PRACTICAL I

CLINICAL BIOCHEMISTRY ASSAYS GLUCOSE PROFILE FBS/RBS (FPG) GTT 2HR PP HbA1c URINE sugar/ketones ALT ALP GGT TOTAL BILIRUBIN Direct Indirect TOTAL PROTEIN Albumin Globulins BUE URIC ACID CREAT CLEAR. Mg2+ CREAT Na+ K+ ClLIVER FUNCTION TEST(LFT) AST KIDNEY FUNCTION TEST(KFT) BUN TOTAL CHOLES TRIG HDL-C LDL-C VLDL LDH CK-MB MYOGLO TROPONIN AST TOTAL IRON TIBC Ferritin ALB Ca2+ PO42ALP CREAT LIPID PROFILE CARDIAC PROFILE IRON STUDIES BONE PROFILE

HORMONAL ASSAYS THYROID TEST T4 T3 TSH FN FERTILITY HORMONES FSH LH PRL PRG E2 TUMOUR MARKERS Total PSA B-HCG CEA AFP CA 125 OTHER HORMONES INSULIN CORTISOL OTHER CHEMISTRIES AMYLASE

TESTOSTERONE CA19-9 /CA15-3

SPECTROPHOTOMETRY Measurement of light intensity of multiple wavelengths

LAMBERT-BEERS LAW: The [substance] is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. C A log 100 %T A=abC Where A= absorptivity, b=length of light path, C= [ ] a and b are constants. C1=A1, C2=A2, C1 = A1 C2 A2 [ Ctest= Cstd A test] Astd Beers law is the basis of all the spectrophotometric measurements. Advantages of spectrophotometric measurements: 1. High sensitivity 2

2. Ease of measurement 3. High degree of specificity 4. Accuracy

Urinalysis is an indispensable part of chemical pathology. It is used to uncover dx anywhere in the urinary tract. TESTS ON URINE COMMONLY INCLUDES A) URINE COLOUR AND APPEARANCE (MACROSCOPY): Its colour is determined by its concentration, the presence of drugs, exogenous and endogenous compounds and its pH. Colourless urine---normal or 20 to diuretic use, high fluid intake,DIorDM. Cloudy/Hazy------phosphates, pyuria,or bacteruria Yellow to orange----- presence of bile, riboflavin , bilirubin, urobilinogen Red----------------presence of haemoglobin, Amber B)CHEMICAL COMPONENTS TEST Specific gravity pH Protein Glucose Ketones Bilirubin REFERENCE 1.003-1.029 4.5-7.8 Negative Negative Negative Negative

Occult blood Leukocyte Nitrite Urobilinogen

Negative Negative Negative 0.1-1.0 EU/dL

C )MICROSCOPY COMPONENTS: Microscopy components consists of epithelial cells, pus cells, RBCs,WBCs,casts. These are examined microscopically.

PRACTICAL II
An essential metabolic fuel and its concentration in the blood are normally tightly controlled

by the action of insulin and the counter regulatory hormones like Glucagon, cortisol, catecholamine, and growth hormone. [Glu] rises after meals and stabilizes at lower levels on fasting. Syno: blood sugar, FBS, FBG sugar. Specimen: plasma: Note: whole blood taking in fluoride oxalate vacutainer. Patient care: Pt should be fasting for 8hours-12hours prior to the test. Source of error: blood not in fluoride oxalate tube. Glycolysis by red cells will consume glu and may lower concentrate in vitro in absence of fluoride. BLOOD & PLASMA GLUCOSE Plasma [glu] is 10-15% higher than whole blood [glu] since red cells contain less water per unit volume than plasma. The discrepancy can be greater them this if [glu] is changing rapidly because glu will not have reached equilibration across the red cell membrane. Therefore plasma yields more reliable results.

