R. K.

LIFE SERVICES PRIVATE LIMITED

(Department of Laboratory Medicine)

STANDARD OPERATING
PROCEDURE FOR CLINICAL
BIOCHEMISTRY
(Prepared As per ISO 15189: 2012)

COPY: Master

HOLDER: Quality manager

ISSUE NO: 04

ISSUE DATE: 09.03.2020

 R. K. LIFE SERVICES PRIVATE LIMITED
(Department of Laboratory Medicine)

Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY

AMENDMENT SHEET
Signature
Section/
Page Date of Amendment Signature of
Sr. No. Clause Reasons
No. Amendment Made of QM Laboratory
No.
Director

Issue No:03
has been Major
withdrawn changes in
01 All All 09.03.2020 and issue no IQC policy
04 is issued and
in the procedures
system

Issue No.:04 Issue Date: 09.03.2020 Prepared By: Copy No. Page 1 of 1

Rev. No.:00 Rev. Date: Nil Approved By: Issued By

 R. K. LIFE SERVICES PRIVATE LIMITED
(Department of Laboratory Medicine)

Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY CHAPTER - I

ESTIMATION OF HbA1C

a. Purpose of examination
Estimation of HbA1C

b. Principle of the Procedure used for Examination

High Performance Liquid Chromatography (HPLC)

c. Performance specifications: 3.8 – 18.5% Hb

d. Primary Sample System

EDTA Blood.

e. Patient preparation:
QMSP 15

f. Type of container & additives

EDTA vials

g. Required equipment and reagents

1. Bio-Rad D-10. 2. Test Tube Racks 3. 10 x 17mm Test Tubes 4. Variable Micro Pipettes 0-100ul and 0-1000 ul.
5. Micro tips 7. HbA1C System Kit

h. Environmental and safety controls

1. Temp: 200C-280C
2. Humidity: not more than 80% -90%
3. Dust free.
Sample storage:
The sample is stored at 2 – 8ºC for 1 day.
Safety and precautions procedure is described in chapter-II

i. Calibration procedure All instruments are calibrated as per NABL 112 requirements. Biorad D10 is calibrated
annually, by manufacturer.

j. Procedural Steps
Procedure for operation of the instrument Biorad D-10 is described in the operating manual.

k. Quality Control Procedure

Commercial control two levels are run daily before testing patient samples. EQAS participation with BIORAD is
done.

l. Interferences
A. Labile A1C – To test the level of interference, patient specimen pools with normal and diabetic levels of A1C
were split into aliquots. These aliquots were supplemented with an addition of a glucose stock solution which
resulted in a glucose concentration of 200 – 700 mg / dl. Sample having other than normal haemoglobin variant
can interfear and give low value .
Normal Patient Diabetic Patient
% Labile A1C % A1C % Labile A1C % A1C
1.3 6.0 1.7 9.8
Issue No. 04 Issue Date: 09.03.2020 Prepared By: Copy No. Page 1 of 3

Rev. No.: 00 Rev. Date: Nil Approved By: Issued By

 R. K. LIFE SERVICES PRIVATE LIMITED
(Department of Laboratory Medicine)

Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY CHAPTER - I

4.0 6.1 4.6 9.7

B. Lipemic Samples -
Triglyceride Levels (mg/dl) Normal Patient % A1C Diabetic Patient % A1C
101 5.1 9.4
1500 5.0 9.7
3000 5.0 9.7
4500 5.0 9.7
568 5.0 9.6

C. Icterus –
Bilirubin Concentration (mg/dl) Normal Patient Diabetic Patient
% A 1C % A1C
0 5.9 10.2
20 5.9 10.2

D. Hemoglobin F (HbF) –
% HbF Spikes Normal Patient Diabetic Patient
% A1C % A1C
0 5.1 8.5
5 5.1 8.6
10 5.3 8.7

E. Carbamylated Hemoglobin –

Normal Patient Diabetic Patient

% Carbamylated % A1C % Carbamylated % A1C
0.0 5.0 0.0 9.6

2.0 4.9 2.2 9.2

(Ref.: - Kit Literature)

 Icterus, as indicated by bilirubin concentrations up to 20 mg/dL, does not interfere with the assay.
 Lipemia, as indicated by triglyceride concentrations up to 5680 mg/dL, does not interfere with the assay.
 Hemoglobin F concentrations up to 10% do not interfere with the assay.
 Labile A1c (L A1c/CHb-1) concentration up to 4% do not interfere with the assay.
 Carbamylated hemoglobin (L A1c/CHb-2) concentration up to 3.5% do not interfere with the assay.

