STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

INDEX
SL.NO INVESTIGATION METHOD NAME SOP PAGE NO
1 GLUCOSE Hexokinase 1
2 UREA Urease 3
3 CREATININE Jaffemod/Alk.Picrate 5
4 URICACID Uricase 7
5 CALCIUM Arsenazo III 9
6 PHOSPHOROUS Phospho molybdate 11
7 CHOLESTROL Enzymatic CHOD-PAP 13
8 TRIGLYCERIDES GPO-PAP 15
9 HDL Accelerator SelectiveDetergent 17
10 LDL Liquid Selective Detergent 19
11 BILIRUBIN TOTAL Diazo Dye 22
12 BILIRUBIN DIRECT Diazo Dye 25
13 TOTAL PROTEIN Biuret 28
14 ALBUMIN BCG 31
15 SGOT NADH w/o P-5’-P 33
16 SGPT NADH w/o P-5’-P 35
17 ALKALINEPHOSPHATASE Para-Nitro phenyl-phosphate 38
18 SODIUM ISE 41
POTASSIUM ISE
CHLORIDE ISE
19 BICARBONATE ISE 44
20 C-REACTIVE PROTEIN Immunoterbidimetric 46
21 RA FACTOR Immunoterbidimetric 49
22 AMYLASE 2-chloro-p-nitrophenyl-a-D- 51
maltotrioside
23 IRON Colorimetric, Ferene 53

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 1KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

S.NO NAME OF THE PERSON DESIGNATION SIGNATURE

1. Dr.P.Pasupathi HOD

2. Mrs. Deepa Senior Biochemist

3. Mrs.Bhuwana Senior Biochemist

4. Mrs.Vanitha Senior Biochemist

5. Mr.Asha Senior Biochemist

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 2KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-GLUCOSE

Summary

Blood glucose determinations are the most frequently performed clinical chemistry laboratory procedures,
commonly used as an aid in the diagnosis and treatment of diabetes. Elevated glucose levels (hyperglycemia)
may also occur with pancreatic neoplasm, hyperthyroidism, and adrenal cortical hyper function as well as other
disorders. Decreased glucose levels (hypoglycemia) may result from excessive insulin therapy or various liver
diseases.

Test Principles

Methodology: Hexokinase/G-6-PDH.

Glucose is phosphorylated by hexokinase (HK) in the presenceof adenosine triphosphate (ATP) and magnesium
ions to produce glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). Glucose-6-phosphate
dehydrogenase (G-6-PDH) specifically oxidizesG-6-P to 6-phosphogluconate with the concurrent reduction of
nicotinamide adenine dinucleotide phosphate (NADP) to nicotinamideadenine dinucleotide phosphate reduced
(NADPH). One micromoleof NADPH is produced for each micromole of glucose consumed. The NADPH
produced absorbs light at 340 nm and can be detected spectrophotometrically as an increased absorbance.

Specimen collection and Preparation

Serum, plasma are acceptable specimens. Collect serum using standard sampling tubes, Heparin plasma is also
suitable.

Reagent Preparation

Glucose is supplied as a liquid, ready-to-use, two-reagent

R1: Ready to use

R2: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 3KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing procedure
Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test GLU .Put the sample loaded rack into the machine it starts running.

Calibration and Quality Controls

Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 12 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/plasma

Fasting: 70-115 mg/dl

Random: 80-140 mg/dl

Post Prandial: 120-180 mg/dl

Child : 30-60 mgs%

References

1.Kit Pack Insert

2. Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry, 2nd ed. Philadelphia, PA: WB
Saunders; 1994:959–60.

3. Tietz NW, editor. Clinical Guide to Laboratory Tests, 3rd ed.Philadelphia, PA: WB Saunders;

1995:268–72.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 4KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-UREA

Summary

Measurements obtained by this test are used in the diagnosis of certain renal and metabolic diseases. The
determination of serum urea nitrogenis is a widely used test for the evaluation of kidney function. The test is
frequently requested in conjunction with the serum creatinine test for the differential diagnosis of prerenal
(cardiac decompensation, waterdepletion, increased protein catabolism), renal (glomerulonephritis,chronic
nephritis, polycystic kidney, nephrosclerosis, tubular necrosis),and postrenal (obstructions of the urinary tract)
hyperuremia.

Test Principles

Methodology: Urease

The Urea Nitrogen assay is a modification of a totally enzymatic procedure first described by Talke and
Schubert.1 The test is performed as a kinetic assay in which the initial rate of the reaction is linear for limited
period of time. Urea in the sample is hydrolyzed by urease to ammonia and carbon dioxide. The second
reaction, catalyzed by glutamate dehydrogenase (GLD) converts ammonia and α-ketoglutarate to glutamate and
water with the concurrent oxidation of reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide
adenine dinucleotide (NAD). Two moles of NADH are oxidized for each mole of urea present. The initial rate
of decrease in absorbance at 340 nm is proportional to the urea concentration in the sample.

Specimen collection and Preparation

Suitable Specimens

Serum, plasma are acceptable specimens. Collect serum using standard sampling tubes, Heparin plasma is also
suitable.

Reagent Preparation

Urea Nitrogen is supplied as a liquid, ready-to-use, two-reagent

R1:Ready to use

R2:Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 5KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID(12digits) in
position from 1st till 5th and select the test UREA .Put the sample loaded rack into the machine it starts running.

Calibration and Quality Controls

Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 12 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/plasma

10-50 mg/dl

References

1.Kit Pack Insert

2. Talke H, Schubert GE. Klinische Wochenschrift 1965;43:174.

3. Tietz NW, editor. Clinical Guide to Laboratory Tests, 3rd ed.Philadelphia, PA: WB Saunders; 1995:622–

4. US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030,
Occupational Exposure to Blood borne Pathogens.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 6KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-CREATININE

Summary

In 1886 Jaffe developed an assay for creatinine based upon the reaction between creatinine and sodium
picrate. In 1904 Folin2 used this reaction for the quantitative determination of creatinine in urine. Kinetic
procedures based on the observed reaction rates of various substances, including creatinine, with alkaline
picrate have been proposed by Fabiny3 and Soldin.4 This improved Jaffe chemistry is a kinetic procedure
which does not require deproteinization of the sampleand is formulated to reduce the interference of serum
proteins.

Test Principles

Methodology: Alkaline Picrate

At an alkaline pH, creatinine in the sample reacts with picrate to form a creatinine-picrate complex. The rate of
increase in absorbance at 500 nm due to the formation of this complex is directly proportional to the
concentration of creatinine in the sample.