Types of glucose test: RBS, 1hr pp, 2hr pp. 2hr Postprandial: glucose levels 2hrs after meal or after measured glucose level. It is used extensively to establish the diagnosis of DM Ref Ranges: FBS : 3.6-6.4 mmol/L RBS :3.3-7.0mmol/L (Dependant on time & meal content)

PRACTICAL III GTT/OGTT

The GTT is a provocation test to examine the efficiency of the body to metabolise glucose and is used to establish the presence of glu intolerance. It is used in pts with borderline fasting and PP glu to support or rule out the diagnosis of DM. It is used to work up glycosuria without hyperglycemia and also used to predict prenatal mortality in pregnancy, to diagnose gestational diabetes. PRECAUTION BEFORE TEST 1. Pt. should fast 12 hours prior to test. 2. Pt. should not smoke due to glu stimulation by nicotine. 3. Pt should not take any drug prior to test. 4. Pt should not be stressed. 5. Pt should not eat nor drink nor smoke during test. 6. Pt should not indulge in unaccustomed amounts of exercise liking jogging. 7. Pt should be sitting up or lying over on the right side so as to facilitate rapid emptying of the stomach. Normal Glucose load: 75G in 300ml H2O Children: Pregnant women: 1.75 x weight ( up to 75g) 100g in 300ml

Take samples of Times 0. FBS 1. 1 hr pp 2. hr pp 3. 3hr pp. with urine samples H2O GLUCOSE LOAD: g in 300 ml H2O RESULTS OF OGTT Vs TIME 5

TIME (Min) 0
FASTING BLOOD SUGAR

BLOOD GLUCOSE CONC (mmol/L)

URINE SUGAR

60 120 180 METHODOLOGY: Glucose oxidase method or Hexokinase method. Principle of method: glucose oxidase (GOD) catalyzes the oxidation of glucose to give hydrogen peroxide (H2O2) and gluconic acid. In the presence of enzyme peroxidase (POD), the hydrogen peroxide is broken down and the oxygen released reacts with 4-aminophenazone (4-aminoantipyrine) and phenol to give a pink colour. The absorbance of the colour produced is measured in a spectrophotometer at 520nm. Glu+O2 + H2O Glucose + phenol + 4Aminophenazone PROCEDURE STD Glucose Rgt STD TEST 10L 10L 10L oxidase 1 ml STD 1ml TEST 1ml BLK 1ml

G Oxidase

Mix well and incubate at room temperature for 10mins or in water bath for 5mins and read absorbance at 520nm against reagent blank. Calculation; [test] = [std] Abstd CAUSES OF ABNORMAL RESULTS HIGH 1. DM a. Type I (IDDM) b. Type II (NIDDM) c. Impaired glucose tolerance d. Gestational diabetes 2. Pancreatitis 3. Endocrine disorders eg. thyrotoxicosis 4. Drugs eg. steroids, oral contraceptives 5. Chronic renal failure 6. Stress 7. I.V. glucose infusion 8. Postprandial 4. Extra-pancreatic neoplasm 5. Serum liver dysfunction 6. Ethanol ingestion 7. Drugs eg. sulfourea, salicylates, insulin 2. Adrenal cortical insufficiency acromegaly, 3. Hypopituitarism LOW 1. Insulinoma Abtest mmol/l

GLYCATED HAEMOGLOBULIN (HbA1c)

A glycated derivative of Hb, formed at a rate dependent on blood [glucose]. Its formation is effectively irreversible so that it persists for the life of the RBC in which it is contained. HbA1c is a valuable indicator of glycaemic control over the previous 4 8 wks in patients with DM. Reference range: Depends on method. ( < 5% ) Specimen: Anticoagulated whole blood (generally EDTA or Fluoride oxalate bottle)

PRACTICAL IV

TOTAL SERUM PROTEIN 1. There are over a hundred individual plasma proteins: many are present in very small concentrations. 2. Approximately half of the total protein is albumin; much of the rest is immunoglobulins (principally IgG) i.e. Total Protein Albumin = Total Immunoglobulin Ref. Range: 60 80g/L SPECIMEN: Serum ( Total plasma protein is slightly higher than total serum protein because of coagulation factor present). SOURCES OF ERROR: 1. Venous stasis during venipuncture can lead to increased values. 2. Haemolysis can falsely elevate (total protein) METHODOLOGY: BIURET REACTION PRINCIPLE: Cu2+ ions react with peptide bonds in alkaline medium to form a purple complex which absorbs maximally at 540nm.The [ Complex] = [ protein ] STD Biuret Rgt. Std Sample 2.5mL 0.05mL STD 2.5mL 0.05mL BLANK 2.5mL T1 2.5mL 0.05mL T2 2.5mL 0.05mL

