A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
METHODS AND PRINCIPLES
Cholesterol
Introduction
The standard procedures for cholesterol and triglyceride measurements are the Monotest Cholesterol procedure (1) and
the GPO Triglyceride procedure (2) of Boehringer Mannheim. Both of these tests are adapted to automatic analysis using
the Hitachi 911 Analyzer By using these reagents and instrumentation we have demonstrated that measurements meet
the criteria of precision and accuracy set forth by CDC. A number of factors are involved in the interference of the
enzymatic determination of plasma lipids (3,4). Among common drugs, only ascorbic acid and `L-methyldopa produced
borderline negative interference at therapeutic plasma concentrations. In Trinder systems (5), bilirubin has been reported
to interact in the peroxidase-catalyzed reaction by competing for hydrogen peroxide, which results in lower lipid levels (6).
The inclusion of hexacyanoferrate (II) into the reagent mixture minimizes this chemical interference, and bilirubin
concentrations of up to 15 mg/dL did not produce significant changes in results. Positive interference may be caused by
hemoglobin, which acts as a pseudoperoxidase. However, no distortion of results was observed by concentrations of up
to 2 g/dL.
Since the proper functioning of the Hitachi 911 Analyzer is essential for the timely analysis of samples, a detailed
maintenance program is in operation, which includes a service contract with 24 hour response time of a field engineer.
Automated Determination of Cholesterol
Principal
Cholesteryl esters are hydrolyzed to free cholesterol and fatty acids by cholesteryl esterase. The free cholesterol produced
and that already present in the sample is oxidized by cholesterol oxidase to form cholest-4-en-3-one and hydrogen
peroxide. Peroxidase catalyzes the reaction of hydrogen peroxide, 4-aminoantipyrine, and phenol (or a substituted
phenol) to produce a quinoneimine dye, with maximum absorbance at 500 nm. The absorbance readings at this
wavelength are proportional to the concentration of total cholesterol in the sample (1).
Materials
Equipment and Supplies
- Boehringer Mannheim/Hitachi 911
- Gilson pipetman
- Sample cups
- Cells for Hitachi 911.
The Hitachi 911 analyzer is a totally self-contained Computerized, Programmable, Discrete, Selective, Sequential,
fully automated Chemistry Analyzer. It is composed of two hardware units:
The analytical unit and the control unit. The 911 analyzer uses several operational systems to
perform required functions.
Cholesterol Reagent:
The Cholesterol / HP reagent is supplied from Boehringer Mannheim Corporation. The reagent is automatically shipped
to the Laboratory once a month. “High Performance” is the acceptable reagent grade. The ingredients for the Cholesterol
Reagent are as follows:
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
0.40 mmol 3,4-Dichlorophenol
0.60 mmol Phenol
0.10 mmol 4-Aminophenazone
 25 U Cholesterol Oxidase (EC 1.1.3.6: Nocardia erythropolis; 25C)
 40 U Cholesterol Esterase (EC 3.1.1.13: microorganism; 25C)
 20 U POD (EC 1.11.1.1.7; horse radish; 25C)
Non Reactive Ingredients: Buffer, Stablizers, and Preservatives
Additional materials that are required are an automated clinical chemistry analyzer, Precical Calibrator Serum, and a
solution of 0.9% NaCl.
Reagent Preparation
Cholesterol / HP reagent comes prepackaged in two forms. One is a premeasured powder that is reconstituted in its
bottle with 100 mL of distilled or deionized water from the tank attached to the Hitachi 911. When mixed gently it is
ready for use in 10 minutes. The other form is a ready-to-use premade solution, which requires no reconstitution. The
degree of accuracy required for measuring reagents is a maximum error for glassware of  5%. It is necessary to use
Class A Volumetric Glassware to meet performance guidelines. Reagents should only be used according to the
manufacturer’s specific guidelines for this specific procedure.
Storage Requirements
Reagent is stored in the bottle that it arrives in from the supplier. It should be stored at 2-8C. Refer to the labels on the
box and bottles for expiration dates on unopened components. Once opened the reagent should be put into the analyzer.