Specimen collection and Preparation

Suitable Specimens

Serum, plasma are acceptable specimens. Collect serum using standard sampling tubes, Heparin plasma is also
suitable.

Reagent Preparation

Creatinine is supplied as a liquid, ready-to-use, two-reagent

R1:Ready to use

R2:Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 7KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test creatinine .Put the sample loaded rack into the machine it starts
running.

Calibration and Quality Controls

Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 12 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range
Serum/plasma

Male : 0.7-1.3 mg/dl

Female : 0.6-1.1 mg/dl

Chile : 0.3-1.0 mg/dl

References

1.Kit Pack Insert

2. Folin O. Beitrag zur Chemie des Kreatinins und Kreatins Im Harne.Hoppe Seylers Z Physiol Chem
1904;41:223–42.

3. Fabiny DL, Ertingshausen G. Automated reaction-rate method for determination of serum creatinine with the
CentrifiChem. Clin Chem 1971;17:696–700.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 8KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-URIC ACID

Summary

Uric acid is a metabolite of purines, nucleic acids, and nucleoproteins. Consequently, abnormal levels may be
indicative of a disorder in the metabolism of these substances. Hyperuricemia may be observed in renal
dysfunction, gout, leukemia, polycythemia, atherosclerosis, diabetes, hypothyroidism, or in some genetic
diseases. Decreased levels are present in patients with Wilson’s disease.

Test Principles

Methodology: Uricase

The Uric Acid assay is based on the methods of Trivedi and Kabasakalian.3,4 Uric acid is oxidized to allantoin
by uricase with the production of hydrogen peroxide (H2O2). The H2O2 reacts with 4-aminoantipyrine (4-
AAP) and 2,4,6-tribromo-3-hydroxybenzoic acid (TBHB) in the presence of peroxidase to yield a quinoneimine
dye. The resulting change in absorbance at 548 nm is proportional to the uric acid concentration in the sample.

Specimen collection and Preparation

Suitable Specimens

Serum, plasma are acceptable specimens. Collect serum using standard sampling tubes, Heparin plasma is also
suitable.

Reagent Preparation

Uric acid is supplied as a liquid, ready-to-use, single -reagent

R1:Ready to use

Testing procedure
Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test uric acid .Put the sample loaded rack into the machine it starts
running.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 9KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Calibration and Quality Controls

Calibration is stable for approximately 26 days (624 hours) and calibration is required with each change in
reagent cartridge and reagent lot number. Verify calibration with at least two levels of controls according to the
established quality control requirements for your laboratory. If control results fall outside acceptable ranges,
recalibration may be necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours.
Two levels namely Bio-Rad L1 & L2 are to be used.

Reference range

Serum/plasma

Male : 3.5-7.2 mg/dl

Female : 2.6-6.0 mg/dl

References

1.Kit Pack Insert

2. Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry, 2nd ed. Philadelphia, PA: WB
Saunders; 1994:1541–2.

3. Tietz NW, editor. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia, PA: WB Saunders; 1995:624–5

4. Trivedi R, Rebar L, Berta E, et al. New enzymatic method for serum

uric acid at 500 nm. Clin Chem 1978; 24(11):1908–11.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 10
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-CALCIUM

Summary

The majority of calcium in the body is present in bones. The remainder of the calcium is in serum and has
various functions. For example, calcium ions decrease neuromuscular excitability, participate in blood
coagulation, and activate some enzymes. Hypercalcemia can result from hyperparathyroidism, hypervitaminosis
D, multiple myeloma, and some neoplastic diseases of bone. Long-term lithium therapy has been reported to
cause hyperparathyroidism in some individuals, with hypercalcemia. Hypocalcemia can result from
hypoparathyroidism, steatorrhea, nephrosis, nephritis, and pancreatitis. Calcium has traditionally been difficult
to measure accurately and precisely, and a large variety of methods have been developed. Among these are
flame photometry, oxalate precipitation with titration, atomic absorption spectrophotometry, EDTA chelation,
and more recently calcium dye complexes, which are measured spectrophotometrically. Examples of calcium
dyes are o-cresolphthalein complexone andArsenazo III, the latter being the dye used for calcium determination
in this method.

Test Principle

Methodology: Arsenazo III

Arsenazo-III dye reacts with calcium in an acid solution to form blue-purple complex. The color developed is
measured at 660 nm and is proportional to the calcium concentration in the sample.

Specimen Collection and preparation

Suitable sample

Serum, plasma are suitable samples. Heparin plasma is also acceptable.

Reagent Preparation

Calcium is supplied as a liquid, ready-to-use, single reagent

R1: ready to use.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 11
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing Procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test calcium .Put the sample loaded rack into the machine it starts running.

Calibration and Quality control

Calibration is stable for approximately 41 days (984 hours) and is required with each change in reagent lot
number. Verify calibration curve with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/plasma

Normal: 8.9-10 mg/dl

Cord: 8.2 to 11.2 mg/dl

Newborn

Premature: 6.2 to 11.0mg/dl

0 to 10 days: 7.6 to 10.4mg/dl

10 days to 24 months: 9.0 to 11.02mg/dl

Child, 2 to 12 years: 8.8 to 10.8 mg/dl

Adult: 8.4 to 10.2 mg/dl

Male > 60 years: 8.8 to 10.0mg/dl

References

1.Kit Pack Insert

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 12
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-PHOSPHOROUS

Summary

The majority of the body phosphorus (80% to 85%) is present in the bones as hydroxyapatite. The
remainder of the phosphate is present as inorganic phosphorus and phosphate esters. Calcium and phosphorus in
serum usually exhibit a reciprocal relationship. Increased serum phosphorus cause hypervitaminosis
hypoparathyroidism, and renal failure. Reduced serum phosphorus levels are seen in rickets (Vitamin
deficiency, hyperparathyroidism, and Fanconi’s syndrome).

Test Principle

Methodology: Phosphomolybdate

Inorganic phosphate reacts with ammonium molybdate to form a heteropolyacid complex. The use of a
surfactant eliminates the need to prepare a protein-free filtrate. The absorbance at 340 nm is directly
proportional to the inorganic phosphorus level in the sample. Sample blanks must be run to correct for any non-
specific absorbance in the sample

Specimen collection and preparation

Suitable specimens

Serum, plasma are suitable samples. Heparin plasma is also acceptable.

Reagent Preparation

Phosphorus is supplied as a liquid, ready-to-use, two-reagent kit

R1: ready to use.

