Glucose

METHOD [LAB] GLUCOSE OXIDASE REFERENCE METHOD GLUCOSE HEXOKINASE

PRINCIPLE

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 70 to 105 mg/dL 𝐴𝐴𝐴𝐴
100 mg/dL 0.0555 × 𝐶𝐶𝐴𝐴 500 nm
S.I. 3.8 to 5.8 mmol/L 𝐴𝐴𝐴𝐴

INTERFERENCES
• Grossly lipemic samples may falsely elevate glucose
• Increased bilirubin, uric acid and ascorbic acid may be oxidized by peroxidase, and therefore decreasing glucose values.

CLINICAL SIGNIFICANCE

Gestational Diabetes
Continuous glucose monitoring for managing Type 1 and Type 2 diabetes.
 TOTAL CHOLESTEROL
3-hydroxy-5,6 cholestene

METHOD [LAB] Cholesterol Oxidase Reaction REFERENCE METHOD Abell, Levy and Brodie Method

PRINCIPLE

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. < 200
200-239 mg/dL 𝐴𝐴𝐴𝐴
700 mg/dL 200 mg/dL 0.026 × 𝐶𝐶𝐴𝐴 500 nm
(borderline 𝐴𝐴𝐴𝐴
S.I. <5.2 mmol/L

INTERFERENCES
• A number of drugs and substances affect concentrations of cholesterol.
• Ascorbic acid is oxidized by peroxidase, therefore falsely reducing total cholesterol values.

CLINICAL SIGNIFICANCE
RODRIGUEZ:
• It evaluates the risk for atherosclerosis, myocardial and coronary arterial occlusions.
• There is a direct relationship between elevated serum cholesterol and myocardial infarction.
• It is used as thyroid, liver, and renal function tests, and for DM studies.
• It is essential in the diagnosis and management of lipoprotein disorders.
• It is also used to monitor effectiveness of lifestyle changes and stress management.
 TRIGLYCERIDES
METHOD [LAB] ENZYMATIC GLYCEROL PHOSPATE OXIDASE REFERENCE METHOD

PRINCIPLE

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 44-148 mg/dL 𝐴𝐴𝐴𝐴
200 mg/dL 200 mg/dL 0.0113 × 𝐶𝐶𝐴𝐴 500 nm
S.I. 0.50 – 1.67 mmol/L 𝐴𝐴𝐴𝐴

INTERFERENCES
• Hemolysis causes transient triglyceride synthesis.
• Detergents can interfere with the action of lipase

CLINICAL SIGNIFICANCE
• For the diagnosis and treatment of
o Liver disease
o Controlling Diabetes Mellitus
o Nephrosis
 HDL
High Density Lipoprotein/ Alpha Lipoprotein (HDL)

METHOD [LAB] ENZYMATIC COLORIMETRIC METHOD REFERENCE METHOD

PRINCIPLE
The HDL cholesterol reagent reacts with LDL and VLDL ,forming an insoluble precipitate at ph=10. The supernatant is obtained by centrifugation and is
measured at 520 nm.

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. Male: 26-63 10 𝑑𝑑𝑑𝑑/𝐿𝐿
mg/dL 𝐴𝐴𝐴𝐴
Female: 33-75 120 mg/dL 50 mg/dL × 𝐶𝐶𝐴𝐴 520 nm
S.I. 0.85 – 1.94 mM/L 386.6 𝑚𝑚𝑚𝑚/𝑚𝑚𝑚𝑚 𝐴𝐴𝐴𝐴

INTERFERENCES
Heparinized plasma
Gentrisic acid
Hemolysis

CLINICAL SIGNIFICANCE
Maintains cholesterol equilibrium
Diagnosis of Coronary heart disease
 BUN
METHOD [LAB] BERTHELOT REACTION REFERENCE METHOD

PRINCIPLE
The Berthelot reaction, in which ammonia reacts with hypochlorite, phenol, a catalyst, and alkali to produce a stable blue complex (indophenol) has been
known for over 100 years but only relatively recently used in a method for serum urea. The use of sodium nitroferricyanide was introduced in 1962 and
the substitution of salicylate for phenol was produced in 1967. This procedure is based upon a modified Berthelot reaction wherein urease hydrolyzes urea
to ammonia and carbamic acid. Carbamic acid spontaneously decomposes into ammonia and carbon dioxide. Ammonia reacts with salicylate,
nitroferricyanide and an alkaline solution of hypochlorite to yield a blue-green chromophore which is measured photometrically and is proportional to the
amount of urea in the sample

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 8-23 mg/dl 𝐴𝐴𝐴𝐴
45 mg/dL 25 mg/dL 0.357 × 𝐶𝐶𝐴𝐴 630 nm
S.I. 2.9-8.2 mmol/L 𝐴𝐴𝐴𝐴

INTERFERENCES
• Ammonium of fluoride salt anticoagulants
• If a sample exceeds the linearity of the test, make a five-fold dilution of sample with ammonia-free reagent grade water and re-run· assay; multiply
result by 5

CLINICAL SIGNIFICANCE

Clinically, BUN rises in response to renal dysf unction.