Mix well and incubate at RT for 10mins and read absorbance at 540nm against rgt. blank. Use Beers Law to calculate the [test]. ABNORMAL LEVELS OF PROTEINS CAN BE DUE TO: HIGH 1. Sarcoidiosis LOW 1. Poor nutrition

2. Chronic inflammation 3. Myeloma 4. Hypergammaglobulinemia

2. Over hydration 3. Hepatic insufficiency 4. Protein losing enteropathy 5. Agammaglobulinemia

ALBUMIN
The major plasma protein and is involved non-specifically in the transport of numerous substances including unconjugated bil, Ca2+, free fatty acids, vitamins A, K and other drugs. It is the most important determinant of oncotic pressure (osmotic pressure due to ptn) and hence the distribution of fluid between the vascular and interstitial spaces. USES: Evaluation of nutritional status. Diagnosis of renal diseases with proteinuria. Diagnosis of chronic diseases. SPECIMEN: Serum Ref. Range: 35 50g/L SOURCES OF ERROR: 1. Stasis during venipuncture can cause an apparent increase. 2. Taking blood sample close to site of intravenous infusion. METHODOLOGY Bromocresol Green (BCG) is widely used. PRINCIPLE: BCG reacts with albumin in acidic medium of PH 4.2 to form a green complex which is measured at 600nm. The reading of the complex has to be done at 30s to avoid the tendency of other proteins reacting with the BCG. STD BCG STD 2.0mL 10L STD 2.0mL 10L BLANK 2.0mL TEST 2.0mL TEST 2.0mL -

BLANK TEST

10L

10L

Mix well and read absorbance at 600nm at exactly 30s. Use Beers law to calculate [test]. ABNORMAL VALUES OF ALBUMIN CAN BE DUE TO: INCREASED CONC 1.Dehydration DECREASED CONC 1. Renal disease 2. Liver insufficiency with decreased albumin synthesis. 3. Severe malnutrition 4. Acute inflammation 5. Chronic inflammation 6. Pregnancy 7. Burns

PRACTICAL V

An orange-yellow pigment, the end product of haem degradation normally metabolized in the liver and excreted into bile. Accumulation causes jaundice which usually become apparent when [plasma] exceed twice normal. Frequently elevated in Hepatobiliary dx of any type when bilirubinuria is usually also present. Differential diagnosis of liver dx requires total and direct bil values as well as other tests. Total bil=Direct (conjugated) bil+Indirect (unconjugated) bil Direct bil is the water soluble fraction and indirect is water insoluble. SPECIMEN: SERUM N.B. Protect samples from since bil is photosensitive. Ref. Range: DBil: (< 3.4mol/l) Total Bil: 2-21mol/l

10

Neonates values vary with age in days, prematurity and maturity. [Total bil] are measured in neonates in mol/l AGE Cord <24hr <48hr 35days 7days Sources of Error: Gross haemolysis Lipaemia JENDRASSIK-GROF METHOD PRINCIPLE: Bil reacts with diazotized sulphanilic acid (DSA) to form a red azo dye. The absorbance of this dye at 546nm is directly proportional to [bil] in the sample. H2O-soluble bil glucuronides react directly with DSA whereas the alb-unconjugated ( indirect) bil will only react with DSA in the presence of an accelerator eg. Na benzoate, caffeine and ethanol. Total Bil = DBil + Indirect Bil PROTOCOL TBil Diazo Rgt. NaNO3 Caffeine Normal saline Serum 1mL 1 drop 1mL 100L Mix well and incubate in the dark for 20mins. Read absorbance at 540nm. 11 DBil 1mL 1 drop 1mL 100L Mix well and incubate in the dark for 5mins. Blank 1mL 100L Premature 49.6 136.8 205.2 256.5 256.5 Full term 42.8 102.6 171 205.2 171

