and anti-AB antisera

at room temperature and in saline medium. We put one drop of each anti serum in its place on

the right around 2 cm apart, and then we put one drop of the patient's serum. Then, we put one drop of the patient's blood and saline opposite to each antiserum and serum of the patient. We

mix them well using the corner of slide, caring not to contaminate one with the other. We

allow the slide to stand for 30 seconds at room temperature and then tilt the slide backward

and forward by 30º. In the one-sided method, we determine the presence/absence of antigens

on RBCs and only that! Hence, one-sided method.

2. Two-sided

In this method, we determine both the Ags on the RBCs and the Abs in the serum of the

patient (hence "two-sided").

We

divide a tile into 2 parts by an imaginary line. We put one drop of anti-A, anti-B and anti-

AB

sera with proper distance from each other on the left side, and mark their places for safety

(see figure on page 55 of the practice book). We then make a 10% suspension of the blood sample in physiological saline and put one drop opposite each of them. On the right side we put 4 drops of the requested serum with sufficient space from each other. We put one drop of known A1, A2, B and O test RBCs opposite the blood sera. We mix them carefully and let them stand for 10 minutes at room temperature. We again tilt it and read the results visually.

Rh determination

The test is performed on a glass slide or white glass bottle filled with hot water and with a

surface temperature of 37-42ºC. We place one drop of anti-D serum, one drop of the appropriate control reagent and to both drops we add one drop of a well-mixed 50% suspension of the investigated RBCs in their own serum or plasma. We then mix the RBC suspension and the reagent, and put it in a wet chamber at 37ºC for 20 minutes. We gently tilt

the slide and observe for agglutination. After 2-3 minutes we record and interpret the results.

We should use Rh+ and Rh- controls with the same method at the same time.

Papain enzyme treatment of RBCs cleaves the sialoglycoproteins from the RBCs, and the net surface charge decreases. This enhances the agglutination reaction. It has a special importance

in cases when allo-Abs or auto-Abs are bound to the surface of the RBCs as a result of

previous incompatible transfusion. Rh+: the suspension is agglutinated and the autocontrol is homogenous. Rh-: if the suspension ad autocontrol are the same and homogenous. In case of an uncertain result (positive autocontrol, weak reaction, etc.), the sample should be sent to special

departments for further examination.

Avi Sayag

Clinical Biochemistry

Topic 18

Coagulopathies, laboratory control of anticoagulant treatment

The coagulopathies can be inherited, acquired, iatrogenic or therapeutic. The coagulopathies can be caused by 3 main mechanisms:

1. Decreased levels or absence of clotting factors:

In this category, the decreased levels can be caused by inherited disorders, acquired disorders

(e.g. in liver failure which leads to decreased synthesis of clotting factors as well as in consumption coagulopathies) and by therapeutic mechanisms (agents leading to thrombolysis).

2. Synthesis of abnormal clotting factors

In this category, the synthesis of abnormal clotting factors can be caused by congenital defects, by acquired disorders (such as in dysfibrinogemia in liver diseases and in vitamin K deficiency), by therapeutic agents (e.g. Syncumar, aka Coumarin) and by iatrogenic factors

(such as administration of cephalosporin).

3. Inhibitors of coagulation

In this category, the coagulopathies can be caused by neutralizing or non-neutralizing antibodies that are directed against clotting factors. This condition can be accompanied by inherited factor deficiency but not necessarily. However, not only antibodies can cause

coagulopathies, but global inhibitors can as well (e.g. heparin). Bearing in mind the coagulation pathway, 4 main screening tests of blood coagulation should be mentioned: APTT, PT, TT and bleeding time.

the coagulation pathway, 4 main screening tests of blood coagulation should be mentioned: APTT, PT, TT

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Clinical Biochemistry

Intrinsic pathway Extrinsic pathway

APTT 28-40 seconds + 9 seconds prolonged After collecting blood samples in vacu-tubes with oxalate or citrate to arrest coagulation by binding calcium, the specimen is then delivered to the laboratory. In order to activate the intrinsic pathway, phospholipid, an activator (such as silica, celite, kaolin, ellagic acid), and calcium (to reverse the anticoagulant effect of the oxalate) are mixed into the plasma sample . The test is termed

