Transcript Clinical Chemistry Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653 [email protected].

Clinical Chemistry
Gregory S. Travlos, DVM, DACVP
National Institute of Environmental Health Sciences
Research Triangle Park, NC 27709
919-541-0653
[email protected]
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
Too numerous to cover
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Photometry
Fluorometry
Nephelometry
Electrophoresis
Isotopic immunoassay
Chromatography
Spectrometry
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
Diet
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NIH-07 v NTP 2000
Fasting
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Glucose
Diurnal variation
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Hormones
Analytical Methods & Sample Collection/Handling Techniques
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Cholinesterase
Creatine Kinase
In vitro Hemolysis
Urine Collection/Handling
Diet: NIH-07 v. NTP-2000
Analyte
ALT (IU/L)
Males
Females
NIH-07
NTP-2000
56.5
47.5
90.0
77.0
20.0
20.5
15.0
14.8
BUN (mg/dL)
Males
Females
Switching diets resulted in an approximately 60% increase in control animal serum ALT activity
and a 26% decrease in serum BUN concentration.
Es tradiol Value s in Cycling Rats and M ice
60
50
F344
Estradiol (ng/mL)
SD
40
B6
CD-1
30
20
10
0
Early Proest
Late Proest
Estrus
Metestrus
Diestrus
Diestrus 2
Se rum M e latonin afte r Five 1-M inute Light Expos ure s in Fe m ale F344 Rats
250.0
12/12 control
1-day exposure
200.0
pg/mL
150.0
100.0
50.0
0.0
06:00
11:30
12:30
13:30
14:30
15:30
16:30
17:30
Time
18:30
19:30
20:30
21:30
22:30
23:30
00:30
Assay Variation: AChE (IU/L)
Propargyl Alcohol
Males
Control
64 ppm
1071
778
Assay Variation: AChE (IU/L)
Propargyl Alcohol
Males
Control
64 ppm
1071
778
Suggested an approximate 30% enzyme inhibition
Assay Variation: AChE (IU/L)
Propargyl Alcohol
Males
Control
64 ppm
1071
778
Suggested an approximate 30% enzyme inhibition
PTC assay
BTC 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/L)
Propargyl Alcohol
Males
Control
64 ppm
1071
778
Suggested an approximate 30% enzyme inhibition
Untreated
0.1 mM
1.0 mM
10.0 mM
PTC assay
BTC assay
876
795
825
836
272
289
299
262
Assays: male rat serum; 2.5 hour incubation; performed in duplicate
Troponin
Comparison of cTn Measurement in the Beagle
20
0.25
Abbott Architect
Tosoh AIA 600 II
Bayer Advia Centaur
Beckman Access
Dade Dimension RxL
OCD Vitros ECi
10
0.15
DPC Immulite
Dog Troponin EIA
0.10
Roche Elecsys 2010
5
0.05
0
0.00
Neg
Low
Med
High
cTnT (ng/mL)
cTnI (ng/mL)
15
0.20
Comparison of cTn Measurement in the Cynomolgus
Monkey
25
1.00
Abbott Architect
Tosoh AIA 600 II
Bayer Advia Centaur
0.75
cTnI (ng/mL)
Beckman Access
Dade Dimension RxL
15
OCD Vitros ECi
DPC Immulite
0.50
Monkey Troponin EIA
10
Roche Elecsys 2010
0.25
5
0
0.00
Neg
Low
Med
High
cTnT (ng/mL)
20
Comparison of cTn Measurement in the
Sprague Dawley Rat
30
6
Abbott Architect
Tosoh AIA 600 II
25
5
Bayer Advia Centaur
20
Dade Dimension RxL
4
OCD Vitros ECi
15
DPC Immulite
3
Rat Troponin EIA
Roche Elecsys 2010
10
2
5
1
0
0
Neg
Low
Med
High
cTnT (ng/mL)
cTnI (ng/mL)
Beckman Access
NTP Core Clinical Chemistry
Profile
Protein
• Total protein
• Albumin
Muscle
• Creatine Kinase
Kidney
• Urea Nitrogen
• Creatinine
Liver
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Alanine Aminotransferase
Sorbitol Dehydrogenase
Alkaline Phosphatase
Total Bile Acids
Evaluation of Liver
Alanine Aminotransferase (ALT, SGPT)
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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)
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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.