R2: ready to use.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 13
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing Procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test phosphorous .Put the sample loaded rack into the machine it starts
running.

Calibration and Quality control

Calibration is stable for approximately 41 days (984 hours) and is required with each change in reagent
lot number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range
Serum/plasma
Normal: 2.3-4.7 mg/dl

Reference

1. US Department of Labor, Occupational Safety and HealthAdministration. 29 CFR Part 1910.1030.

Occupational Exposure to Bloodborne Pathogens.
2. US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories.
HHS Publication (CDC), 4th ed. Washington, DC: US Government Printing Office, May 1999.
3. World Health Organization. Laboratory Biosafety Manual. Geneva: World Health Organization; 2004.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 14
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-CHOLESTEROL

Summary
Measurement of serum cholesterol levels can serve as an indicator of liver function, biliary function, intestinal
absorption, propensity toward coronary artery disease, and thyroid function. Cholesterol levels are important
in the diagnosis and classification of hyperlipoproteinemias. Stress, age, gender, hormonal balance, and
pregnancy affect normal cholesterol levels.

Test Principles
Methodology: Enzymatic
Many investigators have studied the use of enzymes to assay cholesterol. This reagent is based on the
formulation of Allain, et al. and the modification of Roeschlau6 with further improvements to render the reagent
stable in solution. Cholesterol esters are enzymatically hydrolyzed by cholesterol esterase to cholesterol and
free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to
cholest-4-ene-3-one and hydrogen peroxide. The hydrogen peroxide combines with hydroxybenzoic acid
(HBA) and 4-aminoantipyrine to form a chromophore (quinoneimine dye), which is quantitated at 500 nm.

Specimen collection and Preparation

Suitable Specimens
Serum, plasma are acceptable specimens. Heparin plasma is also suitable.

Reagent Preparation
Cholesterol is supplied as a liquid, ready-to-use, single -reagent
R1:Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 15
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test cholesterol .Put the sample loaded rack into the machine it starts
running.

Calibration and Quality Controls

Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range
Serum/plasma
Desirable: <200 mgs%
Borderline high: 200-239 mgs%

High: >240 mgs%

References

1. Kit Pack Insert

2. Burtis CA, Ashwood ER, editors. Tietz Fundamentals of Clinical Chemistry, 5th ed. Philadelphia, PA:
WB Saunders; 2001:480–5.
3. Flegg HM. An investigation of the determination of serum cholesterol by an enzymatic method. Ann Clin
Biochem 1973;10:79–84.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 16
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-TRIGLYCERIDES
Summary
Triglycerides are a family of lipids absorbed from the diet and produced endogenously from carbohydrates and
fatty acids. Measurementof triglyceride is important in the diagnosis and management of hyperlipidemia. These
diseases can be genetic or secondary to other disorders including nephrosis, diabetes mellitus, and endocrine
disturbances. The National Cholesterol Education Program (NCEP) cites evidence that triglycerides are an
independent risk factor for atherosclerosis. Individuals with hypertension, obesity, and/or diabetes are at greater
risk than are those without these conditions

Test Principles
Methodology: Glycerol Phosphate Oxidase
Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is
phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3-phosphate
and adenosine diphosphate (ADP). Glycerol-3-phosphate is oxidized to dihydroxyacetone phosphate (DAP) by
glycerol phosphate oxidase (GPO) producing hydrogen peroxide (H2O2). In a color reaction catalyzed by
peroxidase, the H2O2 reacts with 4-aminoantipyrine (4-AAP) and 4-chlorophenol (4-CP) to produce a red
colored dye. The absorbance of this dye is proportional to the concentration of triglyceride present in the
sample. This analytical methodology is based on the reaction sequence described by Fossati et al.and by
McGowan et al. .In this reagent, 4-chlorophenol is used rather than 2-hydroxy-3, 5dichlorobenzenesulfonate,
used in the Fossati and McGowan studies.

Specimen collection and Preparation

Suitable Specimens
Serum, plasma are acceptable specimens. Heparin plasma is also suitable.
Reagent Preparation
Triglycerides is supplied as a liquid, ready-to-use, single -reagent
R1: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 17
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing procedure
Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test triglycerides .Put the sample loaded rack into the machine it starts
running.
Calibration and Quality Controls
Calibration is stable for approximately 41 days (984 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.
Reference range
Serum/plasma
Normal: < 150 mgs%
Borderline high: 150-200 mgs%
High: 200-500 mgs%
Very high: >500 mgs%
References

1.Kit Pack Insert

2.Rubins HB. Triglycerides and coronary heart disease: implications of recent clinical trials. J Cardiovasc Risk
2000;7(5):339–45.
3.Forrester JS. Triglycerides: risk factor or fellow traveler? Curr Opin Cardiol 2001;16:261–4.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 18
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-HDL

Summary

The role of HDL cholesterol in lipid metabolism is the uptake and transport of cholesterol from peripheral
tissues to the liver through a process known as reverse cholesterol transport (a proposed cardioprotective
mechanism). Low HDL cholesterol levels are strongly associated with an increased risk of coronary heart
disease. Hence, the determination of serum HDL cholesterol is a useful tool in identifying high-risk patients.
The Adult Treatment Panel of the National Cholesterol Education Program (NCEP) recommends that in all
adults 20 years of age and over, a fasting lipoprotein profile (total cholesterol,LDL cholesterol, HDL
cholesterol, and triglyceride) should be obtained once every five years to screen for coronary heart disease risk

Test Principles

Methodology: Accelerator Selective Detergent

The method uses a two-reagent format and depends on the properties of a unique detergent. This method is
based on accelerating the reaction of cholesterol oxidase (CO) with non-HDL unesterified cholesterol and
dissolving HDL cholesterol selectively using a specific detergent. In the first reagent, non-HDL unesterified
cholesterol is subject to an enzyme reaction and the peroxide generated is consumed by a peroxidase reaction
with DSBmT yielding a colorless product. The second reagent consists of a detergent (capable of solubilizing
HDL cholesterol), cholesterol esterase (CE), and chromagenic coupler to develop color for the quantitative
determination of HDL cholesterol.

Specimen collection and Preparation

Suitable Specimens

Serum, plasma are acceptable specimens. Heparin plasma is also suitable.

Reagent Preparation

HDL is supplied as a liquid, ready-to-use, single -reagent

R1: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 19
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing procedure

Architect machine is ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test HDL .Put the sample loaded rack into the machine it starts running.

