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Clinical Chemistry

Clinical Chemistry. Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653 Travlos@niehs.nih.gov. Abbreviations. AChE = acetylcholinesterase ALB = albumin ALP = alkaline phosphatase ALT = alanine aminotransferase

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Clinical Chemistry

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  1. Clinical Chemistry Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653 Travlos@niehs.nih.gov

  2. Abbreviations AChE = acetylcholinesterase ALB = albumin ALP = alkaline phosphatase ALT = alanine aminotransferase AST = aspartate aminotransferase BA or TBA = total bile acids Bili or Bil (T or D) = total or direct bilirubin BUN or UN = urea nitrogen BuChE = butyrylcholinesterase Chol = cholesterol CK or CPK = creatine kinase Cre or Creat = creatinine Glu = glucose Glob = globulin LDH = Lactate dehydrogenase NAG = n-acetyl-glucoaminidase SDH = sorbitol dehydrogenase Tn or cTn = troponin (I or T); c = cardiac T pro = total protein Trig = triglycerides

  3. Clinical Chemistry The analysis of individual constituents, proteins, enzymes, nutrients, waste products, metabolites, hormones, etc. in blood or body fluids that provides information regarding the function or integrity of a tissue, organ or organ system While almost anything may be analyzed, the efficacy of a test depends on its specificity and sensitivity to detect pathological change

  4. Analytical Procedures/Methods Too numerous to cover • Photometry • Fluorometry • Luminometry • Spectrometry • Nephelometry • Electrophoresis • Chromatography • Isotopic (RIA/IRMA) and non-isotopic (ELISA/EIA) immunoassay

  5. Considerations for Blood Collection Serum: whole blood collected in a container without anticoagulant • Plasma is acceptable for some assays (heparin) • Samples from indwelling catheters are usually acceptable For serum: allow blood to clot for 30 to 60 minutes Separate serum for red cells into a clean plastic container • Avoid artifact due to prolonged storage on red cells (e.g., LDH)

  6. Sources of Variation Diet • NIH-07 vs. NTP 2000

  7. = NTP 2000 = NIH-07

  8. Diet: NIH-07 v. NTP-2000 Analyte NIH-07 NTP-2000 ALT (IU/L) Males 56.5 90.0 Females 47.5 77.0 BUN (mg/dL) Males 20.0 15.0 Females 20.5 14.8 Switching diets resulted in an approximately 60% increase in control animal serum ALT activity and a 26% decrease in serum BUN concentration.

  9. Sources of Variation Diet • NIH-07 v NTP 2000 Fasting • Glucose • Alkaline phosphatase

  10. Sources of Variation Diet • NIH-07 v NTP 2000 Fasting • Glucose • Alkaline phosphatase Diurnal variation • Hormones

  11. Sources of Variation Diet • NIH-07 v NTP 2000 Fasting • Glucose • Alkaline phosphatase Diurnal variation • Hormones Analytical Methods & Sample Collection/Handling Techniques • Animal handling - rough handling: increased creatine kinase release • In vitro Hemolysis - assay interference: bilirubin • Method selection - troponin

  12. Troponin

  13. Comparison of cTn Measurement in the Sprague Dawley Rat Abbott Architect Tosoh AIA 600 II Bayer Advia Centaur Beckman Access Dade Dimension RxL OCD Vitros ECi DPC Immulite Rat Troponin EIA Roche Elecsys 2010 30 6 25 5 20 4 cTnI(ng/mL) cTnT (ng/mL) 15 3 10 2 5 1 0 0 Neg Low Med High

  14. Abbott Architect Tosoh AIA 600 II Bayer Advia Centaur Beckman Access Dade Dimension RxL OCD Vitros ECi DPC Immulite Dog Troponin EIA Roche Elecsys 2010 Comparison of cTn Measurement in the Beagle 20 0.25 0.20 15 0.15 cTnI (ng/mL) cTnT (ng/mL) 10 0.10 5 0.05 0 0.00 Neg Low Med High