The analyzer will alert the tech when the reagent has passed its shelf life. Reconstituted reagent is stable for up to 4 weeks
at 2-12C or 7 days at 20-25C when protected from light or contamination by microorganisms.1 Reagent bottles should
be labeled with the following: substance (cholesterol reagent), lot number, date of preparation, expiration date, special
storage instructions. In the event that the reagent does not perform within set parameters, eliminate other potential
problems, prepare a new bottle of reagent and recalibrate. If the problem persists, call the Manufacturer’s technical
support.
Calibration
The Hitachi 911 automatically performs calibration on line. Calibrators are manufactured by Boehringher Mannheim and
are prepared according to manufacturer’s insert
Storage Requirements
Store unopened Precical Calibrator Serum at 2-8 degrees Celsius or 20-25 degrees
Celsius. Do Not Freeze! Each vial bears the expiration date on the label.
No evidence of vacuum in vial upon opening is an indication of possible
deterioration of unreconstituted Precical Calibrator Serum.
If there is any evidence of coloration, cloudiness or turbidity in Precical Diluent
the vial should be discarded.
Reconstituted Precical Calibrator Serum is stable for two days when stored securely
stoppered and protected from light at 28 degrees Celsius.
1Boehringer
Mannheim Cholesterol /HP Application, pp. 2.
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
Opened Precical Calibrator Serum, Precitrol-N and Precitrol-A should be labeled
with date of opening and the initials of the person preparing them.
Controls and Standards
All standards and controls are tested in duplicate for quality control purposes. Various suppliers provide the controls that
are used. Boehringer Mannheim provides the Precitrol-N, for normal control, the Precitrol-A, for abnormal control, and
the Precinorm-L , for normal control that is tested twice each month. Solomon Park provides an additional standard
(Sercal) which is tested daily for the Cholesterol and Triglyceride levels. Pacific Biometrics provides three additional
controls: Lot A,B, and Lot C. These three controls are tested daily for cholesterol, triglyceride, HDL and Creatinine.
Additionally, Incstar provides standards and controls for the Apolipoprotein A-1, Apolipoprotein B and Lipoprotein(a)
tests that are performed when requested.
Calculations
The Hitachi 911 does all Calculations.
Analytical Procedure
Hitachi 911
After performing daily maintenance on the Hitachi 911 pipette Precical calibrator, Precitrol-n, Precitrol-A
and Precimat standard into sample cups and place in appropriate positions on the standard and control
section on the sample disk. Program the Hitachi 911 to perform required calibrations for tests to be calibrated. Test that
have 3 or more calibrators always get a full calibration. Pipette a minimum of 0.5mL
of each plasma specimen into a sample cup (without top) . Specimen are numbered 00001-800. following the loading of
specimens, the number of specimens and quality control standards are programmed into the cholesterol program. Sample
volume3l and reagent volume 300l. Incubation temperature is 37C, and running time is 6 min. Cholesterol content of
samples is calculated by the microprocessor of the instrument using the Autoblank. Endpoint Mode.
References
1. Lieberman C. Ber. 1885;18:1803
2. Burchard H. Chem Zentr. 1890;61:25
3. Abell L. et al. Standard Methods in Clinical Chemistry.
1958;26:2.
4. Flegg HM Ann Clin Bicohem. 1973;10:79.
5. Richmond W. Scand J Clin Lab Invest.
1972;28,Suppl 126.
6. Richmond W. Clin Chem. 1973; 19:1350.
7. Roeschlau P, et al; Bergmeyer HU, ed. Methods of
Enzymatic Analysis. 2nd Eng ed. New York, NY;
Academic Press; 1974:1890.
8. Allain CC, et al. Clin Chem. 1974;20;470
9. Roeschlau P, et al. Z Klin Chem Klim Biochem.
1974;12:226
10. Trinder P. Ann clin Biochem. 1969;6:24.
11. Siedel J, Haegele EO, Ziegenhorn J. Wahlefeld AW.
Clin Chem. 1983;29:1075.
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
12. Wiebe DA, Bernert JT. Clin Chem. 1984;30:352
13. Recommendations for Improving Cholesterol
Measurement: A Report from the Laboratory
Standardization Panel of the National Cholesterol
Education Program. NIH Publication No. 90-2964,
February 1990.