Calibration and Quality Controls

Calibration is stable for approximately 26 days (624 hours) and calibration is required with each change in
reagent cartridge and reagent lot number. Verify calibration with at least two levels of controls according to the
established quality control requirements for your laboratory. If control results fall outside acceptable ranges,
recalibration may be necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours.
Two levels namely Bio-Rad L1 & L2 are to be used.

Reference range

Serum/plasma

Negative risk : >60 mgs%

Normal : 40-60 mgs%

High risk : < 40 mgs%

References

1.Kit Pack Insert

2.Gotto AM.Lipoprotein metabolism and the etiology of hyperlipidemia.Hosp Pract 1988;23(Suppl 1):4–13.

3.Badimon JJ, Badimon L, Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma
fraction in the cholesterol-fed rabbit. J Clin Invest 1990;85(4):1234–41

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 20
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-LDL

Summary

Plasma lipoproteins are spherical particles containing varying amounts of cholesterol, triglycerides,
phospholipids, and proteins. The phospholipid, free cholesterol, and protein constitute the outer surface of the
lipoprotein particle, while the inner core contains mostly esterified cholesterol and triglycerides. These particles
serve to solubilize and transport cholesterol and trigylcerides in the bloodstream. The relative proportions of
protein and lipid determine the density of these lipoproteins and provide a basis on which to begin their
classification. These classes are: chylomicrons, very-low-density lipoprotein (VLDL), low-density lipoprotein
(LDL), and high-density lipoprotein (HDL). Numerous clinical studies have shown that the different lipoprotein
classes have very distinct and varied effects on coronary heart disease (CHD) risk.The studies all point to LDL
cholesterol as the key factor in the pathogenesis of atherosclerosis and CHD,2-8 while HDL cholesterol has-
been observed to have a protective effect. Even within the normal range of total cholesterol concentrations, an
increase in LDL cholesterol can occur with an associated increased risk for CHD.
Test Principle

Methodology: Measured, Liquid Selective Detergent

The Multigent Direct LDL assay is a homogeneous method for directly measuring LDL levels in serum or
plasma, without the need for off-line pretreatment or centrifugation steps. The method is in a two-reagent
format and depends on the properties of a unique detergent. This detergent solubilizes only the non-
LDLparticles. The cholesterol released is consumed by cholesterol esterase and cholesterol oxidase in a non-
color-forming reaction. A second detergent solubilizes the remaining LDL particles and a chromogenic coupler
allows for color formation. The enzyme reaction with LDL in the presence of the coupler produces color that is
proportional to the amount of LDL cholesterol present in the sample

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 21
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Specimen Collection And Preparation

Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable.
Reagent Preparation

LDL is supplied as a liquid, ready-to-use kit

R1: ready to use.

R2: ready to use.

Testing Procedure
Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test LDL.. Put the sample-loaded rack into the machine it starts running.

Calibration And Quality Control

Calibration is stable for approximately 28 days (672 hours) and is required with each change in reagent lot
number. Verify calibration curve with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range
Serum/Plasma
< 100mg/dl: Optimal
100 to 129mg/dl: Near or above optimal
130 to 159mg/dl: Borderline high
160 to 189mg/dl: High
≥ 190mg/dl: Very high

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 22
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Reference

1.Gotto AM. Lipoprotein metabolism and the etiology of hyperlipidemia. Hosp Pract 1988;23(Suppl 1):4.
2.Crouse JR, Parks JS, Schey HM, et al. Studies of low-density lipoprotein molecular weight in human beings
with coronary artery disease. J Lipid Res 1985; 26(5):566.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 23
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- Total Bilirubin

Summary

Red blood cells at the end of their circulating life are broken down in the reticuloendothelial system, mainly the
spleen. The resulting heme, once the iron is removed, is then converted to bilirubin. Once formed, bilirubin is
transported to the liver bound to albumin. This fraction of bilirubin is referred to as indirect or unconjugated
bilirubin. In the liver, bilirubin is conjugated to glucuronic acid (mono- anddi- glucuronides) to form conjugated
bilirubin by the enzyme uridyldiphosphate glucuronyl transferase. Conjugated bilirubin or direct bilirubin is
excreted via the biliary system into the intestine whrer it is metabolizedby bacteria to a group of products
known collectively as stercobilinogen . Elimination is almost complete and serum levels are normally
negligible. Total bilirubin is the sum of the unconjugated and conjugated fractions. Total bilirubin is elevated in
conditions causing hepatic obstruction, hepatitis, cirrhosis, hemolytic disorders, and several inherited enzyme
deficiencies.
Test Principle
Methodology: Diazo Reaction
Traditional methods of measuring bilirubin are based on the reaction of bilirubin with a diazo reagent to form
the colored compound: azobilirubin. The diazo reaction can be accelerated by the addition of various
chemicals. For example, Malloy-Evelyn1 used methanol, Jendrassik-Gróf2 used caffeine, and Walters-Gerarde3
used dimethyl sulfoxide (DMSO). Modifications of these methods included the addition of surfactants as
solubilizing agents.4Total (conjugated and unconjugated) bilirubin couples with a diazoreagent in the presence
of a surfactant to form azobilirubin. The diazoreaction is accelerated by the addition of surfactant as a
solubilizing agent. The increase in absorbance at 548 nm due to azobilirubin is directly proportional to the total
bilirubin concentration.

Specimen Collection And Preparation

Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 24
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Reagent Preparation

Total Bilirubin is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use
R2: Ready to use
Testing Procedure

Architect machine is ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Total Bilirubin. Put the sample loaded rack into the machine it starts
running
Calibration And Quality Control
Calibration is stable for approximately 14 days (336 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range
Serum/Plasma
Adult :0.2 to 1.2mg/dl.