➢ Low BUN are not generally considered abnormal renal f unction.
➢ Serum-urea levels drop in severe hepatic disease because of a decline in the capacity of the liver to
generate urea f rom ammonia.
 BUA
METHOD [LAB] URICASE METHOD REFERENCE METHOD

PRINCIPLE

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 2.5 -7.7 mg/dL 𝐴𝐴𝐴𝐴
20 mg/dL 6.3 mg/dL 0.0595 × 𝐶𝐶𝐴𝐴 520 nm
S.I. mM/L 𝐴𝐴𝐴𝐴

INTERFERENCES
Bilirubin falsely
Salicylates increases uric acid

CLINICAL SIGNIFICANCE

Diagnosis and management of gout

Monitoring uric acid levels for patients undergoing chemotherapy.
 CREATININE
METHOD [LAB] DIRECT JAFFE METHOD REFERENCE METHOD

PRINCIPLE
In the Jaffe reaction. creatinine reacts with picric acid under alkaline conditions to form a red-colored product that can be measured at 510 nm.
The red color intensity is directly proportional to creatinine concentration

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. Male: 0.9-1.3
mg/dl
Female: 0.6-1.1 𝐴𝐴𝐴𝐴
20 mg/dL 6 mg/dL 88.4 × 𝐶𝐶𝐴𝐴 510 nm
S.I. Male: 80-115 𝐴𝐴𝐴𝐴
μmol/L
Female: 53-97

INTERFERENCES
Antibiotics such as cephalosporin falsely increases values.
Bilirubin and hemoglobin falsely decrease values.

CLINICAL SIGNIFICANCE
Monitoring renal function

Diagnosis of Acute Kidney Injury (AKI)

 AST
METHOD [LAB] Karmen Method REFERENCE METHOD Karmen Method

PRINCIPLE
AST catalyzes the reversible transfer of an amino group from aspartate to alpha-ketoglutarate forming glutamate and oxaloacetate. The oxaloacetate
produced is reduced to malate-by-malate dehydrogenase and NADH. The rate of decrease in concentration of NADH, measured photometrically is
proportional to the catalytic concentration of AST in the sample.
𝐴𝐴𝐴𝐴𝐴𝐴
𝐿𝐿 − 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴+ ∝ −𝐾𝐾𝐴𝐴𝐴𝐴𝐾𝐾𝑚𝑚𝑑𝑑𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 → 𝐺𝐺𝑑𝑑𝐴𝐴𝐴𝐴𝐴𝐴𝑚𝑚𝐴𝐴𝐴𝐴𝐴𝐴 + 𝑂𝑂𝑂𝑂𝐴𝐴𝑑𝑑𝐾𝐾𝐴𝐴𝑂𝑂𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴
𝑚𝑚𝑁𝑁𝑁𝑁
𝑂𝑂𝑂𝑂𝐴𝐴𝑑𝑑𝐾𝐾𝐴𝐴𝑂𝑂𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 + 𝑁𝑁𝐴𝐴𝑁𝑁𝑁𝑁 + 𝑁𝑁 𝑚𝑚𝐴𝐴𝑑𝑑𝐴𝐴𝐴𝐴𝐴𝐴 + 𝑁𝑁𝐴𝐴𝑁𝑁
→

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 5-37 U/L None (Multiply by 𝐴𝐴𝐴𝐴
0 to 467 U/L Multiply by 16.67 × 𝐶𝐶𝐴𝐴 340 nm
S.I. 1750) 𝐴𝐴𝐴𝐴

INTERFERENCES
Anticoagulants such as heparin, EDTA, oxalate and fluoride do not affect assay results n
Hemolysis interferes with assay.

CLINICAL SIGNIFICANCE
AMI
. The test may help diagnose or monitor liver problems
Chronic liver disorders