14. Tietz NW. Clinical Guide to Laboratory Texts. 2nd ed.
Philadelphia, Pa: WB Saunders Co; 1990:122,124.
15. Beaucamp K, et al, in manuscript.
16. Young DS. Effects of Drugs on Clinical laboratory
Tests. 3rd ed. Washington, DC: AACC Press; 1990.
17. Friedman RB, Young DS. Effects of Disease on
Clinical Laboratory Tests 2nd ed. Washington, DC:
AACC Press; 1989.
18. Report of the National Cholesterol Education Program
Expert Panel on Detection, Evaluation, and Treatment
of High Blood Cholesterol in Adults. Arch of Int Med.
Jan 1988; vol 148, No 1:36-69.
19. Statland BE. Clinical Decision Levels for Laboratory
Tests. 2nd ed. Oradell, NJ: Medical Economics
Books, 1987.
20. Data on file at Boehringer Mannheim.
L A B O R A T O R Y
M A N U A L
O F
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
Triglycerides
Principal
Triglycerides are hydrolyzed to glycerol and fatty acids by lipase. The glycerol formed is then phosphorylated in the
presence of ATP. Oxidation of the resulting glycerol-3-phosphate to dihydroxyacetone phosphate and hydrogen peroxide
is catalyzed by glycerol phosphate oxidase. An intense red chromogen is produced by the peroxidase catalyzed coupling
of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzene-sulfonate with hydrogen peroxide. The absorbance
readings at 510 nm are proportional to the concentration of total glycerol content (free and esterified) in the samples (1).
Materials
Equipment and Supplies
- Boehringer Mannheim/Hitachi 911
- Gilson pipetman
- Sample cups
- Cells for Hitachi 911.
The Hitachi 911 analyzer is a totally self-contained Computerize, Programmable, Discrete, Selective, Sequential,
fully automated Chemistry Analyzer. It is composed of two hardware units:
The analytical unit and the control unit. The 911 analyzer uses several operational systems to perform required
functions.
Reagents
The Triglyceride reagent is supplied from Boehringer Mannheim Corporation (Cat. No. 7474029). It is a mixture of R1
Buffer, 4-Chlorophenol and enzymes. The reagent is automatically shipped to the Laboratory once a month with a
standing order. “High Performance” is the acceptable reagent grade. The ingredients for the Triglyceride Reagent are as
follows:
3.5 mmol/L 4-Chlorophenol
0.5 mmol/L ATP
0.35 mmol/L 4-Aminophenazone
3 U/mL Lipase (EC 3.1.1.13; pseudomonas species; 25C)
2.5 U/mL Glycerol phosphate oxidase (EC 1.1.3.21)
0.2 U/mL Glycerokinase (EC 2.7.1.30; Candida mycoderma; 25C)
0.15 U/mL Peroxidase (EC 1.11.1.7; horseradish; 25C)
Nonreactive ingredients: Buffer, preservative, detergent
Additional materials that are required are an automated clinical chemistry analyzer, Precical Calibrator Serum, and a
solution of 0.9% NaCl.
Reagent Preparation
Triglycerides/GPO reagent can mixed in the shipping bottles. Connect Bottle 1 containing buffer and 4-Chlorophenol to
Bottle 1a containing enzymes using the enclosed adapters. Mix by gentle inversion. Disconnect Bottle 1a and connect the
second Bottle 1a. Mix again by gentle inversion until the lyophilizate is completely dissolved in the buffer. Reagent
should only be used according to the manufacturer’s specific guidelines for this specific procedure.
Storage Requirements
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
Reagent is stored in the shipping bottles. It should be stored at 2-8C. Refer to the labels on the box and bottles for
expiration dates on unopened components. Once opened the reagent should be put on board the analyzer. The analyzer
will alert the tech when the reagent has passed its shelf life. Reconstituted reagent is stable for up to 14 days at 2-12C or
2 days at 15-25C when protected from light or contamination by microorganisms. Reagent bottles should be labeled with
the following: substance (triglyceride reagent), lot number, date of preparation, expiration date, special storage instructions
(Initial of Technician). In the event that the reagent does not perform within set parameters, eliminate other potential
problems, prepare a new bottle of reagent and recalibrate. If the problem persists, call the Manufacturer’s technical
support.