Reference
1. Malloy HT, Evelyn KA. The determination of bilirubin with the
photoelectric colorimeter. J Biol Chem 1973;119:481–90.
2. Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry,
3rd ed. Philadelphia, PA: WB Saunders, 1999:1136–7.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 25
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- DIRECT BILIRUBIN

Summary
Red blood cells at the end of their circulating life are broken down in the reticuloendothelial system, mainly the
spleen. The resulting heme, once the iron is removed, is then converted to bilirubin. This process accounts for
about 80% of the 500 μmol (300 mg) of bilirubin formed daily. Other sources of bilirubin include the
breakdown of myoglobin and cytochromes and the catabolism of immature red blood cells in the
bonemarrow.Once formed, bilirubin is transported to the liver bound to albumin. This fraction of bilirubin is
referred to as indirect or unconjugated bilirubin. In the liver, bilirubin is conjugated to glucuronic acid (mono-
and diglucuronides) to form conjugated bilirubin by the enzyme uridyldiphosphate glucuronyl transferase.
Conjugated bilirubin or direct bilirubin is excreted via the biliary system into the intestine, where it is
metabolized by bacteria to a group of products known collectively as stercobilinogen. Elimination is almost
complete and serum levels are normally negligible.Direct bilirubin is the sum of the conjugated fractions. Direct
bilirubin is elevated in conditions causing hepatic obstruction, hepatitis, cirrhosis,several inherited enzyme
deficiencies, and inherited defects in canalicular excretion.
Test Principle

Methodology: Diazo Reaction

Bilirubin determination is generally based on the reaction of bilirubin with a diazotized sulfanilic acid,
described by Ehrlich. In this method, direct (conjugated fractions) bilirubin couples with a diazonium salt in the
presence of sulfamic acid to form the colored compound azobilirubin.The increase in absorbance at 548 nm due
to azobilirubin is proportionalto the direct bilirubin concentration.
Specimen collection and preparation
Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable
Reagent Preparation

Direct Bilirubin is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use
R2: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 26
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing Procedure

Architect machine is ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Direct Bilirubin. Put the sample loaded rack into the machine it starts
running.
Calibration And Quality Control
Calibration is stable for approximately 14 days (336 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used .
Reference range
Serum/Plasma
0.0-0.5mg/dl
Reference
1. Burtis CA, Ashwood ER, editors. Tietz Textbook of ClinicalChemistry, 3rd ed. Philadelphia, PA: WB
Saunders; 1999:1136.
2. US Department of Labor, Occupational Safety and HealthAdministration. 29 CFR Part 1910.1030,
Occupational Exposure to Bloodborne Pathogens.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 27
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-TOTAL PROTEIN
Summary
Plasma proteins are derived primarily from synthesis in the liver, plasmacells, lymph nodes, spleen, and bone
marrow. In diseased state, both the total plasma protein level and the ratio of the individual fractions may be
dramatically altered from their normal values. Hypoproteinemia may be caused by such conditions as nephrotic
syndrome, extensivebleeding, sprue (deficient protein absorption), severe burns, salt retention syndromes, and
Kwashiorkor (acute protein starvation). Hyperproteinemia may be observed in cases of severe dehydration and
disease states such as multiple myeloma. Changes in the proportions of the plasma proteins may occur in one or
several of the protein fractionsand often without alterations in the quantity of the total protein. The A/Gratio has
commonly been used as an index of the distribution betweenthe albumin and globulin fractions. This ratio can
be significantly altered in such conditions as cirrhosis of the liver,glomerulonephritis, nephroticsyndrome, acute
hepatitis, lupus erythematosis, and in some acute and Chronic infections.
Test Principle
Methodology: Biuret
Polypeptides containing at least two peptide bonds react with biuretReagent. In alkaline solution, cupric ion
forms a coordination complex with protein nitrogen with very little difference between albumin andGlobulin on
a protein-nitrogen basis.
Specimen collection and preparation
Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable
Reagent Preparation

Total protein is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 28
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing Procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Total Protein .Put the sample loaded rack into the machine it starts
running.

Calibration and Quality control

Calibration is stable for approximately 23 days (552 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range(serum)

Premature: 3.6 to 6.0 g/dl

Newborn: 4.6 to 7.0g/dl

Cord: 4.8 to 8.0 g/dl

1 week: 4.4 to 7.6 g/dl

7 months to 1 year: 5.1 to 7.3g/dl

1 to 2 years: 5.6 to 7.5g/dl

≥ 3 years: 6.0 to 8.0 g/dl

Adult, Ambulatory: 6.4 to 8.3 g/dl

Adult, Recumbent: 6.0 to 7.8 g/dl

> 60 years: lower by ~ 0.2g/dl

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 29
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Reference

1. US Department of Labor, Occupational Safety and HealthAdministration. 29 CFR Part 1910.1030,

Occupational Exposure toBloodborne Pathogens.
2. US Department of Health and Human Services. Biosafety inMicrobiological and Biomedical Laboratories.
HHS Publication(CDC), 4th ed. Washington, DC: US Government Printing Office,
May 1999

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 30
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP-ALBUMIN
Summary

Albumin is the major serum protein in normal individuals. Elevated serum albumin levels are usually the result
of dehydration. Decreased albumin levels are found in a wide variety of conditions, including Kidney disease,
liver disease, malabsorption, malnutrition, severe burns, infections, and cancer.

Test Principle

Methodology: Bromcresol Green

The Albumin BCG procedure is based on the binding of bromcresolgreen specifically with albumin to produce
a colored complex. The absorbance of the complex at 628 nm is directly proportional to the Albumin
concentration in the sample
.Specimen collection and preparation

Suitable Sample

Serum, plasma are suitable samples. Heparin plasma is also acceptable.

Reagent Preparation

Albumin BCG is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use

Testing Procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Albumin .Put the sample loaded rack into the machine it starts
running.
Calibration and Quality control

Calibration is stable for approximately 41 days (984 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 31 KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used

Reference range
Serum/Plasma

0 to 4 days: 2.8 to 4.4 g/dl

4 days to 14 years: 3.8 to 5.4 g/dl

Adult: 3.5 to 5.0 g/dl

60 years: 3.4 to 4.8 g/dl

Reference

1. US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030.
Occupational Exposure toBlood borne Pathogens.

2. US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories.
5th ed. Washington, DC: US Government Printing Office; January 2007.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 32
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP - Aspartate aminotransferase (AST)

Summary

Aspartate aminotransferase (AST), also referred to as glutamateoxaloacetate transaminase (GOT), is one of a

group of enzymes which catalyzes the interconversion of amino acids and α-keto acids by transfer of amino
groups. Both AST and alanine aminotransferase (ALT) are normally found in most body fluids, but not in urine
except in instances of kidney lesions. The greatest concentrations of ASTare found in heart, liver, muscle, and
kidney tissues. Damage to these tissues can greatly elevate serum AST levels. Following myocardial infarction,
AST in serum begins to increase within 6 to 8 hours of onset of pain, reaching a peak within 18 to 24 hours and
falling to normal by the fourth or fifth day. Serum values may increase to 10 to 15 time normal levels and the
increase is roughly proportional to the degree of tissue damage.

Test Principle

Methodology: NADH (without P-5'-P)

AST present in the sample catalyzes the transfer of the amino group from L-aspartate to α-ketoglutarate,
forming oxaloacetate and L--glutamate. Oxaloacetate in the presence of NADH and malatedehydrogenase
(MDH) is reduced to L-malate. In this reaction, NADH is oxidized to NAD. The reaction is monitored by
measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH to NAD.