  15. Sources of Variation Diet • NIH-07 v NTP 2000 Fasting • Glucose • Alkaline phosphatase Diurnal variation • Hormones Analytical Methods & Sample Collection/Handling Techniques • Animal handling - rough handling: increased creatine kinase release • In vitro Hemolysis - assay interference: bilirubin • Method selection - troponin • Multiple factors - cholinesterase

  16. Cholinesterase Assay Two types of cholinesterase acetylcholinesterase (AChE; RBCs) butyrylcholinesterase (BuChE; liver) For rat plasma: AChE:BuChE ratio approximately 1.2:1 Spectrophotometric assay: but different assays use different substrates dinitrobenzoic acid acetylcholine propionylthiocholine butyrylthiocholine And the different cholinesterases have varying affinities for the different substrates There are species differences For RBC and serum: human > dog > rat; platelets: rat > human There can be sex differences For rat: female > male

  17. Serum AChE (IU/L) Propargyl Alcohol Control 64 ppm Males 1071 778

  18. Serum AChE (IU/L) Propargyl Alcohol Control 64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition

  19. Serum ChE (IU/L) Propargyl Alcohol Control 64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition PTC assayBTC assay Untreated 0.1 mM 1.0 mM 10.0 mM Assays: normal male rat serum; 2.5 hour incubation; performed in duplicate

  20. Serum ChE (IU/L) Propargyl Alcohol Control 64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition PTC assayBTC assay Untreated 876 272 0.1 mM 795 289 1.0 mM 825 299 10.0 mM 836 262 Assays: normal male rat serum; 2.5 hour incubation; performed in duplicate

  21. NTP Core Clinical Chemistry Profile Liver • Alanine Aminotransferase • Sorbitol Dehydrogenase • Alkaline Phosphatase • Total Bile Acids Kidney • Urea Nitrogen • Creatinine Protein • Total protein • Albumin Muscle • Creatine Kinase

  22. Evaluation of Liver Alanine Aminotransferase (ALT, SGPT) • Greatest activity - hepatocytes; also found in skeletal/cardiac muscle • Biological half-life - varies (~48-60 hours) • Sample stability - stabile at room, refrigerated and frozen temperatures • Can be induced (eg., glucocorticoids) • Increased - hepatocellular injury, induction, muscle injury • Decreased - enzyme inhibition (cyclosporin) Sorbitol Dehydrogenase (SDH) • Greatest activity - hepatocytes; also found in testes • Biological half-life - short (≤6 hours) • Sample stability - not as stabile; in rats, stabile refrigerated (~2 days) • Not known to be induced • Only known cause for serum increase - hepatocellular injury or leakage

  23. Evaluation of Liver - cont. Aspartate Aminotransferase (AST, SGOT) • Greatest activity - found in numerous tissues (not specific for liver injury) • Biological half-life - short (~15-24 hours) • Sample stability - stabile at room, refrigerated and frozen temperatures • Red blood cells contain significant amounts (hemolysis - falsely elevates) • Used in past to detect hepatocellular injury (still used for large animals); used for muscle injury Alkaline Phosphatase (ALP) • Greatest activity - liver, bone intestine, kidney, placenta • Biological half-life - isoenzymes of different tissues highly variable • Sample stability - stabile in serum; not in urine • Can be induced (eg., glucocorticoids, phenobarbital, dieldrin) • Increased - cholestasis, drug induction, increased osteoblastic activity, cancer • Decreased - decreased food intake (rats)

  24. Evaluation of Liver - cont. Bilirubin, direct (conjugated) and total (Dbili & Tbili) • Breakdown product of hemoglobin • Liver removes unconjugated bilirubin (insoluble) from plasma, conjugates it (glucuronide - renders bilirubin water soluble) and secreted into bile • Sample stability - stabile serum and urine • Increased - Retention-type (hemolysis, decreased hepatic uptake); Regurgitation-type (cholestasis) Bile Acids (TBA) • Produced by liver - cholic and chenodeoxycholic (primary bile acids) • Taurine or glycine conjugated and secreted into bile • Intestinal bacterial modification produces deoxycholic and lithocholic acids • Increased - cholestasis, decreased hepatic uptake/conjugation, hepatic injury • Decreased - altered enterohepatic recirculation

