Clinical laboratory Tests :

1. Clinical laboratory Tests : Tests which determine the presence (/ absence) or concentrations of various parameters in body fluids i.e., blood, urine and or other body fluids. Frequently, the concentrations of some parameter(s) is(are) altered in the body fluids in disease condition. Urea levels in blood increases in kidney disease.

2. Qualitative tests Tests which determine the presence / absence of a substance - positive or negative . Quantitative tests Tests which determine the concentration of a substance. Clinical laboratory Tests : Functions : • Diagnosis and prognosis of disease. • Evaluation of functions of organs.

3. Organ function Tests A group of tests that reflect distinct organ dysfunction / disease . • Liver function Tests (LFT) : Blood : bilirubin, Plasma proteins, Prothrombin time and some enzymes • Renal function Tests (RFT): Blood Urea, Creatinine, electrolytes Proteinuria

4. Thyroid function Tests (TFT) : T3, T4 and TSH • Reproductive function Tests : FSH, LH, Prolactin and sex hormones.

5. Liver function Tests (LFT) : Definition : are a set of biochemical investigations done to diagnose and monitor diseases of liver and the biliary tract. Selection of the tests : Based on the nature of the clinical problem. They should reflect different aspects of liver function

6. Indications of liver function tests • Differential diagnosis of Jaundice Monitoring/Follow up of Chronic Liver Failure Screening of suspected cases during outbreak of infective hepatitis, Suspected liver metastasis, • Alcoholic liver disease, Coagulation disorders and follow up of therapy with hepatotoxic drugs.

7. Classification of LFT • Based on laboratory findings : Group I Tests of hepatic excretory function i) Serum Bilirubin- total; conjugated; unconjugated. ii) Bromosulphthalein excretion test. iii) With urine – Tests for Bile pigments, bile salts and urobilinogen. Group II Markers of liver injury / cholestasis Group III Enzyme panel – ALT; AST; ALP; GGT Group IV

8. Group III Tests for synthetic function Serum albumin Prothrombin time (PTT). Group IV : Special tests such as -fetoprotein (AFP)- tumor markers.

9. 1. Test based on detoxification and excretion functions of the liver : Serum Bilirubin: Estimation : Colorimetric method Reaction based on : van den Bergh reaction Reaction : Conjugated bilirubin + diazotized sulphanilic acid → Azobilirubin (purple colored complex). “ DIRECT BILIRUBIN”

10. Conjugated bilirubin is water soluble, reacts directly without adding any solubilizing agents like methanol. “ DIRECT BILIRUBIN” Reaction : Bilirubin + diazotized sulphanilic acid + Methanol → Azobilirubin (purple colored complex). “ TOTAL BILIRUBIN” Total Bilirubin- Direct Bilirubin= Indirect bilirubin (UNCONJUGATED BILIRUBIN)

11. Biphasic reaction Methanol Indirect Bilirubin Direct Bilirubin Total Bilirubin Normal values : • Serum Total bilirubin – up to 1 mg/dl (0.2 – 0.8 mg / dl) • Serum unconjugatedbilirubin – up to 0.7 mg/dl (0.2 – 0.7 mg / dl) • Serum conjugated bilirubin – up to 0.3 mg/dl (0.1 – 0.4 mg / dl).

12. Interpretation of serum bilirubin values: Pre-hepatic / Hemolytic Jaundice ↑ in unconjugatedbilirubin Hepatic Jaundice Biphasic Reaction (since both forms of bilirubin are ↑) Post hepatic / Cholestatic / Obstructive jaundice : ↑ in conjugatedbilirubin

13. Estimation of serum bilirubin assesses the detoxificationand excretion functionsof the liver Tests done with Urine Bilirubin Fouchets Test +ve in Hepatic and Post hepatic / obstructive Jaundice Bile salts Hays Test - ↑ in Pre hepatic. Variable in hepatic diseases. • Urobilinogen • Ehrlich Test • ↓ /absent in obstructive jaundice.

14. 2.Tests Based on Metabolic Capacity of Liver: Galactose tolerance test: Principle :Galactose is metabolized to glucose, exclusively in the liver. In liver failure, this process is impaired, leading to high plasma levels of galactose after an overload. Indication: This test is indicated in patients with suspected liver failure, even in absence of jaundice.

15. Procedure : A load of galactose is administered intravenously, and its level in blood is measured at frequent intervals. Interpretation : In cirrhosis of the liver, the galactose is retained in the blood for a longer time.

16. 3. Tests Based on Synthetic Function of Liver: All plasma proteins, except -globulins, are synthesized in liver • Important tests under this category include: • Measurement of Serum Albumin • Prothrombin Time. Estimation of serum albumin: Normal serum protein levels : Albumin : 3.6 – 5.4 g /dl Globulin : 1.8 - 3.6 g / dl

17. Interpretation liver diseases : liver cirrhosis chronic active hepatitis ↓serum albumin ↑ Immunoglobulins Note: ↓ Albumin - is often the rule of cirrhosis Reason : due to ↓↓↓ synthesis of albumin and the associated Hypergammaglobulinemia - because of the infective element.

18. In acute liver disease neither albumin nor globulin is altered because of its half life. Serum albumin - the only parameter that can assess the progress of the liver disease because of the associated oedema. Serum electrophoresis of plasma proteins :

19. Prothrombin time (PTT): Time taken for the plasma to clot on addition of thromboplastin and CaCl2 0.1 ml plasma - in glass tube and the tube is placed in a water bath 37 C Add 0.1 ml thromboplastin, mix, place in water bath 37 C Add 0.1 ml CaCl2. Mix. Start the stop watch End point : when clots are formed.