Specimen Collection And Preparation

Suitable Sample

Serum, plasma are suitable samples. Heparin plasma is also acceptable.

Reagent Preparation

Alanine Aminotransferase is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use

R2: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 33
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing Procedure

Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Aspartate Aminotransferase. Put the sample loaded rack into the
machine it starts running.

Calibration And Quality Control

Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/Plasma

Adult :5-34 U/L

Reference

1. Burtis CA, Ashwood ER, editors. Tietz Textbook of ClinicalChemistry, 2nd ed. Philadelphia, PA: WB
Saunders; 1994:790–1.

2. Friedman RB, Young DS. Effects of Disease on Clinical LaboratoryTests. Washington, DC: AACC Press;
1989:3-38–3-41.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 34
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- ALANINE AMINOTRANSFERASE (ALT)

Summary
Alanine Aminotransferase (ALT), also referred to as glutamate pyruvate transaminase (GPT), is an enzyme
involved in amino acid metabolism. It is found in many tissues, but the highest levels are found in the liver and
kidney. Tissue destruction leads to the release of the intracellularenzyme into the circulating blood. Markedly
elevated serum ALT levelsmay be found in a variety of diseases of the liver, such as hepatitis,mononucleosis,
and cirrhosis. These very high levels of ALT are notusually observed in other disease processes, e.g.,
myocardial infarction;thus, ALT is regarded as a reasonably specific indicator of liver disease.
Test Principle
Methodology: IFCC
ALT present in the sample catalyzes the transfer of the amino group from L-Alanine to 2-Oxoglutarate, in the
presence of Pyridoxal-5'-Phosphate, forming Pyruvate and L-Glutamate. Pyruvate in the presence of NADH and
Lactate Dehydrogenase (LD) is reduced to L-Lactate. In this reaction NADH is oxidized to NAD. The reaction
is monitored by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH to NAD

ALT, P-5'-P
L-Alanine + 2-Oxoglutarate Pyruvate + L-Glutamate
LD
Pyruvate + NADH L-Lactate + NAD
The Activated ALT reagent is based on the optimized formulation as recommended by the International
Federation of Clinical Chemistry (IFCC).
Specimen Collection And Preparation
Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable.

Reagent Preparation

Alanine Aminotransferase is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 35
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

R2: Ready to use

Testing Procedure

Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Alanine Aminotransferase. Put the sample loaded rack into the
machine it starts running

Calibration And Quality Control

Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established qualitycontrol
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary.Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/Plasma

Adult :0 to 55 U/L

Reference

1. Bergmeyer HU, Horder M, Rej R. Approved recommendation (1985) on IFCC methods for the measurement
of catalytic concentration of enzymes. J Clin Chem Clin Biochem 1986; 24(7):481–95.

2. Schumann G, Bonora R, Ceriotti F, et al. IFCC primary reference procedures for the measurement of
catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical

3.Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic
concentration of alanine aminotransferase. Clin Chem Lab Med 2002;40(7):718–24.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 36
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- ALKALINE PHOSPHATES

Summary

Human alkaline phosphatase consists of a group of at least five tissue-specific isoenzymes, which catalyzes the
hydrolysis of Phosphate monoesters at alkaline pH. A variety of disease processes can result in the release of
increased quantities of alkaline phosphatase into the blood.

Test Principle

Methodology: Para-nitrophenyl Phosphate

Several substrates have been used to measure alkaline phosphatase activity such as glycerophosphate,1 phenyl
phosphate,1 and-nitro phenyl phosphate. Bowers and McComb improved the method of Bessey et al. to include
a kinetic measurement. Tietz et al.4optimized this method to include a chelated metal-ion buffer of zinc,
magnesium, and HEDTA. This Alkaline Phosphatase procedure is a modification of this method.Alkaline
phosphatase in the sample catalyzes the hydrolysis of colorlessp-nitrophenyl phosphate (p-NPP) to give p-nitro
phenol and inorganic phosphate. At the pH of the assay (alkaline), the p-nitro phenol is in theYellow phenoxide
form. The rate of absorbance increase at 404 nm is directly proportional to the alkaline phosphatase activity in
the sample. Optimized concentrations of zinc and magnesium ions are present to activate tha alkaline
phosphatase in the sample.

Specimen collection and preparation

Suitable Sample

Serum, plasma are suitable samples. Heparin plasma is also acceptable

Reagent Preparation

Alkaline Phosphatase is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 37
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

R2: Ready to use

Testing Procedure

Architect machine is ready mode than give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Alkaline phosphotase .Put the sample loaded rack into the machine it
starts running.

Calibration and Quality control

Calibration is stable for approximately 10 days (240 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established qualitycontrol
requirements for your laboratory. If control results fall outside acceptable range, recalibration may be necessary.
Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-Rad L1 &
L2 are to be used.

Reference range

Male

1 to 12 years: < 500U/L

12 to 15 years: < 750U/L

> 20 years: 40 to150U/L

Female

1 to 12 years: < 500U/l

> 15 years: 40 to 150U/L

Reference

1. King EJ, Armstrong AR. A convenient method for determining serum and bile phosphatase activity. Can
Med Assoc J 1934;31:376–81.
2. Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five
cubic millimeters of serum. J Biol Chem 1946;164:321–9.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 38
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP -Electrolytes
Summary
Sodium is the major cation of extra cellular fluid; it plays an essential role in the normal distribution of water
and in the maintenance of osmotic pressure in extra cellular fluid compartments. Decreased levels of sodium
may be caused by an excessive use of diuretics, prolonged vomiting, a decrease in the intake of sodium in the
diet, and metabolic acidosis. Increased levels of sodium may be found in Cushing’s Syndrome, severe
dehydration, or in high levels of salt intake without an adequate supply of water.
Potassium is the major intracellular cation. The concentration of potassium in the erythrocytes is
approximately 23 times the concentration in plasma. For this reason, only unhemolyzed samples must be used.
Decreased levels of extra cellular potassium are characterized by weakness in the muscles, irritability, paralysis,
accelerated heartbeat, and eventually cardiac arrest, and may be caused by a poor intake of potassium in the
diet, by a redistribution ofextracellular potassium, and by an increased loss of body fluids rich in potassium.1
Abnormally elevated levels of extra cellular potassium produce mental confusion, general weakness, numbness,
flaccid paralysis in the extremities, a slowed heart rate, and eventually collapse of the peripheral vascular
system and cardiac arrest. Causes of increased potassium levels may be linked to inappropriate intravenous
therapy, dehydration, shock, diabetic Ketoacidosis, and severe burns.
Chloride is the major extra cellular anion. The majority of ingested chloride is absorbed, and the
excess is excreted along with other ions into the urine. Low levels of chloride are observed in the caseof
prolonged vomiting accompanied by the loss of hydrochloric acid(HCl), in some cases of metabolic acidosis in
which there is an increased accumulation of organic anions, in critical cases of Addison’sdisease, and in kidney
disease resulting in loss of salt.
Elevated levels of chloride are observed in metabolic acidosis associated with prolonged diarrhea and
with loss of sodium bicarbonate (NaHCO3), and in the case of renal tubular diseases in which there is a
decreased excretion of hydrogen ion (H+), which causes in turn a decrease in the reabsorption of bicarbonate
ion (HCO3–). Elevated levels of serum chloride are also implicated in certain cases of hyperparathyroidism.
Test Principle
Methodology: Ion-selective electrode diluted (Indirect)
Ion-selective electrodes for sodium, potassium, and chloride utilize membranes selective to each of these ions.
An electrical potential (voltage) is developed across the membranes between the reference and measuring
NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 39
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