Monitoring therapy with hepatotoxic drugs

 ALT
METHOD [LAB] Enzymatic Method REFERENCE METHOD Kinetic UV Method

PRINCIPLE
ALT catalyzes the reversible transfer of an amino group from alanine to alpha-ketoglutarate forming glutamate and pyruvate. The pyruvate produced is
reduced to lactate by lactate dehydrogenase and NADH. The rate of decrease in concentration of NADH when measured spectrophotometrically is
proportional to the catalytic concentration of ALT present in the sample.
𝐴𝐴𝐿𝐿𝐴𝐴
𝐿𝐿 − 𝐴𝐴𝑑𝑑𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴+ ∝ −𝐾𝐾𝐴𝐴𝐴𝐴𝐾𝐾𝑚𝑚𝑑𝑑𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐺𝐺𝑑𝑑𝐴𝐴𝐴𝐴𝐴𝐴𝑚𝑚𝐴𝐴𝐴𝐴𝐴𝐴 + 𝑃𝑃𝑃𝑃𝐴𝐴𝐴𝐴𝑃𝑃𝐴𝐴𝐴𝐴𝐴𝐴
→
𝐿𝐿𝑁𝑁𝑁𝑁
𝑃𝑃𝑃𝑃𝐴𝐴𝐴𝐴𝑃𝑃𝐴𝐴𝐴𝐴𝐴𝐴 + 𝑁𝑁𝐴𝐴𝑁𝑁𝑁𝑁 + 𝑁𝑁 𝐿𝐿𝐴𝐴𝑂𝑂𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 + 𝑁𝑁𝐴𝐴𝑁𝑁
→

PROCEDURE

ALT has no R1 and

R2 based on the
SOP of the Lab. So
1000 uL of reagent
should be added.

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 5-38 U/L None (Multiply by ∆𝐴𝐴𝐴𝐴𝐴𝐴
0-400 U/L Multiply by 16.67 𝑂𝑂1750 340 nm
S.I. 83.35-633.46 nKat/L 1750 or 1768) 𝑚𝑚𝐴𝐴𝐴𝐴

INTERFERENCES
Anticoagulants such as heparin, EDTA, oxalate and fluoride do not affect assay results n
Hemolysis interferes with assay.

CLINICAL SIGNIFICANCE
. The test may help diagnose or monitor liver problems

Diagnosis of acute hepatitis

Obstructive jaundice
Hepatic cancer
 TOTAL PROTEIN
METHOD [LAB] Biuret Method REFERENCE METHOD Biuret Method

PRINCIPLE
Proteins in the serum forms a blue-violet colored complex when mixed with copper ions in alkaline solution with the unshared electrons of the nitrogen and
oxygen atoms of the protein peptide bonds. The amount of color produced is proportional to the protein concentration and is measured
spectrophotometrically at 550 nm.

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 6.6-8.7 g/dL 𝐴𝐴𝐴𝐴
Up to 15 g/dL 8.0 g/dL 10 (g/L) × 𝐶𝐶𝐴𝐴 550 nm
S.I. 66-87 g/L 𝐴𝐴𝐴𝐴

INTERFERENCES
• Hemolysis should be avoided (7). The color produced by 1 mg of hemoglobin is equivalent to 1.9 mg of serum protein.

• Lipemic samples and lyophilized control sera should have serum blanks.
• Bromsulfophthalein will give falsely high results.

• Bilirubin up to Principle 25 mg I dL does not interfere.

CLINICAL SIGNIFICANCE
Assessment of nutritional status such as malabsorption and malnutrition
For evaluations of kidney, liver and bone marrow disease
 ALBUMIN
METHOD [LAB] Bromcresol Green REFERENCE METHOD Bromcresol Green

PRINCIPLE
The principle of the test is based on the dye-binding principle of albumin to the dye bromcresol green. The absorbance of the blue-green colored complex at
630 nm is proportional to albumin concentration.

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. 3.5 to 5.5 g/dL Multiply by 10 (Unit: 𝐴𝐴𝐴𝐴
0.5 – 6.0 g/dL 4.0 g/dL × 𝐶𝐶𝐴𝐴 630 nm
S.I. g/L) 𝐴𝐴𝐴𝐴

INTERFERENCES
Young et al (5) have reviewed drug ef(ects on serum albumin. These include insulin, steroids, and hormones.

CLINICAL SIGNIFICANCE
Evaluation of kidney ad liver function
 HBA1C
METHOD [LAB] REFERENCE METHOD

PRINCIPLE
A hemolyzed preparation of the whole blood is mixed continuously for 5 minutes with a weakly binding caution-exchange resin. During this time, HbAOI
binds to the resin. After the mixing period, a filter separator is used to remove the resin from the supernatant liquid which absorbances at 415 nm for the
glycohemoglobin fraction and the total hemoglobin fraction. Calculating the ratio of the substances and comparing this ratio to that of a calibrator carried
through the separation procedure.

PROCEDURE

CONCENTRATION CONVERSION
REFERENCE VALUES LINEARITY FORMULA ABSORBANCE
(Standard) FACTOR
I.U. Normal: 4.2 – 6.2
Good Control: 5.5-6.8% 𝐴𝐴𝐴𝐴
Fair Control: 6.8-7.6% 4-20 % × 𝐶𝐶𝐴𝐴 415 nm
Poor Control: >7.6% 𝐴𝐴𝐴𝐴
S.I.
 INTERFERENCES

CLINICAL SIGNIFICANCE