Calibration
The Hitachi 911 automatically performs calibration on line. Calibrators are manufactured by Boehringer Mannheim and
are prepared according to manufacturer’s insert.
Storage Requirements
Store unopened Precical Calibrator Serum at 2-8 degrees Celsius or 20-25 degrees
Celsius. Do Not Freeze! Each vial bears the expiration date on the label.
No evidence of vacuum in vial upon opening is an indication of possible
deterioration of unreconstituted Precical Calibrator Serum.
If there is any evidence of coloration, cloudiness or turbidity in Precical Diluent
the vial should be discarded.
Reconstituted Precical Calibrator Serum is stable for two days when stored securely
stoppered and protected from light at 28 degrees Celsius.
Opened Precical Calibrator Serum, Precitrol-N and Precitrol-A should be labeled
with date of opening and the initials of the person preparing them.
Controls and Standards
All standards and controls are tested in duplicate for quality control purposes. Various suppliers provide the controls that
are used. Boehringer Mannheim provides the Precitrol-N, for normal control, the Precitrol-A, for abnormal control, and
the Precinorm-L , for normal control that is tested twice each month. Solomon Park provides an additional standard
(Sercal) which is tested daily for the Cholesterol and Triglyceride levels. Pacific Biometrics provides three additional
controls: Lot A,B and Lot C. These three controls are tested daily for cholesterol, triglyceride and HDL. Additionally,
Incstar provides standards and controls for the Apolipoprotein
A-1, Apolipoprotein B and Lipoprotein(a) tests that are performed when requested.
Calculations
The Hitachi 911 does all Calculations.
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
Analytical Procedure
Hitachi 911
After performing daily maintenance on the Hitachi 911 pipette Precical calibrator, Precitrol-n, Precitrol-A
and Precimat standard into sample cups and place in appropriate positions on the standard and control
section on the sample disk. Program the Hitachi 911 to perform required calibrations for tests to be calibrated. Test that
have 3 or more calibrators always get a full calibration. Pipette a minimum of 0.5mL
of each plasma specimen into a sample cup (without top) . Specimen are numbered 00001-800. following the loading of
specimens, the number of specimens and quality control standards are programmed into the
cholesterol program. Sample volume 3l and reagent volume 300l. Incubation temperature is 37C, and running time is
6 min. Triglyceride content of samples is calculated by the microprocessor of the instrument using the Autoblank.
Endpoint Mode.
References
Triglycerides/GPO:
1. Eggstein M, Kreutz F. Klin Wschr. 1966;44:262,267.
2. Buccolo G, David H. Clin Chem. 1973; 19:476.
3. Wahlefeld A; Bergmeyer HU, ed. Methods of Enzymatic Analysis. 2nd English ed. New York, NY: Academic Press Inc;
1974:1831.
4. Tietz NW. Clinical Guide to Laboratory Tests. 2nd ed. Philadelphia, PA: WB Saunders Co; 1990:554.
5. Shepard MDS, Whiting MJ. Clin Chem. 1990; 36(2):325-329.
6. Young DS. Effects of Drugs on Clinical Laboratory Tests. 3rd ed. Washington, DC: AACC Press;
1990.
7. Friedman RB, Young DS. Effects of Disease on Clinical Laboratory Tests. 2nd ed. Washington, D.C:
AACC Press; 1989.