PT 8-12 seconds + 4 seconds prolonged The prothrombin time is most commonly

measured using blood plasma. Blood is drawn into a test tube containing liquid citrate. The blood is mixed, then centrifuged

to separate blood cells from plasma. An

excess of calcium is added (thereby reversing

the effects of citrate), which enables the blood to clot again. Tissue factor (also known as factor III or thromboplastin) is added, and

"partial" due to the absence of tissue factor in

the

time the sample takes to clot is measured

the reaction mixture. The time is measured

optically or using the KC-1. TF is both the

until a thrombus forms. If the clotting does not occur within 200 seconds, the result is said to be APTT > 200 sec.

receptor and activator of FVII. If the clotting does not occur within 100 seconds, the result is said to be PT > 100 sec. Factors II, V, VII,

Causes of prolonged APTT:

IX

and X are vit.-K dependent, therefore the

1. Deficiency or decreased "intrinsic

PT

test is good to monitor coumarin therapy.

factors": hemophilia A and B

If so, the result should be given as

2. Presence of heparin in the sample

3. Presence of inhibitors directed against clotting factors or phospholipids (lupus anticoagulant)

4. Inappropriate ratio of Na- citrate:blood (citrate in excess)

5. Consumption coagulopathies

When do we use APTT?

1. To monitor unfractionated heparin therapy (UFH) 2

2. Control of fibrinolytic therapy:

before thrombolytic therapy, screening for hemorrhagic diathesis should be performed.

3. Diagnosis of DIC

4. Diagnosis of thrombophilia

5. In liver diseases

6. If a patient with severe bleeding is treated only with RBC concentrate,

and does not receive the plasma clotting factors.

INR =

PT

pt

PT

contorl

ISI

. ISI is the International

Sensitivity Index. The smaller the ISI is, the more sensitive the reagent is. The INR should

be kept between 2-3. INR of patients with

prosthetic heart valve should be between 2.5- 3.5. PT determination and INR should be performed every 2 weeks during the first 6 weeks of therapy, and then, if the INR is stable, once in a month. If INR is > 5, there is

a risk of spontaneous bleeding. Causes of prolonged PT:

1. Coumarin therapy

2. Hereditary/acquired absence of "extrinsic" factors/abnormal synthesis

3. Inappropriate ratio of Na-

citrate:blood (citrate in excess)

4. Fibrinolytic therapy

When do we use PT?

1. To monitor coumarin therapy

2. Before thrombolytic therapy:

screening for hemorrhagic diathesis should be performed.

3. Diagnosis of DIC

4. Liver disease

2

APTT taken after:

0.5-1 hr

2-3 hr

4-6 hr

UFH continuous

X

   

I.V bolus heparin

 

X

 

Subcutaneous heparin

   

X

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Clinical Biochemistry

Thrombin time (TT): TT evaluates the last phase of the clotting cascade and represents the time (in seconds) that elapses between the addition of thrombin (usually bovine thrombin) and the onset of clotting. The values of both the control's and the patient's plasma are reported. The reference interval is 14-22Avi Sayag Clinical Biochemistry seconds. TT is considered prolonged if the patient's TT exceeds the control

seconds. TT is considered prolonged if the patient's TT exceeds the control value by 8 seconds. If clotting does not occur within 100 seconds, the result is given as TT > 100 seconds. Causes of prolonged TT:

i. Heparin treatment

ii. Pathologic levels of fibrinogen/fibrin split products (acute DIC, primary hyperfibrinolysis, dissolution of a thrombus) that inhibit the thrombin activity and fibrin polymerization

iii. Severe hypofibrinogenemia or afibrinogenemia

iv. Dysfibrinogenemia

v. Certain hepatic diseases due to hypo- or dysfibrinogenemia.

vi. If a patient with severe bleeding is treated only with RBC

concentrate, and does not receive the plasma clotting factors.