Bilirubin conc. [mg/dL] = A540 x 13.0 or [mol/L] = A540 x 17.1x 13 ABNORMAL VALUES (CAUSES) TOTAL 1. Liver insufficiency 2. Extra-hepatic obstruction 3. Haemolysis 4. Neonate from a variety of caused including neonatal physiological hyperbilirubinemia. 5. Gilbert syndrome 6. Erythroblastosis fetalis 7. Dubin-Johnson syndrome DIRECT 1. Neonatal jaundice due to atresia of bile ducts. 2. Obstructive jaundice

PRACTICAL VI LIVER ENZYMES

ALKALINE PHOSPHATASE (ALP) An enzyme occurring particularly in osteoblasts (bone-forming cells) and the liver. Plasma activities occur in various bone and Hepatobiliary disease. Measured as part of bone and liver profiles.

SPECIMEN: Serum

12

Ref. Range: 53 128 U/L (in adults). Levels up to 3x this may be normal in children. SOURCE OF ERROR: None. CAUSES OF ABNORMAL VALUES Increased levels are characteristics of bone disease associated with increased osteoblastic activity and Hepatobiliary disease with partial or complete biliary obstruction. Increased ALP due to bone disease 1. Paget disease of bone 2. Hyperparathyroid bone dx 3. Ricketts 4. Renal osteodystrophy 5. Healing fractures 6. Osteomalacia 7. Bone tumours (1o and 2o) Increased ALP can occur in late pregnancy due to secretions of the enzyme in the placenta. Decreased ALP 1. Hypophosphotasemia 2. Hypothyroidism 3. Scurvy PROCEDURE Working Rgt 20mL of buffer + substrate TEST 1 Sample Working Rgt. 10L 500L TEST 2 10L 500L Increased ALP due to Hepatobiliary disease 1. Cirrhosis 2. Hepatic tumours (1o and 2o) 3. Extrahepatic biliary obstruction( eg. carcinoma of pacreas)

Mix well and read absorbance and start time simultaneously. Read again after 1,2 and 3 min [Conc. In U/L] = 2760 x A405 13

GAMMA-GLUTAMYL TRANSFERASE (GGT) GGT is an enzyme that is mainly in the liver, often measured as part of a panel of LFT. Increased plasma activities of GGT can occur in all types of liver disease but it is a particularly sensitive (though not specific) indicator of excessive consumption of alcohol. Ref. Range (12-64) U/L SPECIMEN: Serum CAUSES OF ABNORMAL RESULTS Increased GGT 1. Hepatitis (alcoholic) 2. Excessive alcohol consumption 3. Cholestatic liver disease of any cause. 4. Treatment with certain anticonvulsant and other drugs. 5. Fatty liver (eg. in obesity, poorly controlled diabetes). PRACTICAL VII ALANINE TRANSAMINASE (ALT, GPT) A soluble cytoplasmic enzyme widely distributed in body tissues but present in particularly high amounts in the liver. Released into the plasma when there is tissue damage (eg. Hepatitis) ALT activity is most often measured as one of LFT as a measure of hepatocellular damage since it is more specific indicator than AST. Parallel measurement of ALT and AST is therefore applied to distinguish liver from heart or applied to distinguish liver from heart or skeletal muscle damages, the AST/ALT is used for differential diagnosis in liver dx. <1 >1 SPECIMEN: Serum Ref. Range : 0 50 U/L SOURCE OF ERROR: None METHODOLOGY mild liver damage severe or chronic dx.

14

Enzymatic kinetic method + Enzymatic end point method. CAUSES OF ABNORMAL RESULTS HIGH LEVELS 1. Hepatitis 2. Cirrhosis 3. Cholestatic Jaundice 4. Congestive cardiac failure (due to hepatic congestion) ASPARTATE TRANSAMINASE (AST/GOT) A soluble cytoplasmic and mitochondrial enzyme widely distributed in body tissues, particularly in the liver, skeletal and cardiac muscles. Tends to released more than ALT in chronic hepatocellular dx. e.g. cirrhosis. LOW LEVELS 1. Renal hemodialysis or renal insufficiency 2. End stage of liver disease

SPECIMEN: Serum SOURCES OF ERROR: Haemolysis METHODOLOGY Enzymatic end point reaction/ enzymatic kinetic reactions CAUSES OF ABNORMAL TESTS: 1. Liver dx a. Hepatitis b. Preceded jaundice c. Less elevation in cholestatic dx. d. Excessive alcohol, obesity, drug toxicity, DM 2. Myocardial infarction 3. Skeletal muscle damage dx.