  25. Liver Case Examples Ref Value 1 ALT 30-55 IU/L 34 SDH 10-20 IU/L 16 ALP 250-350 IU/L 157 TBA 25-35 µmol/L 31 Tbili 0.1-0.5 mg/dL 0.2 Dbili 0.05-0.2 mg/dL 0.1

  26. Liver Case Examples Ref Value 1 ALT 30-55 IU/L 34 SDH 10-20 IU/L 16 ALP 250-350 IU/L 157 TBA 25-35 µmol/L 31 Tbili 0.1-0.5 mg/dL 0.2 Dbili 0.05-0.2 mg/dL 0.1 • Case 1: Decreased ALP

  27. Liver Case Examples Ref Value 1 ALT 30-55 IU/L 34 SDH 10-20 IU/L 16 ALP 250-350 IU/L 157 TBA 25-35 µmol/L 31 Tbili 0.1-0.5 mg/dL 0.2 Dbili 0.05-0.2 mg/dL 0.1 • Case 1: Decreased ALP - decreased food intake?

  28. Liver Case Examples Ref Value 1 2 ALT 30-55 IU/L 34 130 SDH 10-20 IU/L 16 13 ALP 250-350 IU/L 157 321 TBA 25-35 µmol/L 31 27 Tbili 0.1-0.5 mg/dL 0.2 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1

  29. Liver Case Examples Ref Value 1 2 ALT 30-55 IU/L 34 130 SDH 10-20 IU/L 16 13 ALP 250-350 IU/L 157 321 TBA 25-35 µmol/L 31 27 Tbili 0.1-0.5 mg/dL 0.2 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1 • Case 2: Increased ALT

  30. Liver Case Examples Ref Value 1 2 ALT 30-55 IU/L 34 130 SDH 10-20 IU/L 16 13 ALP 250-350 IU/L 157 321 TBA 25-35 µmol/L 31 27 Tbili 0.1-0.5 mg/dL 0.2 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1 • Case 2: Increased ALT - suspect enzyme induction

  31. Liver Case Examples Ref Value 1 2 3 ALT 30-55 IU/L 34 130 450 SDH 10-20 IU/L 16 13 63 ALP 250-350 IU/L 157 321 279 TBA 25-35 µmol/L 31 27 43 Tbili 0.1-0.5 mg/dL 0.2 0.3 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1 0.1

  32. Liver Case Examples Ref Value 1 2 3 ALT 30-55 IU/L 34 130 450 SDH 10-20 IU/L 16 13 63 ALP 250-350 IU/L 157 321 279 TBA 25-35 µmol/L 31 27 43 Tbili 0.1-0.5 mg/dL 0.2 0.3 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1 0.1 • Case 3: Increased ALT, SDH, TBA

  33. Liver Case Examples Ref Value 1 2 3 ALT 30-55 IU/L 34 130 450 SDH 10-20 IU/L 16 13 63 ALP 250-350 IU/L 157 321 279 TBA 25-35 µmol/L 31 27 43 Tbili 0.1-0.5 mg/dL 0.2 0.3 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1 0.1 • Case 3: Increased ALT, SDH, TBA - suspect hepatocellular injury

  34. Liver Case Examples Ref Value 4 ALT 30-55 IU/L 44 SDH 10-20 IU/L 18 ALP 250-350 IU/L 257 TBA 25-35 µmol/L 31 Tbili 0.1-0.5 mg/dL 9.3 Dbili 0.05-0.2 mg/dL 0.3

  35. Liver Case Examples Ref Value 4 ALT 30-55 IU/L 44 SDH 10-20 IU/L 18 ALP 250-350 IU/L 257 TBA 25-35 µmol/L 31 Tbili 0.1-0.5 mg/dL 9.3 Dbili 0.05-0.2 mg/dL 0.3 • Case 4: Increased Tbili