electrodes in accordance with the Nernst equation. The voltage is compared to previously determined calibrator
voltages and converted into ion concentration.
Specimen Collection And Preparation
Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable.
Reagent Preparation

Ready to use.
Testing Procedure

Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test sodium or potassium or chloride accordingly. Put the sample-loaded
rack into the machine it starts running.
Calibration And Quality Control
Calibration is stable up to 24 hours and calibration is required with each change in reagent lot number. The
laboratory may choose any calibration interval up to 24 hours. The use of a particular calibration time interval is
dependent on individual laboratory policy or preference. Verify calibration curve with at least two levels of
controls according to the established quality control requirements for your laboratory. If control results fall
outside acceptable ranges, recalibration may be necessary. Two levels of controls (normal and abnormal) are to
be run every 24 hours. Two levels namely Bio-Rad L1 & L2 are to be used.
Reference range
Serum/Plasma
Sodium-136-145
Potassium-3.5-5.1
Chloride-98-107

Reference

1. US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030. Blood
borne Pathogens.
2. US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories,
5th ed. Washington,DC: US Government Printing Office, January 2007.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 40
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- CARBON DIOXIDE

Summary
The determination of serum carbon dioxide total (CO2) in conjunction with other clinical and
laboratory information is necessary for the evaluation of acid-base status. A high CO2 content may be
observedin compensated respiratory acidosis and metabolic alkalosis. A lowCO2 content may be observed in
compensated respiratory alkalosis and metabolic acidosis. Additional laboratory determinations will permit
differentiation between metabolic and respiratory conditions.
Test Principle
Methodology: PEP Carboxylase
Carbon dioxide, as bicarbonate (HCO3–), and phospho(enol)pyruvate(PEP) are converted to oxalacetate
and phosphate in the reaction catalyzed by phospho(enol)pyruvate carboxylase (PEPC). Malatedehydrogenase
(MDH) catalyzes the reduction of oxalacetate tomalate with the concomitant oxidation of reduced nicotinamide
adeninedinucleotide (NADH) analog.2 The resulting decrease in absorbance at404 nm is proportional to the
CO2 content in the sample.
Specimen Collection And Preparation
Suitable Sample
Serum, plasma are suitable samples.
Reagent Preparation
Carbon dioxide is supplied as a liquid, ready-to-use kit.
R1: ready to use.
Testing Procedure
Architect machine is in ready mode then give program, Choosing the rack number and give ID (12
digits) in position from 1st till 5th and select the test carbon dioxide. Put the sample-loaded rack into the
machine it starts running.
Calibration And Quality Control
Calibration is stable for approximately 14 days (336 hours) and is required with each change in reagent
lot number. Verify calibration curve with at least two levels of controls according to the established quality

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 41
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

control requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range
Serum/Plasma
Child: 20-28mEq/l
Adult: 22-29mEq/l
>60years: 23-31mEq/l
Reference
1.US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030.
Occupational Exposure toBloodborne Pathogens.
2.US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories.
HHS Publication (CDC),4th ed. Washington, DC: US Government Printing Office, May 1999.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 42
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- C-REACTIVE PROTEIN

Summary
C-reactive protein was discovered in 1930, and named for its reaction with the C-polysaccharide of the
pneumococcal cell wall. CRP is acyclic pentamer with an approximate molecular mass of 118 kDa. Each

subunit is composed of 206 amino acids. CRP is synthesized in the liver and released into the circulatory system
in response to proinflammatorystimuli, the strongest of which is interleukin-6 (IL-6). It is this positive response
to inflammation from which CRP derives its diagnostic utility and categorizes it with “acute phase reactants”.
CRP has been called the archetype of acute phase proteins due to its rapid (24 to 28 hours)and marked response
to a wide variety of inflammatory conditions and diseases. These stimuli include bacterial and fungal
components, as well as damaged cell membranes from injured tissue arising fromtrauma, arthritis, vasculitides,
and a spectrum of autoimmune processes. A reactant common to these stimuli is phosphocholine, which shows
calcium-dependent binding to CRP and is the major ligand. CRP

shares some of the immunological functions of IgG. It activates the classical complement pathway, binds to Fc
receptors, and acts as an opsonin. These CRP activities make it a critical component of

the innate immunological system that provides early defense against infectious agents. As stated by Volanakis,
the main biological function of CRP appears to be host defense against bacterial pathogens and

clearance of apoptotic and necrotic cells.1 CRP is clinically useful due to its 1,000-fold rise during an
inflammatory response, which provides a nonspecific indication that an inflammatory process is present. Its

concentration falls rapidly when the condition resolves, due to a short half-life (19 hours).

Test Principle

Methodology: Immunoturbidimetric
C-Reactive Protein is an in vitro diagnostic assay for the quantitative determination of CRP in human serum
and plasma. When an antigen-antibody reaction occurs between CRP in a sample andpolyclonal anti-C-reactive
protein antibody, which has been adsorbed to latex particles, agglutination results. This agglutination is detected

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 43
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

as an absorbance change, with the magnitude of the change being proportional to the quantity of CRP in the
sample. The actual concentration is then determined by interpolation from a calibration curve prepared from

Specimen Collection And Preparation

Suitable Sample
Serum, plasma are suitable samples. Heparin plasma is also acceptable.
Reagent Preparation

C-Reactive Protein is supplied as a liquid, ready-to-use kit.

