Clinical chemistry
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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

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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


Clinical chemistry

= NTP 2000

= NIH-07


Diet nih 07 v ntp 2000

Diet: NIH-07 v. NTP-2000

AnalyteNIH-07NTP-2000

ALT (IU/L)

Males56.5 90.0

Females47.5 77.0

BUN (mg/dL)

Males20.0 15.0

Females20.5 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


Clinical chemistry

Troponin


Clinical chemistry

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


Clinical chemistry

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 AlcoholControl64 ppm

Males1071 778


Serum ache iu l1

Serum AChE (IU/L)

Propargyl AlcoholControl64 ppm

Males1071 778

Suggested an approximate 30% enzyme inhibition


Serum che iu l

Serum ChE (IU/L)

Propargyl AlcoholControl64 ppm

Males1071 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 AlcoholControl64 ppm

Males1071 778

Suggested an approximate 30% enzyme inhibition

PTC assayBTC assay

Untreated876 272

0.1 mM795 289

1.0 mM825 299

10.0 mM836 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 Value1

ALT 30-55 IU/L34

SDH 10-20 IU/L16

ALP 250-350 IU/L157

TBA 25-35 µmol/L31

Tbili 0.1-0.5 mg/dL0.2

Dbili 0.05-0.2 mg/dL0.1


Liver case examples1

Liver Case Examples

Ref Value1

ALT 30-55 IU/L34

SDH 10-20 IU/L16

ALP 250-350 IU/L157

TBA 25-35 µmol/L31

Tbili 0.1-0.5 mg/dL0.2

Dbili 0.05-0.2 mg/dL0.1

  • Case 1: Decreased ALP


Liver case examples2

Liver Case Examples

Ref Value1

ALT 30-55 IU/L34

SDH 10-20 IU/L16

ALP 250-350 IU/L157

TBA 25-35 µmol/L31

Tbili 0.1-0.5 mg/dL0.2

Dbili 0.05-0.2 mg/dL0.1

  • Case 1: Decreased ALP - decreased food intake?