  36. Liver Case Examples Ref Value 4 ALT 30-55 IU/L 44 SDH 10-20 IU/L 18 ALP 250-350 IU/L 257 TBA 25-35 µmol/L 31 Tbili 0.1-0.5 mg/dL 9.3 Dbili 0.05-0.2 mg/dL 0.3 • Case 4: Increased Tbili - suspect hemolytic disease

  37. Liver Case Examples Ref Value 4 5 ALT 30-55 IU/L 44 51 SDH 10-20 IU/L 18 20 ALP 250-350 IU/L 257 301 TBA 25-35 µmol/L 31 13 Tbili 0.1-0.5 mg/dL 9.3 0.3 Dbili 0.05-0.2 mg/dL 0.3 0.1

  38. Liver Case Examples Ref Value 4 5 ALT 30-55 IU/L 44 51 SDH 10-20 IU/L 18 20 ALP 250-350 IU/L 257 301 TBA 25-35 µmol/L 31 13 Tbili 0.1-0.5 mg/dL 9.3 0.3 Dbili 0.05-0.2 mg/dL 0.3 0.1 • Case 5: Decreased TBA

  39. Liver Case Examples Ref Value 4 5 ALT 30-55 IU/L 44 51 SDH 10-20 IU/L 18 20 ALP 250-350 IU/L 257 301 TBA 25-35 µmol/L 31 13 Tbili 0.1-0.5 mg/dL 9.3 0.3 Dbili 0.05-0.2 mg/dL 0.3 0.1 • Case 5: Decreased TBA - suspect impaired enterohepatic recirculation

  40. Liver Case Examples Ref Value 4 5 6 ALT 30-55 IU/L 44 51 87 SDH 10-20 IU/L 18 20 28 ALP 250-350 IU/L 257 301 987 TBA 25-35 µmol/L 31 13 104 Tbili 0.1-0.5 mg/dL 9.3 0.3 4.7 Dbili 0.05-0.2 mg/dL 0.3 0.1 3.1

  41. Liver Case Examples Ref Value 4 5 6 ALT 30-55 IU/L 44 51 87 SDH 10-20 IU/L 18 20 28 ALP 250-350 IU/L 257 301 987 TBA 25-35 µmol/L 31 13 104 Tbili 0.1-0.5 mg/dL 9.3 0.3 4.7 Dbili 0.05-0.2 mg/dL 0.3 0.1 3.1 • Case 6: Increased ALT, SDH, ALP, TBA, T & Dbili

  42. Liver Case Examples Ref Value 4 5 6 ALT 30-55 IU/L 44 51 87 SDH 10-20 IU/L 18 20 28 ALP 250-350 IU/L 257 301 987 TBA 25-35 µmol/L 31 13 104 Tbili 0.1-0.5 mg/dL 9.3 0.3 4.7 Dbili 0.05-0.2 mg/dL 0.3 0.1 3.1 • Case 6: Increased ALT, SDH, ALP, TBA, T & Dbili - suspect biliary obstruction

  43. Evaluation of Kidney Need ~75% of nephrons non-functional for alterations in serum markers to occur Urea Nitrogen (UN, BUN) • Method of ammonia excretion • Liver converts ammonia to urea; kidney excretes urea • Sample stability - stabile serum and urine • Increased - renal and non-renal (e.g., dehydration) causes • Decreased - hepatic insufficiency Creatinine (Cre, Creat) • Waste product of muscle metabolism • Excreted by kidney • Sample stability - stabile serum and urine • Increased - renal injury • Decreased - decreased muscle mass

  44. Evaluation of Kidney - cont. Urine indicators • Urine contains most constituents found in plasma (except molecules >70,000 daltons) • But concentration varies due to water conserving ability of kidney • When interpreting data must account for kidney’s concentrating ability (per time or per mg creatinine basis) • Sample stability - concentrated salt solution (some enzymes are not stabile in urine) • Urine specific gravity - estimates concentrating ability; alterations when 66% of nephrons affected • Chemical constituents - creatinine, glucose, protein, ALP, LDH, AST, NAG, glucuronidase, electrolytes When evaluating quantitative urine chemistry data, always normalize (e.g., creatinine, urine volume)

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