15

PRACTICAL VIII
CREATININE Creatinine is used to diagnose impaired renal function. Creatinine is a catabolic product of creatinine phosphate, which is used in skeletal muscle contraction. It is excreted entirely by the kidney s and is directly proportional to renal excretory function. Age-Related Concerns: The elderly and young children normally have lower creatinine levels as a result of reduced muscle mass. This may potentially muscle renal dx in patients of these age groups. Creatinine is the most common clinical RFT providing a rough approximation of glomerular filtration. GFR must fall to approximately half normal before serum creatinine becomes reliably increased. SPECIMEN: Serum Ref. Ranges: Adult males: < 106mol/L Adult females: < 97mol/L PRINCIPLE OF THE METHOD Creatinine reacts with picric acid in an alkaline medium. The absorbance of the yellow-red colour produced is measured at 490nm wavelength. A number of other compounds similarly react with picric acid giving artificially high results for creatinine if one simply measures the total yellow-red colour produced. A second reading is therefore made after making the solution acid. The colour produced by creatinine is quickly destroyed by acid whereas that given by non-creatinine chromogens is destroyed more slowly. By subtracting the second reading which is due to noncreatinine substances from the first reading (due to creatinine and non-creatinine substances) the colour produced by the true creatinine can be obtained.

PROTOCOL 16

WORKING REAGENT: EQUAL VOLUMES OF REAGENT 1 & 2 ABSORBANCE:490nm on spectrophotometer. STD WORKING RGT STD/ SAMPLE 1 mL 100L BLK 1 mL TEST 1 mL 100L

Mix well and incubate for 20seconds and read A1 at 490nm against rgt blank and incubate at room temperature for exactly 80 seconds and read A2. CALCULATION: [CREAT] in mol/L Increased levels Dx affecting renal function, such as glomerulonephritis, pyleronephritis, acute tubular necrosis, U.T obstruction, reduced renal blood flow (eg. shock, dehydration, congestive heart failure, and atherosclerosis), diabetic nephropathy, and nephritis. Renal function impaired and creatinine increases. Acromegaly Gigantism: Associated with increased muscle mass. Decreased levels 1. Debilitation 2. Decreased muscle mass (e.g. muscular dystrophy) CREATININE CLEARANCE [CrCl] CrCl is a measure of the GFR. Urine and serum creatinine levels are assessed and the clearance rate is calculated as : CrCl = UV P U = [urine creatinine] over 24 hrs (mol/L) V = volume of urine in mL/min= vol of urine 24 x 60 P = [serum creatinine] in mol/L. PATIENT PREPARATION = 176.8 mol/L ASTD X ASAMPLE

17

24 hr urine collection. Discard the initial specimen and start 24hr timing at that point. Collect all the urine passed during the test. Keep urine sample refrigerated. Indicate the starting time. Patient should drink fluids during the test. Avoid vigorous exercise during the test. Collect last specimen as close as possible to the end of the 24hr collection. UREA: urea is the major end product of nitrogen (protein) metabolism and is synthesized in the liver and excreted by the kidneys. Urea increases in renal failure but can be increased in dehydration and in individuals on a high protein in take in the absence of renal impairment. Often increased as part of renal profile i.e BUE but creatinine provides a better index of renal function for most purposes. SPECIMEN: serum/plasma REF. RANGE: 2.5-6.7mmol/l none SOURCES OF ERROR:

METHODOLOGY: Berthelot Reaction Causes of abnormal results HIGH LEVELS 1. Dehydration 2. Renal failure 3. Gastrointestinal bleeding (due to catabolism of retained blood) 4. Recent protein intake LOW LEVELS 1. Starvation