R1: ready to use.
R2: ready to use.
Testing Procedure

Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test C-Reactive Protein. Put the sample-loaded rack into the machine it
starts running.
Calibration And Quality Control
Calibration is stable for approximately 30 days (720 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.
Reference range
Serum/Plasma
Adult -0.01 to 0.82
Reference

1. Volanakis JE. Human C-reactive protein: expression, structure, andfunction. Mol Immunol 2001;38:189–97.
2. Bienvenu J, Whicher JT, Aguzzi F. C-reactive protein, CRP. In:Ritchie RF, editor. Serum Proteins in Clinical
Medicine, Vol 1.AACC, 1996:7.01-1–7.01-5.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 44
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- RA FACTOR
Summary

Rheumatoid factor (RF) is an auto antibody against humanimmunoglobin G (IgG) commonly present at a high
concentration in sera of patients with certain conditions, particularly in patients with Rheumatoid arthritis. The
measurement of rheumatoid factor is useful in evaluating the diagnosis, effects of therapy, and prognosis of
diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren’s syndrome, cryoglobulinemia,
and chronic hepatopathy. This assay is designed to accurately and reproducibly measures serum rheumatoid
factor using latex agglutination.

Test Principle

Methodology:Immunoturbidimetric

Rheumatoid Factor is an in vitro diagnostic assay for the quantitative determination of rheumatoid factor in
human serum. The Rheumatoid Factor assay is latex enhanced immunoturbidimetric assay thatinvolves an
antigen-antibody reaction between rheumatoid factor in the sample and denatured human IgG, which has been
adsorbed to latex particles. The resulting agglutination is detected as an absorbance change (572 nm), with the
magnitude of the change being proportional to the quantity of rheumatoid factor in the sample. The actual
concentration is then determined by interpolation from a calibration curve prepared from calibrators of known
concentration.

Specimen Collection And Preparation

Suitable Sample

Serum, plasma are suitable samples.

Reagent Preparation

RA Factor is supplied as a liquid, ready-to-use kit.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 45
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

R1: ready to use.

R2: ready to use.

Testing Procedure

Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test RA Factor. Put the sample-loaded rack into the machine it starts
running.

Calibration And Quality Control

Calibration is stable for approximately 60 days (1,440 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/Plasma

Adult-<30 IU/ml.

Reference

1.US Department of Labor, Occupational Safety and HealthAdministration. 29 CFR Part 1910.1030.
Occupational Exposure toBloodborne Pathogens.

2.US Department of Health and Human Services. Biosafety inMicrobiological and Biomedical Laboratories.
HHS Publication (CDC),4th ed. Washington, DC: US Government Printing Office, May 1999.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 46
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- AMYLASE
Summary

Normal individuals have low but measurable serum and urine α-amylase activity, which is produced in the
pancreas and parotid glands. Measurement of α-amylase activity is of value in diagnosing Pancreatitis

and other pancreatic disorders which result in elevation of serum and urine α-amylase activity. Numerous
methods have been used for clinical analysis.

Test Principle

Methodology: CNPG3 Substrate

α-Amylase hydrolyzes the 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) to release 2-chloro-4-

nitrophenol (CPNP) and form2-chloro-4-nitrophenyl-α-D-maltoside (CNPG2), maltotriose, and glucose. The
rate of formation of the 2-chloro-4-nitrophenol can be detected spectrophotometrically at 404 nm to give a
direct measurement of α-amylase activity in the sample.

Specimen collection and preparation

Suitable Sample

Serum, plasma are suitable samples. Heparin plasma is also acceptable

Reagent Preparation

Amylase is supplied as a liquid, ready-to-use, two-reagent kit.

R1: Ready to use

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 47
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

Testing Procedure

Architect machine is ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Amylase .Put the sample loaded rack into the machine it starts
running.

Calibration and Quality control

Calibration is stable for approximately 19 days (456 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/Plasma

Newborn: 5 to 65

Adult: 25 to 125

70 years: 20 to 160

Reference

1. US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030.
Bloodborne Pathogens

2 US Department of Health and Human Services. Biosafety inMicrobiological and Biomedical Laboratories. 5th
ed. Washington,DC: US Government Printing Office, January 2007

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 48
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

SOP- IRON
Summary

Measurement of iron is used in the diagnosis and treatment of various iron anemias, iron overload, and iron
poisonings. Serum iron has been determined by several spectrophotometric methods, including the use of
FERROZINE, bathophenanthroline, and FERENE.1,2.

Test Principle

Methodology: FERENE without a protein removal step

The Iron assay utilizes an acidic media to release ferric iron fromtransferrin. The ferric iron is converted to the
ferrous form by the action of hydroxylamine hydrochloride. The released ferrous iron reacts with FERENE to
produce a colored iron-FERENE complex. The absorbance of the iron-FERENE complex is measured at 604
nm and is proportional to the concentration of iron present in the sample. Thiourea and detergent are added to
prevent copper interference and turbidity, respectively.

Specimen Collection And Preparation

Suitable Sample

Serum, plasma are suitable samples.

Reagent Preparation

Iron is supplied as a liquid, ready-to-use kit.

R1: ready to use.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 49
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

R2: ready to use.

Testing Procedure

Architect machine is in ready mode then give program, Choosing the rack number and give ID (12 digits) in
position from 1st till 5th and select the test Iron. Put the sample-loaded rack into the machine it starts running.

Calibration And Quality Control

Calibration is stable for approximately 27 days (648 hours) and is required with each change in reagent lot
number. Verify calibration with at least two levels of controls according to the established quality control
requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be
necessary. Two levels of controls (normal and abnormal) are to be run every 24 hours. Two levels namely Bio-
Rad L1 & L2 are to be used.

Reference range

Serum/Plasma

Adult, Male: 31 – 144 µg/dl.

Adult, Female: 25 –156 µg/dl.

Reference

1.Higgins T. Novel chromogen for serum iron determinations. ClinChem 1981; 27(9): 1619.

2.Artiss JD, Vinogradov S, Zak B. Spectrophotometric study of several sensitive reagents for serum iron. Clin
Biochem 1981; 14:311–5.

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 50
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01

 STANDARD OPERATING PROCEDURES (Sop’s)

DATE OF ISSUE:
Department: of Biochemistry

NABH/CDRL/LS/SOP/BIO 7/03/2022Ref:
Page 51
KGH/LS/SOP
Prepared by: Approved by: HOD Date of Issue:

Mrs.Deepa Dr.P.Pasupathi Ph.D., Version No: 01