Bleeding time: apply 40 mmHg tourniquet pressure to the upper arm and maintain it during the entire process. Wipe the inner surface of the forearm with ethanol and, by avoiding larger visible veins, cut the skin using a special disposable device. The device is attached to the forearm without pressure, and pushing a trigger two 5-mm blades are released that make two 1-mm- deep cuts. Dry the blood with a sterile blotting paper every 30 seconds without touching the wound. The time when the last drop of blood is visible on the blotting paper is the bleeding time. The reference interval is 2.5-9.5and does not receive the plasma clotting factors. minutes. If bleeding does not stop within 20

minutes. If bleeding does not stop within 20 minutes, the result is given as bleeding time > 20 minutes. Unfortunately, in most labs and hospitals the bleeding time is determined by pricking the fingertip. This is a completely unreliable method of no clinical significance, for the bleeding time in this case depends on the thickness of the skin of the fingertip and on the depth of the pricking, rather than on platelet function. Bleeding time is the most important screening test of platelet function, as bleeding of a small wound

stops when platelets adhere to the injured vessel wall forming a primary platelet plug. Bleeding time is normal in coagulopathies with the exception of afibrinogenemia (i.e. it is normal in hemophilias!) Diseases that cause prolonged bleeding time include:

i. Thrombocytopenia

ii. DIC

iii. Aspirin and other cyclooxygenase inhibitors can prolong bleeding time significantly.

iv. While warfarin and heparin have their major effects on coagulation factors, an increased bleeding time is sometimes seen with use of these medications as well.

v. People with von Willebrand disease usually experience increased bleeding time, as von Willebrand factor is a platelet agglutination protein, but this is not considered an effective diagnostic test for this condition.

vi. It is also prolonged in hypofibrinogenemia.

Several coagulopathies should be mentioned:

1. Hemophilia A

2. Hemophilia B

3. Afibrinogenemia and dysfibrinogenemia

4. Other factor deficiencies

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Clinical Biochemistry

Hemophilia A

This bleeding disorder is caused by a mutation in FVIII gene, which is located on chromosome X (Xq28) and is the most common hemophilia. FVIII is a big glycoprotein synthesized in the liver, and perhaps in endothelial cells. It is a cofactor of FX activation (along with FIX). Thrombin is required to activate FVIII. The deficiency in FVIII can be inherited (a mutated FVIII gene) or acquired (antibodies directed against FVIII). Symptoms: hemophilia leads to a severely increased risk of bleeding from common injuries.

The first symptoms that should suspect of hemophilia are bleeding at labor and during delivery, circumcision, vaccination and onset of walking. The sites of bleeding include the GI and the brain (less common) and the joints and muscles (most common). Hemarthrosis occurs primarily in the knee and elbow joints. As RBCs are lysed, iron is deposited in the synovium. This leads to chronic synovitis, synovial fibrosis, joint stiffness, limited motion and pain. Diagnosis: APTT is elevated, PT is normal, TT is normal and bleeding time is normal. Factor assay can also be performed. Hemophilia A should be differentially diagnosed from:

1.

von Willenbrand disease;

2.

Combined FVIII and FV deficiency; and

3.

Consumption coagulopathies.

4.

Hemophilia B

5.

Vitamin K deficiency

6.

Vitamin K antagonist drugs.

Treatment: administration of FVIII.

Hemophilia B

Hemophilia B is caused by a mutation in the FIX gene located on the X chromosome (Xq27). FIX is synthesized in the liver and its function depends on vitamin K. Its cleavage is carried via FXI and FVII. In the presence of FVIII and Ca +2 it cleaves FX. Symptoms: Factor IX deficiency leads to an increased propensity for hemorrhage. This is in response to mild trauma or even spontaneously, such as in joints (haemarthrosis) or muscles. Diagnosis: APTT is elevated, with normal PT, normal TT and normal bleeding time. Factor assay can also be carries out. Hemophilia B should be differentially diagnosed from:

1.

Liver disease

2.

Vitamin K deficiency

3.

Vitamin K antagonist drugs.

4.

von Willebrand disease

5.

Afibrinogenemia/dysfibrinogenemia

6.