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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. Clinical Chemistry.

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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

clinical chemistry2
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

analytical procedures methods
Analytical Procedures/Methods

Too numerous to cover

  • Photometry
  • Fluorometry
  • Nephelometry
  • Electrophoresis
  • Isotopic immunoassay
  • Chromatography
  • Spectrometry
considerations for blood collection
Considerations for Blood Collection

Whole blood collected in a container without anticoagulant

  • Samples from indwelling catheters are usually acceptable

Allow blood to clot for 30 to 60 minutes

Separate serum for red cells into a clean plastic container

  • Glucose
  • Enzyme leakage
sources of variation
Sources of Variation

Diet

  • NIH-07 v NTP 2000

Fasting

  • Glucose

Diurnal variation

  • Hormones

Analytical Methods & Sample Collection/Handling Techniques

  • Cholinesterase
  • Creatine Kinase
  • In vitro Hemolysis
  • Urine Collection/Handling
diet nih 07 v ntp 2000
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.

assay variation ache iu l
Assay Variation: AChE (IU/L)

Propargyl Alcohol Control 64 ppm

Males 1071 778

assay variation ache iu l11
Assay Variation: AChE (IU/L)

Propargyl Alcohol Control 64 ppm

Males 1071 778

Suggested an approximate 30% enzyme inhibition

assay variation ache iu l12
Assay Variation: AChE (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: male rat serum; 2.5 hour incubation; performed in duplicate

assay variation ache iu l13
Assay Variation: AChE (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: male rat serum; 2.5 hour incubation; performed in duplicate

slide15

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

slide16

Comparison of cTn Measurement in the Cynomolgus Monkey

Abbott Architect

Tosoh AIA 600 II

Bayer Advia Centaur

Beckman Access

Dade Dimension RxL

OCD Vitros ECi

DPC Immulite

Monkey Troponin EIA

Roche Elecsys 2010

25

1.00

20

0.75

15

cTnI (ng/mL)

cTnT (ng/mL)

0.50

10

0.25

5

0

0.00

Neg

Low

Med

High

slide17

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

ntp core clinical chemistry profile
NTP Core Clinical Chemistry Profile

Protein

  • Total protein
  • Albumin

Muscle

  • Creatine Kinase

Kidney

  • Urea Nitrogen
  • Creatinine

Liver

  • Alanine Aminotransferase
  • Sorbitol Dehydrogenase
  • Alkaline Phosphatase
  • Total Bile Acids
evaluation of liver
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
evaluation of liver cont
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)
evaluation of liver cont21
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
liver case examples
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

liver case examples25
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

evaluation of kidney
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 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
evaluation of kidney cont
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
proteinuria
Proteinuria

Detection of protein in urine (plasma, genitourinary)

In general:

>20 mg/kg/day

Persistent

types
Types

Functional - reversible

  • Stress
  • Exercise
  • Fever/exposure to temp extremes
  • Seizures
  • Congestion of kidneys

Glomerular overload - Hyperproteinemia

Glomerular - may result in hypoalbuminemia

Tubular overload - Hgb, Mgb, Bence-Jones

Tubular - defective resorption

methods
Methods

Tougher to do in urine v. serum

  • Small quantities
  • Sample-to-sample variation
  • Origin of protein
  • Protein degradation products

Sample: Fresh or refrigerated

  • Screening (dipstick) - uncentrifuged
  • Quantitative or semiquantitative - centrifuged
methods cont
Methods - cont.

Dipstick

  • Screening - based on pH dyes
  • Albumin gives stronger results

Spectrophotometric

  • Quantitative - timed collection
  • Toluene
  • Ur prot/Ur creatinine ratios

SSATT - semiquantitative

Bence Jones - heat precipitation

reference values
Reference Values

Dog

  • <20mg/kg/day
  • 0.67 - 0.96 mg prot/mg creat

F344 rats (adult male)

  • ~141 mg/dL (67 - 213 mg/dL)
  • ~5.5 mg/16 hr
  • ~0.87 mg prot/mg creat (0.68 - 1.01 mg prot/mg creat)

F344 rats (adult female)

  • 10 mg/dL (7 - 16 mg/dL)
  • ~0.7 mg/16 hr
  • ~0.11 mg prot/mg creat (0.09 - 0.13 mg prot/mg creat)
pgmbe urinalysis raw data
PGMBE Urinalysis: raw data

Analyte Control 1200 ppm

SG 1.017 1.013

Volume (mL) 12.2 26.8

Creat (mg/dL) 68.4 34.0

Gluc (mg/dL) 8.0 5.0

Prot (mg/dL) 65.0 54.0

AST (IU/L) 6 26

LDH (IU/L) 27 54

NAG (IU/L) 10 9

pgmbe urinalysis converted data
PGMBE Urinalysis: converted data

Analyte Control 1200 ppm

Gluc (ug/mg creat) 117 147

Prot (ug/mg creat) 950 1588

AST (mU/mg creat) 9 76

LDH (mU/mg creat) 39 159

NAG (mU/mg creat) 15 26

other markers
Other Markers

Proteins

  • Total
  • Albumin
  • Globulin

Carbohydrate Metabolism

  • Glucose

Lipid Metabolism

  • Cholesterol
  • Triglycerides

Muscle

  • Creatine Kinase or Phosphokinase (CK, CPK) - total and isoenzymes
  • Troponin T and I
other markers37
Other Markers

Electrolytes

  • Sodium
  • Potassium
  • Chloride
  • Bicarbonate
  • Calcium
  • Phosphorus

Hormones

  • Insulin
  • Thyroxine (T4)
  • Triiodothyronine (T3)
  • Thyroid Stimulating Hormone (TSH)
  • Estradiol (E2)
  • Progesterone (P10)
  • Testosterone