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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 [email protected] Abbreviations. AChE = acetylcholinesterase ALB = albumin ALP = alkaline phosphatase ALT = alanine aminotransferase

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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

[email protected]

abbreviations
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

clinical chemistry1
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
  • Luminometry
  • Spectrometry
  • Nephelometry
  • Electrophoresis
  • Chromatography
  • Isotopic (RIA/IRMA) and non-isotopic (ELISA/EIA) immunoassay
considerations for blood collection
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)
sources of variation
Sources of Variation

Diet

  • NIH-07 vs. NTP 2000
slide9

= NTP 2000

= NIH-07

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.

sources of variation1
Sources of Variation

Diet

  • NIH-07 v NTP 2000

Fasting

  • Glucose
  • Alkaline phosphatase
sources of variation2
Sources of Variation

Diet

  • NIH-07 v NTP 2000

Fasting

  • Glucose
  • Alkaline phosphatase

Diurnal variation

  • Hormones
sources of variation3
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
slide19

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

slide20

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

sources of variation4
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
cholinesterase assay
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

serum ache iu l
Serum AChE (IU/L)

Propargyl Alcohol Control 64 ppm

Males 1071 778

serum ache iu l1
Serum AChE (IU/L)

Propargyl Alcohol Control 64 ppm

Males 1071 778

Suggested an approximate 30% enzyme inhibition

serum che iu l
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

serum che iu l1
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

ntp core clinical chemistry profile
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
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 cont1
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

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

liver case examples1
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
liver case examples2
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?
liver case examples3
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

liver case examples4
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
liver case examples5
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
liver case examples6
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 examples7
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
liver case examples8
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
liver case examples9
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

liver case examples10
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
liver case examples11
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
liver case examples12
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

liver case examples13
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
liver case examples14
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
liver case examples15
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

liver case examples16
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
liver case examples17
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
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 (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
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

When evaluating quantitative urine chemistry data, always normalize (e.g., creatinine, urine volume)

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

normalization calculations
Normalization Calculations
  • Using creatinine
    • Assay Variable (units/vol.) / Creatinine (mg/dL)
creatinine normalization calculation
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
creatinine normalization calculation1
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
      • 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat
creatinine normalization calculation2
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
      • 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat or 117 g/mg
creatinine normalization calculation3
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
      • 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat or 117 g/mg
    • Urine LDH
creatinine normalization calculation4
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
      • 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat or 117 g/mg
    • Urine LDH
      • (27 U/L / 68.4 mg/dL) = 0.39 U LDH/mg creat
creatinine normalization calculation5
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
      • 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat or 117 g/mg
    • Urine LDH
      • (27 U/L / 68.4 mg/dL)/10 = 0.039 U LDH/mg creat
creatinine normalization calculation6
Creatinine Normalization Calculation
  • Assay Variable (units/vol.) / Creatinine (mg/dL)
  • Examples
    • Urine glucose
      • 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat or 117 g/mg
    • Urine LDH
      • (27 U/L / 68.4 mg/dL)/10 = 0.039 U LDH/mg creat or 39 mU/mg
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

normalization calculations1
Normalization Calculations
  • Using creatinine
    • Assay Variable (units/vol.) / creatinine (mg/dL)
  • Using volume
    • Assay Variable (units/vol.) x volume (mL/time)
volume normalization calculation
Volume Normalization Calculation
  • Assay Variable (units/vol.) x Volume (mL/time)
  • Example
    • Urine glucose
      • 24.0 mg/dL x 5.6 mL/16 hr x dL/100 mL = 1.34 mg Glu/16 hr
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 markers1
Other Markers

Electrolytes

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

Hormones

  • Insulin
  • Thyroxine (T4)
  • Triiodothyronine (T3)
  • Thyroid Stimulating Hormone (TSH)
  • Estradiol (E2)
  • Progesterone (P10)
  • Testosterone
slide70

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

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)
ad