PRACTICAL IX
18

ELECTROLYTES Sodium (Na+), Potassium (K+) and Chloride (Cl-) SPECIMEN: Serum/Plasma SOURCES OF ERROR: Haemolysis Delay in separation of serum Dont allow patient to clench and unclench Minimal venestasis Dont force blood through needle or syringe

METHODOLOGY: Iron selective Electrode and Flame Photometry Potassium (K+) K+ is the principal intracellular cation but its plasma concentration has a major effect on the excitability of nerve and muscle membranes. REF. RANGE: 3.6-5.0 mmol/l

CAUSES OF HYPOKALAEMIA Gastroinstinal loss (diarrhoea Renal loss Renal failure Accumulation of potassium supplements

Hyperkalaemia

Sodium (Na+) Principal extracellular cation and determinant of extracellular fluid osmolality and volume. Abnormalities of potassium concentration can be related to abnormal water or sodium heamostasis. REF. RANGE: 135-145mmol/

19

CAUSES OF ABNORMAL RESULTS HYPONATREMIA 1. constant diuretic treatment 2. cancer 3. diarrhea and vomiting 4. congestive cardiac failure 5.chronic liver dysfunction 6.chronic renal failure Chloride (Cl-) The principal negatively charged ion in the ECF Provides additional diagnostic information on plasma [ Na+] HYPERNATREMIA 1. Dehydration

Ref. range: 95-105 mmol/l Methods used 1. Flame photometry 2. Ion selective electrode (ISE) URIC ACID Urate is the end product of nucleic acid metabolism Increased urate can predispose of gout, due to the deposition of monosodium urate crystals in joints. Hyperuricaemia does not always cause gout, but gout is usually related to hyperuricaemia Specimen: serum (ideally fasting) Ref. Range :( 208-428) mol/L ABNORMAL RESULTS Increased [urate] Decreased [urate]

20

1. Gout 2. Renal failure 3. Diuretic therapy 4. Lesch-Nyhan syndrome 5. Polycystic kidney 6. Leukaemia,lymphoma,polycythaemia 7. Diet(high protein intake) 8. Drugs(uricostatic drugs e.g. diuretic) 9. Lactate excess e.g. ethanol ingestion 10. Chronic lead nephropathy

1. Administration of uricosuric drugs( high doses of salicylates,cortisone,allopurinol) 2. Renal tubular defects(Fanconis syndrome, Wilson disease)

PRACTICAL X
LIPID PROFILE Acts as energy stores (triglyceride) and structural component. Insoluble in water are transparent in pleasure as perimeter complexes the proteins the lipoprotein It includes total cholesterol,triglycerides,HDL choles, LDL choles andVLDL choles PATIENT CARE PREPARETION: Patients should be on stable diet ideally for 2-3 weeks prior to collection of blood. Patients should fast for at least 12hours before collection of blood. Patients must abstain from alcohol at least 72hours. Posture may be a significant factor: cholesterol value may be 10% to 15% hour after 20 min in a recumbent position. From standing to a sitting position values are about 6% hours after 20mins. Increases of 2-5% in cholesterol may be seen if tourniquet is applied for 2mins during sampling

21

 Cholesterol is the most prominent member of steroid family and is an important component of many cell membranes Procure of 3 major classes of steroids: steroid hormones and bile acid High serum (choles) are an important risk factor for atherosclerosis (esp. affecting the coronary ) Serum Sources of error: Avoid recent food intake Ideal conc. TOTAL <5.2mmol/l LDL <3.5 mmol/l HDL >1.1 mmol/l Methodology: enzymatic and Lieberman-Burchardt rxn (L&B) LDL CALCULATION Friedwald Formula is based on assumption that the total triglycerides divided by 2.2 (or 5) is approximately the value of VLDL cholesterol. The formula is not reliable if [triglyceride] > 4.5mmol/L (>400mg/dL). Calculation of LDL formula LDL= TC- (HDL+0.46 trigly) mmol/l =T C- (HDL+0.20 trigly) mg/dl Acquired [Choles] 1. Hyperthyroidism 2. the cholestatic liver dx 3. renal failure 4. nephrotic syndrome Inherited [Choles] 1. hypercholesterolemia 2. common or polygenic hypercholesterolemia 3. Hyperlipidaemia