8. NIH Consensus Conference (1984). JAMA. 251:1196.
9. Data on file at Boehringer Mannheim.
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
Measurement of HDL-Cholesterol
Principal
Methods to quantify plasma levels of HDL include various kinds of ultracentrifugation (1-3), electrophoretic techniques
(4), and procedures which rely on precipitation of lipoproteins other than HDL (5). While the classical ultracentrifugation
techniques are impractical for analyzing such large numbers of samples as required in this program, precipitation
techniques have been proven useful in previous epidemiological studies (6-9). In the majority of these studies, VLDL and
LDL were precipitated by heparin and Mn2+, and the cholesterol of HDL remaining in solution was measured by a
modified Lieberman Burchard method. The use of heparin and Mn2+ is, however, inadequate when cholesterol is
determined by enzymatic procedures, since Mn2 + interferes in the determination (10). To overcome this drawback,
Mg2+ and dextran sulfate was used and evaluated to precipitate VLDL and LDL (5,11). The method was found highly
reliable and reproducible, but revealed, on the average, 5% lower plasma levels of HDL-cholesterol, when compared with
the LRC-procedure (11). The explanation for this negative bias may be that a more complete precipitation of Apo B
containing lipoproteins (11) and precipitation of Apo E-containing lipoproteins (12) had occurred. The supernate
contained less than 10% of total apo(a) immunoreactivity (13). These data are similar to a previous study in which about
10% of apo(a) immunoreactivity was found in the lipoprotein-free fraction subsequent to sequential ultracentrifugation
(14). Thus about 10% of apo(a) is associated with little or no lipid.
Mixtures of dextran sulfate (molecular weight 50,000), an analog of heparin, and magnesium ions (Mg2+) cause
precipitation of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) while the high density
lipoproteins (HDL) remain in solution. Precipitated lipoproteins are separated by centrifugation, and the supernate
containing HDL is analyzed for cholesterol content.
Materials
Equipment and Supplies
- Boehringer Mannheim/Hitachi 911
- Gilson pipetman
- Sample cups
- Cells for Hitachi 911.
The Hitachi 911 analyzer is a totally self-contained Computerize, Programmable, Discrete, Selective, Sequential,
fully automated Chemistry Analyzer. It is composed of two hardware units:
The analytical unit and the control unit. The 911 analyzer uses several operational systems to perform required
functions.
Reagents
The HDL-Cholesterol reagent is supplied from Boehringer Mannheim Corporation. The reagent is automatically shipped
to the Laboratory once a month with a standing order. “High Performance” is the acceptable reagent grade. The
Precipitant for the reagent is a combination of:
0.55 mmol/L Phosphotungstic acid
25 mmol/L Magnesium chloride
The ingredients for the HDL-Cholesterol Reagent are as follows:
75 mmol/L PIPES buffer, pH 6.8
10 mmol/L Mg2+
0.2 mmol/L Sodium cholate
0.15 mmol/L 4-Aminophenazone
4.2 mmol/L Phenol
1% Fatty alcohol-polyglycol ether
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
0.5 U/mL Cholesterol esterase (EC 3.1.1.13; pseudomonas species; 25C)
0.15 U/mL Cholesterol oxidase (EC 1.1.3.6; E. coli; 25C)
0.25 U/mL Peroxidase (POD) (EC 1.11.1.7; horseradish; 25C)
Nonreactive Ingredients: Buffer, stabilizers, preservative
Additional materials that are required are an automated clinical chemistry analyzer, Precimat 50mL Cholesterol Calibrator,
and a solution of 0.9% NaCl.
Reagent Preparation
HDL-Cholesterol reagent comes in two bottles; one containing the precipitant and the other containing the working
solution. The precipitant is ready for use. The working solution should be mixed by gently swirling before use. Reagent
should only be used according to the manufacturer’s specific guidelines for this specific procedure.
Storage Requirements
Reagent and precipitant are stored in the shipping bottles. Reagent should be stored at 2-8C. The precipitant should be
stored at 15-25C. Refer to the labels on the box and bottles for expiration dates on unopened components. Once
opened the reagent and precipitant should be put on board the analyzer. The analyzer will alert the tech when the reagent
or precipitant has passed its shelf life. Reconstituted reagent is stable for up to 28 days at 2-12C or 7 days at 15-25C
when protected from light or contamination by microorganisms. Precipitant will remain stable until the expiration date on
the bottle when stored at 15-25C. Reagent and precipitant bottles should be labeled with the following: substance (HDLCholesterol reagent or precipitant), lot number, date of preparation, expiration date, special storage instructions. In the
event that the reagent does not perform within set parameters, eliminate other potential problems, prepare a new bottle of
reagent and recalibrate. If the problem persists, call the Manufacturer’s technical support.