20. Samples : Patients and Healthy subjects (controls). Samples : citrated blood – Ca++ is chelated by citrate Interpretation : PTT is prolonged in chronic liver disease. NOTE : good indicator of chronic liver diseases (cirrhosis of liver) and not of acute conditions. Reason : fairly long half-life (slow turnover) PTT is prolonged only when 80% of the reserve capacity of the liver is lost.

21. In obstructive Jaundice, the PTT may be prolonged. On administration of vitamin K, PTT comes back to normal. 4. Tests Based on Detoxification by Liver: Hippuric acid synthesis test: Principle : benzoic acid + Glycine → benzoylglycine (Hippuric acid) which is excreted in urine Procedure : Benzoic acid is orally administered. Hippuric acid levels in urine is monitored. Interpretation: In chronic liver diseases, excretion of hippuric acid is substantially ↓↓↓.

22. 5. Measurements of certain Serum Enzymes. to assess hepatobiliary disease • Those that measure hepatocellular damage Serum alaninetransaminase (ALT) 1 – 45 IU/L Serum aspartatetransaminase (AST) 8 – 40 IU/L Conditions : in hepatocellular necrosis / hepatic jaundice (e.g. acute hepatitis). Reason : hepatic cells are rich in these enzymes.

23. Those that measure biliary obstruction / cholestasis : Serum alkaline phosphatase (ALP) 30 – 85 IU/L (3 – 13 KA units) Gamma glutamyltransferase (GGT) 10 – 30 IU /L ALP - ↑↑in biliary obstruction. Reason : Cells lining biliarycanaliculi and bile are rich in this enzyme. GGT : important clinically because of its sensitivity to detect alcohol abuse.

24. The degree of elevation of these enzymes may reflect the extent of hepatocellular necrosis / cholestasisand the gradual lowering of the levels indicate recovery. A sudden fall from a very high level may indicate poor prognosis.

25. Applications of LFT : 1. Differential diagnosis of jaundice.

26. 2. For diagnosis and monitoring the progress in liver diseases (cirrhosis of liver): Tests especially useful in monitoring chronic liver disease are – serum bilirubin, serum albumin and prothrombin time. 3. For screening suspected cases during out break of infective (viral) hepatitis: Serum transaminases↑↑↑ in infected patients even before jaundice manifests. 4. For screening persons exposed to hepatotoxic drugs: • Serum bilirubin and transaminases are useful.

27. Renal Function Tests (RFT)

28. Functions of the kidney : elimination of waste materials and foreign bodies. • Excretory function • Maintain water and electrolyte balance . : Production of erythropoietin, prostaglandins and active form of Vitamin D3. • Endocrine function

29. Renal Function Tests (RFT) RFT is a group of tests used to assess kidney function. • Glomerular filtration rate (GFR) : amount of ultrafiltrate formed per minute when plasma passesthrough the kidney. It is a useful general index for the assessment of the severity of kidney damage. ↓↓ in renal function is due to the loss of number of functional nephrons.

30. Renal failure (↓↓ of GFR) may be seen in a) Pre-renal causes : • when renal blood flow decreases as in hemorrhage, dehydration, cardiac failure, etc. b) Renal causes : • Diseases of the kidneys - acute and chronic glomerulonephritis, polycystic kidney, etc. c) Post-renal causes : • Obstruction to urine flow in the urinary tract as in renal stones, prostate enlargement, etc.

31. Indications for renal function tests • Diagnosis of renal diseases and assessment of prognosis of renal disease • Monitoring renal function during treatment using nephrotoxic drugs.

32. Classification of RFT • Classified under the following categories: Tests to assess 1. Glomerular function : Clearance tests General tests With blood Creatinine, Urea and electrolytes. With Urine Complete urine analysis, proteinuria. 2. Tubular function • Concentration and dilution studies. • Other tests include – reabsorption, secretion and renal acidification.

33. Assessment of glomerular function (GFR) : 1. General Tests : (a) with Blood Plasma creatinine and blood urea levels levels : Normal plasma creatinine levels : 0.7 – 1.5 mg /dl (the values are lower in Females ) Normal blood urea levels • : 15 - 40 mg / dl Interpretation ↓↓↓ GFR is usually associated with ↑↑↑ plasma creatinine and blood urea levels.

34. Creatinine is a better index The limitation with blood urea level is - it is affected by changes in dietary proteins. General Tests that assess GFR: (b) with Urine b1. Urine analysis : examination of the urine • physical appearance, volume, odor, color, specific gravity. Physical analysis : Chemical analysis : • Protein, blood. • Microscopic examination :

35. Presence of Damage to the glomerular membrane, hence these substances leak into the filtrate. • red cells and protein indicates bleeding in the urinary tract. • blood an indication of glomerular disease. • Renal casts • presence of urinary tract infection. • Leukocytes and bacteria

36. Increased excretion of proteins in urine is called as proteinuria. B2) Proteinuria : Cause : Damage of the glomerular membrane, resulting in increased permeability . Because of its small size, albumin is the protein largely lost in urine - Albuminuria. Conditions : Chronic nephritis; Acutenephritis; Nephrotic syndrome and Chronic renal failure.

37. Clearance tests Definition : Volume of plasma that contains the amount of substance excreted by the kidneys in one minute. Formula : Clearance = mg. of substance excreted per min / mg. of substance per ml plasma (U  V) / P ml / minute

38. U = concentration of the substance in urine ; • V = the volume (in ml) of urine excreted per minute. • P = concentration of the substance in plasma • or serum. Normal GFR • Young adults 125 – 130 ml / min. Measurement of GFR by using a filtration marker.