22

HDL AND LDL The 2 principal lipoproteins which contain cholesterol are HDL and LDL. The risk of coronary disease is associated with increased levels of LDL, while [HDL]s are inversely correlated with risk,ie are beneficial. LDL is normally present in [high] and thus has the greater effect on total serum cholesterol. LDL is a major atherogenic particle and its [plasma] can therefore function as a key for 1. Identifying those with high risk of CHD 2. Clinical decision making with respect to cholesterol- lowering therapy. METHODOLOGY The methodology for total cholesterol and HDL cholesterol. 1. TOTAL CHOLESTEROL STD SAMPLE/STD REAGENT/BLK 10L 1mL BLK 1mL TEST1 10L 1mL TEST 2 10L 1mL

Mix well and incubate for 10mins at RT and read absorbance at 500nm. Use Beer Lamberts Law to calculate the [test]. HDL CHOLESTEROL The chylomicrons, VLDL and LDL are precipitated by addition of phosphotungstic acid and MgCl2. After centrifugation, the supernatant contains HDL fraction. Test 1 Sample/serum Precipitant 200L 500L Std. Supernatant Cholesterol rgt. 100L 1mL Blk 1mL Test 1 100L 1mL Test 2 200L 500L Test 2 100L 1mL

Mix well and incubate for 10mins at RT and centrifuge at High speed for 5mins.

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Mix well and incubate at RT for 10mins and read absorbance at 500nm against rgt. blk. Use Beers law to calculate. LOW [HDL] can be due to 1. Obesity 2. NIDDM 3. Physical inactivity 4. Oestrogen deficiency

TRIGLYCERIDES Triglyceride is the principal form in which energy is stored in the body. They are the major constituent of adipose tissue Triglycerides circulate in the plasma as component of various lipoproteins, particularly VLDL and chylomicron(CM) VLDLs are made in the liver and are always present in the plasma. CM transport dietary trig from the gut and are not normally present in the plasma in fasting state. SPECIMEN: Serum Reference range: <1.8mml/L Sources of error: Recent food intake METHODOLOGY: Enzymatic method STD SAMPLE/STD REAGENT 10L 1mL BLANK 1mL TEST1 10L 1mL TEST 2 10L 1mL

Mix well and incubate for 10mins at room temperature and read against reagent blank at 500nm. Calculation = 200 x A sample (mg/mL ) = 2.28 x A sample ( mmol/L) A std CAUSES OF ABNORMAL RESULTS A std

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High Triglycerides 1. Inherited a. Familial combined hyperlipidaemia (common) b. Familial hypertriglyceridemia (uncommon) 2. Acquired a. Drugs b. Corticosteroids c. DM d. Obesity e. Excess alcohol ingestion

Low Triglycerides

PRACTICAL XI
FERTILITY HORMONES It consists of LH, FSH, PRL, PRG, TESTO, ESTROGEN. LH and FSH are anterior-pituitary hormones which regulates male and female gonadal function. Measurement of FSH and LH in serum or plasma can be of value in suspected disorders of gonadal function. SPECIMEN: Serum: For females, the LMP should be taken. Ref. range: for females depends on the menstrual cycle thus luteal, follicular midcycle, premenopausal and post menopausal. SOURCE OF ERROR: 1. In pregnancy 2. In tumor secreting hCG CAUSES OF ABNORMAL VALUES High FSH low or normal LH = primary gonadal failure High FSH = azoospermia, ovulatory failure. LH > FSH = PCOS( polycystic ovary syndrome). PROLACTIN

25

 A hormone secreted by the anterior pituitary which stimulates lactation and inhibits the action of gonadotropins. SPECIMEN: Serum Ref. range: Female: (1.35 24.59)ng/ml Male: (1.5 18.75)ng/ml SOURCES OF ERROR 1. Stress by venipuncture 2. Fasting sample 3. Draw sample between 8.00am to 10.00am. INCREASED LEVELS 1. Hyperthyroidism 2. PCOS 3. Chronic renal failure (CRF) 4. Adenomas (micro and macro adenomas) 5. Drug treatment eg. methyldopa, oestrogen PROGESTRONE PRG is made by the corpus luteum. Its major source in pregnancy is the placenta. SPECIMEN: Serum Ref. Range: Depends upon the menstrual cycle. OESTRADIOL E2 is a principal female gonadal steroid and its measurements with other gonadal hormones. It is useful in menstrual disorders. Ref. Range: Depends on the stage of menstrual cycle. TESTOSTERONE