Calibration
The Hitachi 911 automatically performs calibration on line. Calibrators are manufactured according to manufacturer’s
insert.
Storage Requirements
Store unopened Precical Calibrator Serum at 2-8 degrees Celsius or 20-25 degrees
Celsius. Do Not Freeze! Each vial bears the expiration date on the label.
No evidence of vacuum in vial upon opening is an indication of possible
deterioration of unreconstituted Precical Calibrator Serum.
If there is any evidence of coloration, cloudiness or turbidity in Precical Diluent
the vial should be discarded.
Reconstituted Precical Calibrator Serum is stable for two days when stored securely
stoppered and protected from light at 208 degrees Celsius.
Opened Precical Calibrator Serum, Precitrol-N and Precitrol-A should be labeled
with date of opening and the initials of the person preparing them.
Controls and Standards
All standards and controls are tested in duplicate for quality control purposes. Various suppliers provide the controls that
are used. Boehringer Mannheim provides the Precitrol-N, for normal control, the Precitrol-A, for abnormal control, and
the Precinorm-L , for normal control that is tested twice each month. Solomon Park provides an additional standard
(Sercal) which is tested daily for the Cholesterol and Triglyceride levels. Pacific Biometrics provides three additional
A T H E R O S C L E R O S I S
C L I N I C A L R E S E A R C H
O P E R A T I O N S
L A B O R A T O R Y
M A N U A L
O F
controls: Lot A, B and Lot C. These three controls are tested daily for cholesterol, triglyceride and HDL. Additionally,
Incstar provides standards and controls for the Apolipoprotein A-1, Apolipoprotein B and Lipoprotein(a) tests that are
performed when requested.
Calculations
The Hitachi 911 does all Calculations.
Analytical Procedure
Hitachi 911
After performing daily maintenance on the Hitachi 911 pipette Precical calibrator, Precitrol-n, Precitrol-A
and Precimat standard into sample cups and place in appropriate positions on the standard and control
section on the sample disk. Program the Hitachi 911 to perform required calibrations for tests to be calibrated. Test that
have 3 or more calibrators always get a full calibration. Pipette a minimum of 0.5mL
of each plasma specimen into a sample cup (without top) . Specimen are numbered 00001-800. following the loading of
specimens, the number of specimens and quality control standards are programmed into the
cholesterol program. Sample volume20l and reagent volume 350l. Incubation temperature is 37C, and running time is
6 min. Cholesterol content of samples is calculated by the microprocessor of the instrument using the Autoblank
Endpoint Mode.
References
1.
2.
3.
4.
5.
6.
7.
Barr, et al. Am J Med. 1951;11:480.
Harvel RJ, Eder HA, Bragdon JH. J Clin Invest. 1955;34:1345.
Lopes-Virella MF, Stone P, Ellis S, et al. Clin Chem. 1960;5:609.
Wilson DE, Spizer MJ. J Lab Clin Med. 1973;82:473.
Burstein M, Samaille. J Clin Chem Acta. 1960;5:609.
Assman G, Schriewer H, Schmitz G, et al. Clin Chem. 1983;29:2026.
Recommedations for Improving Cholesterol Measurement: A Report from the Laboratory
Standardization Panel of the National Cholesterol Education Program. NIH Publication No. 90-2964,
February 1990.
8. Tietz NW. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia, PA: WB Saunders Co; 1995:334.
9. Tietz NW. Clinical Guide to Laboratory Tests. 2nd ed. Philadelphia, PA: WB Saunders Co;
1990:122,124.
10. Young DS. Effects of Drugs on Clinical Laboratory Tests. 3rd ed. Washington, DC: AACC Press;
1990.
11. Friedman RB, Young DS. Effects of Drugs on Clinical Laboratory Tests. 2nd ed. Washington, DC:
AACC Press; 1989.
12. Data on file at Boehringer Mannheim.
13. Statland BE. Clinical Decision Levels for Laboratory Tests. 2nd ed. Oradell, NJ: Medical Economics
Books; 1987.