Liver case examples3

Liver Case Examples

Ref Value12

ALT 30-55 IU/L34130

SDH 10-20 IU/L1613

ALP 250-350 IU/L157321

TBA 25-35 µmol/L3127

Tbili 0.1-0.5 mg/dL0.20.3

Dbili 0.05-0.2 mg/dL0.10.1


Liver case examples4

Liver Case Examples

Ref Value12

ALT 30-55 IU/L34130

SDH 10-20 IU/L1613

ALP 250-350 IU/L157321

TBA 25-35 µmol/L3127

Tbili 0.1-0.5 mg/dL0.20.3

Dbili 0.05-0.2 mg/dL0.10.1

  • Case 2: Increased ALT


Liver case examples5

Liver Case Examples

Ref Value12

ALT 30-55 IU/L34130

SDH 10-20 IU/L1613

ALP 250-350 IU/L157321

TBA 25-35 µmol/L3127

Tbili 0.1-0.5 mg/dL0.20.3

Dbili 0.05-0.2 mg/dL0.10.1

  • Case 2: Increased ALT - suspect enzyme induction


Liver case examples6

Liver Case Examples

Ref Value123

ALT 30-55 IU/L34130450

SDH 10-20 IU/L161363

ALP 250-350 IU/L157321279

TBA 25-35 µmol/L312743

Tbili 0.1-0.5 mg/dL0.20.30.3

Dbili 0.05-0.2 mg/dL0.10.10.1


Liver case examples7

Liver Case Examples

Ref Value123

ALT 30-55 IU/L34130450

SDH 10-20 IU/L161363

ALP 250-350 IU/L157321279

TBA 25-35 µmol/L312743

Tbili 0.1-0.5 mg/dL0.20.30.3

Dbili 0.05-0.2 mg/dL0.10.10.1

  • Case 3: Increased ALT, SDH, TBA


Liver case examples8

Liver Case Examples

Ref Value123

ALT 30-55 IU/L34130450

SDH 10-20 IU/L161363

ALP 250-350 IU/L157321279

TBA 25-35 µmol/L312743

Tbili 0.1-0.5 mg/dL0.20.30.3

Dbili 0.05-0.2 mg/dL0.10.10.1

  • Case 3: Increased ALT, SDH, TBA - suspect hepatocellular injury


Liver case examples9

Liver Case Examples

Ref Value4

ALT 30-55 IU/L44

SDH 10-20 IU/L18

ALP 250-350 IU/L257

TBA 25-35 µmol/L31

Tbili 0.1-0.5 mg/dL9.3

Dbili 0.05-0.2 mg/dL0.3


Liver case examples10

Liver Case Examples

Ref Value4

ALT 30-55 IU/L44

SDH 10-20 IU/L18

ALP 250-350 IU/L257

TBA 25-35 µmol/L31

Tbili 0.1-0.5 mg/dL9.3

Dbili 0.05-0.2 mg/dL0.3

  • Case 4: Increased Tbili


Liver case examples11

Liver Case Examples

Ref Value4

ALT 30-55 IU/L44

SDH 10-20 IU/L18

ALP 250-350 IU/L257

TBA 25-35 µmol/L31

Tbili 0.1-0.5 mg/dL9.3

Dbili 0.05-0.2 mg/dL0.3

  • Case 4: Increased Tbili - suspect hemolytic disease


Liver case examples12

Liver Case Examples

Ref Value45

ALT 30-55 IU/L4451

SDH 10-20 IU/L1820

ALP 250-350 IU/L257301

TBA 25-35 µmol/L3113

Tbili 0.1-0.5 mg/dL9.30.3

Dbili 0.05-0.2 mg/dL0.30.1


Liver case examples13

Liver Case Examples

Ref Value45

ALT 30-55 IU/L4451

SDH 10-20 IU/L1820

ALP 250-350 IU/L257301

TBA 25-35 µmol/L3113

Tbili 0.1-0.5 mg/dL9.30.3

Dbili 0.05-0.2 mg/dL0.30.1

  • Case 5: Decreased TBA


Liver case examples14

Liver Case Examples

Ref Value45

ALT 30-55 IU/L4451

SDH 10-20 IU/L1820

ALP 250-350 IU/L257301

TBA 25-35 µmol/L3113

Tbili 0.1-0.5 mg/dL9.30.3

Dbili 0.05-0.2 mg/dL0.30.1

  • Case 5: Decreased TBA - suspect impaired enterohepatic recirculation


Liver case examples15

Liver Case Examples

Ref Value456

ALT 30-55 IU/L445187

SDH 10-20 IU/L182028

ALP 250-350 IU/L257301987

TBA 25-35 µmol/L3113104

Tbili 0.1-0.5 mg/dL9.30.34.7

Dbili 0.05-0.2 mg/dL0.30.13.1


Liver case examples16

Liver Case Examples

Ref Value456

ALT 30-55 IU/L445187

SDH 10-20 IU/L182028

ALP 250-350 IU/L257301987

TBA 25-35 µmol/L3113104

Tbili 0.1-0.5 mg/dL9.30.34.7

Dbili 0.05-0.2 mg/dL0.30.13.1

  • Case 6: Increased ALT, SDH, ALP, TBA, T & Dbili


Liver case examples17

Liver Case Examples

Ref Value456

ALT 30-55 IU/L445187

SDH 10-20 IU/L182028

ALP 250-350 IU/L257301987

TBA 25-35 µmol/L3113104

Tbili 0.1-0.5 mg/dL9.30.34.7

Dbili 0.05-0.2 mg/dL0.30.13.1

  • 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

AnalyteControl1200 ppm

SG1.0171.013

Volume (mL)12.226.8

Creat (mg/dL)68.434.0

Gluc (mg/dL)8.05.0

Prot (mg/dL)65.054.0

AST (IU/L)626

LDH (IU/L)2754

NAG (IU/L)109


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

AnalyteControl1200 ppm

Gluc (ug/mg creat)117147

Prot (ug/mg creat)9501588

AST (mU/mg creat)976

LDH (mU/mg creat)39159

NAG (mU/mg creat)1526


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


Clinical chemistry

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)


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