Aspartate Aminotransferase (AST, SGOT)
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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)
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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 - 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)
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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
Ref Value
ALT
SDH
ALP
TBA
Tbili
Dbili
1
2
3
30-55 IU/L
34
130
450
10-20 IU/L
16
13
63
250-350 IU/L
157
321
279
25-35 µmol/L
31
27
43
0.1-0.5 mg/dL
0.2
0.3
0.3
0.05-0.2 mg/dL
0.1
0.1
0.1
Liver Case Examples
Ref Value
ALT
SDH
ALP
TBA
Tbili
Dbili
4
5
6
30-55 IU/L
44
51
87
10-20 IU/L
18
20
28
250-350 IU/L
257
301
987
25-35 µmol/L
31
13
104
0.1-0.5 mg/dL
9.3
0.3
4.7
0.05-0.2 mg/dL
0.3
0.1
3.1
Evaluation of Kidney
Need ~75% of nephrons non-functional for alterations in serum
markers to occur
Urea Nitrogen (UN, BUN)
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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)
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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.
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
Detection of protein in urine (plasma,
genitourinary)
In general:
>20 mg/kg/day
Persistent
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
Tougher to do in urine v. serum
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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.
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
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
Analyte
SG
Volume (mL)
Creat (mg/dL)
Gluc (mg/dL)
Prot (mg/dL)
AST (IU/L)
LDH (IU/L)
NAG (IU/L)
Control
1.017
12.2
68.4
8.0
65.0
6
27
10
1200 ppm
1.013
26.8
34.0
5.0
54.0
26
54
9
PGMBE Urinalysis: converted data
Analyte
Gluc (ug/mg creat)
Prot (ug/mg creat)
AST (mU/mg creat)
LDH (mU/mg creat)
NAG (mU/mg creat)
Control
117
950
9
39
15
1200 ppm
147
1588
76
159
26
Urine constituent unit conversions for the 1-chloro-2-propanol study
Conversions were performed using treatment group mean values.
Tx. Grps.
ppm
Body Wght.
g
Ur. Vol.
mL/16 hr
Ur. Gluc.
mg/dL
Ur. Prot.
mg/dL
Ur. Gluc.
mg/16 hr
Ur. Prot.
mg/16 hr
Ur. Gluc.
mg/100 g/16 hr
Ur. Prot.
mg/100 g/16 hr
Males (day 15):
0
33
100
330
1000
3300
193
191
198
192
189
155
5.6
8
6.6
5
4.5
0.9
24
23
22
29
33
89
73
62
60
53
63
81
1.34
1.84
1.45
1.45
1.49
0.80
4.09
4.96
3.96
2.65
2.84
0.73
0.70
0.96
0.73
0.76
0.79
0.52
2.12
2.60
2.00
1.38
1.50
0.47
Males (W k 13):
0
33
100
330
1000
3300
383
372
373
378
373
312
5.9
6.8
6.2
5.6
4.7
2.7
31
25
27
29
33
73
68
69
70
81
80
95
1.83
1.70
1.67
1.62
1.55
1.97
4.01
4.69
4.34
4.54
3.76
2.57
0.48
0.46
0.45
0.43
0.42
0.63
1.05
1.26
1.16
1.20
1.01
0.82
Other Markers
Proteins
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Total
Albumin
Globulin
Carbohydrate Metabolism
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Glucose
Lipid Metabolism
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Cholesterol
Triglycerides
Muscle
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Creatine Kinase or Phosphokinase (CK, CPK) - total and isoenzymes
Troponin T and I
Other Markers
Electrolytes
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Sodium
Potassium
Chloride
Bicarbonate
Calcium
Phosphorus
Hormones
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Insulin
Thyroxine (T4)
Triiodothyronine (T3)
Thyroid Stimulating Hormone (TSH)
Estradiol (E2)
Progesterone (P10)
Testosterone