26

 It is the major androgen responsible for sexual differentiation and male secondary sexual characteristics. It is carried in the blood by the sex hormone binding globulin (SHBG). SPECIMEN: Serum Ref. Range:

PRACTICAL XII TUMOUR MARKERS

Tumour markers are substances secreted into the body fluids or expressed on cell surface which are characteristic of the presence of a tumour. They are of potential value in screening for cancer, for diagnosis, in prognosis, in assessing the response to treatment and in long term follow up. The most frequently used TM are substances secreted by tumours, and measured in either in the blood or urine. PSA It is a glycoprotein secreted by normal prostate and [Serum] increases with increasing age and are increased in BPH (Benign Prostatic Hypertrophy) SPECIMEN: Serum Ref. Range: 0 4.0ng/mL (male). Female: < 0.5 SOURCES OF ERROR 1. Constipation 2. Rectal examination 48hrs before test. ASSAY -HCG CA 125 Cancer antigen 125 Significance Molar pregnancy, Choriocarcinoma, gestational trophoblastic Ovarian cancer (0 - 35) U/ml (0 - 5) mlU/ml REFERENCE

27

CA 15-3 Carbohydrate antigen CA 19-9 Carbohydrate antigen CEA Carcinoembryonic antigen

Breast carcinoma Hepatocellular carcinoma Colorectal rectal Hepatoma, teratoma, neoplasm,

(0 - 37)

U/ml

(25 30) U/ml

(0 2.5) ng/ml

AFP

neural tube defect(spina bifide)

(2 16) ng/ml

PSA

Prostate cancer

(0 4) ng/ml

THYROID FUNCTION TEST (T3, T4, TSH) Thyroid hormones (thyroxine {T4} and tri-iodothyronine {T3 } are extensively protein bound in the plasma and measurements of total hormone concentrations can give misleading information if the concentration of binding proteins are abnormal. Measurements of the free ( and physiologically active) hormones (fT4, fT3, but particularly the former) are now replacing total hormone measurements. Measurement of thyroid stimulating hormone (TSH) is valuable since in primary thyroid disease ( which is far commoner than thyroid disease secondary to a pituitary cause), its concentration inversely reflects thyroid activity. Reference ranges TSH Thyroxine (total) Tri-iodothyronine (total) Thyroxine (free) Tri-iodothyronine (free) 0.3-5.0 mIU/L 60-150 nmol/L 1.2-2.9 nmol/L 9.0-26 pmol/L 3.0-8.8 pmol/L

28

T3

T4

TSH Hyperthyroidism T3 Thyrotoxicosis Thyrotoxicosis Hypothyroidism

PRACTICAL XIII
RADIONIMMUNOASSAY (RIA) AND ELISA RIA and ELISA are widely used because these tests are extremely sensitive. RIA and ELISA are methods used for the determination of the concentration of the

hormones, glycoproteins, peptide hormones and other hormones. PRINCIPLE For both the RIA and ELISA tests, the human antigen is put into a plastic test tube or on a plastic microtitre plate. Some of the antigen will adsorb to the surface of the tube or plate and the excess is then washed away. The serum to be tested is added to the tube or plate and incubated for a short time. If antibodies are present, they will bind the Ag. For the RIA, the excess Ab is washed away and a radioactively labeled Ag that can react with the Ab is added. The excess radioactive Ag is washed away and the tubes are then counted using a gamma counter. The ELISA is done in a similar manner except instead of using a radioactive Ag, the Ag is coupled to an enzyme such as peroxidase. The Ag binds to the test Ab, excess ligand is washed away, and chromogen is added. Chromogen is a colorless substrate that produces a colour end point when acted upon by an enzyme such as peroxidase. The colour change can then be observed and measured using a microplate reader.

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