Limitation
HbA1c values determined using the D-10 Hemoglobin A1c program for HbS trait and HbC trait specimens showed
no clinically significant difference from values determines by an NGSP certified boronate affinity method. In the
rare homozygous forms (SS or CC), there is no HbA present; no HbA1c value can be determined.
Other abnormal hemoglobin variants have not been evaluated on the D-10 Hemoglobin A1c program. For the
positive confirmation of any particular hemoglobin variant, alternative separation methods are required.

Issue No. 04 Issue Date: 09.03.2020 Prepared By: Copy No. Page 2 of 3

Rev. No.: 00 Rev. Date: Nil Approved By: Issued By

 R. K. LIFE SERVICES PRIVATE LIMITED
(Department of Laboratory Medicine)

Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY CHAPTER - I

m) Principle of Procedure for calculating result including, relevant, the measurement uncertainty of
measured quality values
Instrument automatically calculates the result.CV% is calculated from internal QC data. MU% is calculated as CV%
X 1.96.

n. Biological Reference Interval

Normal - <5.7%
Prediabetes 5.7% - 6.4%
Diabetes ≥ 6.5%

o. Reportable Interval: Normal: 6 hours

Urgent: 2 hours

p. Instruction for determining quantitative results when a result is not within the measurement interval
Not Applicable

q. Critical Alert Value: not applicable.

r. Laboratory Interpretation: as explained in biological reference interval.

s. Potential source of variability

Demographic variations and wrong sampling, transcription error.

t. Reference: Kit Literature

Biological Reference Interval: ADA Guidelines

Issue No. 04 Issue Date: 09.03.2020 Prepared By: Copy No. Page 3 of 3

Rev. No.: 00 Rev. Date: Nil Approved By: Issued By

 R. K. LIFE SERVICES PRIVATE LIMITED
(Department of Laboratory Medicine)

Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY CHAPTER - II

SAFETY AND PRECAUTIONS PROCEDURE:

 Avoid direct contact with blood/any test material.

 Direct contact with any corrosive reagent.

 Disposable of all waste material should be in accordance with local guideline.

WARNING AND PRECAUTIONS

For in vitro diagnostic use

Reagents: Store as per instruction in the product insert..
 Follow universal precautions, and handle all components as if capable of transmitting
infectious agents. Source materials derived from human blood were tested and found
nonreactive for syphills; for antibodies to HIV 1 and 2; for hepatitis B surface antigens; and
for antibodies to hepatitis C
 Sodium-azide, at concentrations less than 0.1 g/dL, has been added as a preservative. On
disposal flush with large volumes of water to prevent the buildup of potentially explosive
metal azides in lead and copper plumbing.
 Some kits contain products of human origin. No known analysis method can totally
guarantee the absence of transmissible pathologenic agents. It is therefore recommended
that these products be treated as potentially infectious and handled observing the usual
safety precautions
 Do not use the SPRs if the pouch is pierced.
 Do not use visibly deteriorated STs.
 Do not use reagents after the expiration date indicated on the label.
 Do not interchange components between different kits.
 When using the kit check that the reagent solutions are clear. Any cloudiness or order is
indicative of contamination.
 Avoid cross-contamination between serum specimens.

Issue No. 04 Issue Date: 09.03.2020 Issued By: Copy No. Page 1

Rev. No.: 00 Rev. Date: Nil Approved By: Prepared By:

 R. K. LIFE SERVICES PRIVATE LIMITED
(Department of Laboratory Medicine)

Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY CHAPTER - II

 Avoid cross-contamination between conjugate and substrate.

 Do not use the kit after the expiration date.
 Treat all specimens and kit reagents as potentially infectious. Although the human sera
utilized in preparing the kit reagents have been tested and found to be negative to Hepatitis
B and negative to HIV, there is no test method available that can offer complete assurance
that hepatitis B virus, HIV or other infectious agents and absents should be handled at the
Biosafety Level 2 as recommended by the Centers for Disease Cobtrol/U.S. Institutes of
Health publication “Biosafety in Microbiological and Biomedical Laboratoris”, 1984.
 Never pipette by mouth, and use gloves when handling human blood or serum specimens
and other potentially infectious materials.
 The use of disposable glass or plastic-ware is recommended in order to avoid contamination.
 The wash solutions and reagents utilized in the assay should be discarded into a disinfectant
solution such as sodium hypochlorite (i.e. 50 ml household bleach in 950 ml water) before
disposal. Other materials should be treated as biohazard waste and also disposed according
to recommended procedure.

Issue No. 04 Issue Date: 09.03.2020 Issued By: Copy No. Page 2

Rev. No.: 00 Rev. Date: Nil Approved By: Prepared By: