STANDARD OPERATING

PROCEDURE
(BIOCHEMISTRY – DIASYS
RESPONS – 910)
PREPARED AS PER ISO 15189:2012

COPY NO: MASTER COPY

HOLDER: QUALITY MANAGER

ISSUE NO: 04

ISSUE DATE: 09.03.2020

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Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY

AMENDMENT SHEET
Sl. Page Section/ Date of Amendment Reasons Signature Signature
No. No. Clause Amendment Made of
No. of QM Laboratory
Director
01 All All 09.03.2020 Issue No: 03 Change in
has been automated
withdrawn analyser
and issue no
04 is issued in
the system

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

TABLE OF CONTENTS
LAST
CHAPTER SL. REVISION
TITLE REVISION PAGE No.
No. No. NO
MADE
LAST
CHAPTER SL. REVISION
TITLE REVISION PAGE No.
No. No. NO
MADE
AMYLASE
18 00 00 2–4
I LDH
20 00 00 5-7

MACHINE OPERATION
II 00 00 1–1
PROCEDURE

III QUALITY CONTROL PROCEDURE 00 00 1–4

IV CALIBRATION PROCEDURE 00 00 1–1

SAFETY PRECAUTION
V 00 00 1–1

VI LIST OF CRITICAL ALERT VALUE 00 00 1–1

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NAME OF TEST: LDH

a) Purpose of
examination
Quantitative in vitro determination of lactate dehydrogenase (LDH) in serum
or plasma on DiaSys respons 910.

b) Principle of the Procedure used for Examination

LDH

L-Lactate + NAD+ Pyruvate + NADH + H+

Method
Optimized UV-test according to IFCC and DGKC

c) Performance characteristics
Test has a linearity range of 5 - 1200 U/L

d) Primary Sample System

Serum.

e) Patient preparation:
QMSP 15

f) Type of container & additives

Plain Vial.

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g) Required equipment and reagents

1. Diasys 2. Centrifuge, 3. Sample cups, 4.LDH reagent.

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.

i) Calibration procedure
Periodic hardware calibration is done by the manufacturer. Parameter
specific calibration with standards is done in the following cases:
1. L-J Chart shows rejection Multi QC rule.
2. Lot change of reagent.
3. EQAS result shows outliers.
All auxiliary equipments are calibrated as per NABL 112.

j) Procedural steps
Assay procedure is performed automatically by the instrument.
Operation of instrument
Sample is collected, processed as per requirement and then the examination
is carried out following the operating procedure as described in chapter –II.

k) Quality Control Procedure

For internal quality assessment commercial control is run daily.2
different levels i.e level 1 and level 2 are run daily. The laboratory
participates in Bio Rad monthly EQAS programme.

l) Interferences
No interference was observed by ascorbic acid up to 30 mg/dL, bilirubin up
to 40 mg/dL and lipemia up to 2000 mg/dL triglycerides. Hemolysis
interferes because LDH is released by erythrocytes.

m) Principle of Procedure for calculating result including, relevant, the

measurement uncertainty of measured quality values
Analyser automatically calculates the test result. CV% is calculated from L-J
Chart and MU% is calculated as CV% X 1.96.

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

n) Biological Reference intervals

Adult: 0 – 480 U/L

o) Reportable interval of examination results:

Regular: 8 hrs
Emergency: 2 hrs

p) Instruction for determining quantitative results when a result is not

within the measurement interval
Sample having LDH value more than 1200 U/L should be auto diluted and
re-run.

q) Alert/Critical values, where applicable

As described in chapter VI.

r) Laboratory clinical interpretation: Not done

s) Potential source of variability: N.A.

t) Reference:
1. Principle and procedure:
a. Kit Instruction.
b. John Bernard Henry, M.D.
2. Biological reference Interval :
a. Kit Literature.
b. Ref: Jacques Wallach, Md
Interpretation of diagnostic test 8th edition page
26-27. Lippincort.

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

NAME OF TEST: AMYLASE

a) Purpose of examination
Quantitative in-vitro determination of α-Amylase in serum or plasma in
diasys response-910

b) Principle of the Procedure used for Examination

Enzymatic photometric test, in which the substrate 4,6-ethylidene-(G7)-
pnitrophenyl-(G1)-α-D-maltoheptaoside (EPS-G7) is cleaved by α-amylases
into various fragments. These are further hydrolyzed in a second step by
α-glucosidase producing glucose and p-nitrophenol. The increase in
absorbance represents the total (pancreatic and salivary) amylase activity
in the sample.

5 EPS-G7 + 5 H2O α-Amylase 2 Ethylidene-G5 + 2 G2PNP

+ 2 Ethylidene-G4 + 2 G3PNP
+ Ethylidene-G3 + G4PNP

2 G2PNP + 2 G3PNP + G4PNP + 14 H2O α-Glucosidase 5 PNP + 14 G

(PNP = p-Nitrophenol, G =Glucose)

Method
ENZYMATIC PHOTOMETRIC

c) Performance characteristics
Test has a linearity range of 3-2000 U/L.

d) Primary Sample System

Serum.

e) Patient preparation:
QMSP 15

f) Type of container & additives

Plain Vial.

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

g) Required equipment and reagents

1. DIASYS Respons-910 2. Centrifuge 3. Sample cups 4. Micro pipettes of
variable volume 5. Micro tips 6. Amylase 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.

i) Calibration procedure
Periodic hardware calibration is done by the manufacturer. Parameter
specific calibration with standards is done in the following cases:
1. L-J Chart shows rejection Multi QC rule.
2. Lot change of reagent.
3. EQAS result shows outliers.
All auxiliary equipments are calibrated as per NABL 112.

j) Procedural steps
Assay procedure is performed automatically by the instrument.
Operation of instrument
Sample is collected, processed as per requirement and then the examination
is carried out following the operating procedure as described in chapter –II.

k) Quality Control Procedure

For internal quality assessment commercial control is run daily. 2 different
levels i.e level 1 and level 2 are run daily. The laboratory participates in Bio
Rad monthly EQAS programme.

l) Interferences
Icterus: Interference less than 10% up to 20 mg/dl bilirubin.
Hemolysis: Interference less than 10% up to 2.5 g/l haemoglobin.
Lipemia: Interference less than 5% up to 1000 mg/dl intralipid.

m) Principle of Procedure for calculating result including, relevant, the

measurement uncertainty of measured quality values
Analyser automatically calculates the test result. CV% is calculated from L-J
Chart and MU% is calculated as CV% X 1.96.
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Doc No. RKLS/SOP/01 SOP BIOCHEMISTRY CHAPTER - I

n) Biological Reference intervals

Serum / Plasma : 22 - 80 U/L
Urine : 42 - 321 U/L

o) Reportable interval of examination results:

Regular: 8 hrs
Emergency: 2 hrs

p) Instruction for determining quantitative results when a result is not

within the measurement interval
Sample having AMY value more than 2000 U/L should be auto diluted and
re-run.

q) Alert/Critical values, where applicable

As described in chapter VI.

r) Laboratory clinical interpretation: Not done

s) Potential source of variability

Demographic variations and diseased conditions.
It can reflect biliary or gastrointestinal disease and other upheavals.

t) Reference
1. Principle and procedure:
a. Kit Instruction.
b. John Bernard Henry, M.D.
2. Biological reference Interval :
a. Kit Literature.
b. Ref: Jacques Wallach, Md
Interpretation of diagnostic test 8th edition page
26-27. Lippincort.

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

MACHINE OPERATING PROCEDURE

1. Diasys Respons-910

START THE ANALYZER

GO FOR ROTOR BLANKING

CHECK REAGENT INFORMATION

CHECK WATER & WASTE CONTAINER

REQUIEST FOR CALIBRATION

RUN CONTROL

GO FOR ROUTINE SAMPLE RUNS

EVALUTE THE RESULTS IN DISPLAY

REPORT PRINT OUT

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

QUALITY CONTROL PROCEDURE:

Purpose:
Quality control (QC) is concerned with the analytical phase of QA. The purpose
of this document is to monitor the overall reliability and monitoring the validity
of laboratory results.
Scope:
All routine biochemical tests under the scope of Accreditation

RESPONSIBILITY: Consultant Pathologist

Following Procedure is followed in Biochemistry.

A. Regular use of QC material of renowned QC kit manufacturer
B. Participation in EQAS / inter-laboratory comparison
C. Retesting of retained sample
D. Correlations of results

A. Regular use of QC material of renowned QC kit manufacturer:

Laboratory runs QC as per Quality Control Plan (RKLS/FM/70) i.e. daily two level of control for all
the parameters under scope. Laboratory interprets QC results by using statistical technique such
as L-J chart. Where any parameter value is found to deviate Westgard Multi QC rules, cause is
identified & corrective action is taken. Consultant reviews it and records are maintained
accordingly.
Calculation of CV %: The standard deviation (SD) of a set of results divided by the mean result is
expressed as a percentage CV. In automated analyzer percentage CV is auto calculated along with
L-J chart.

GENERAL PROCEDURE AND TERMINOLOGY:

Running commercial control:

Diasys Respons-910
Two level controls are run once daily for all of the parameters under the scope of accreditation.

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PROCEDURE FOR HANDLING INTERNAL COMMERCIAL CONTROL:

 Receive the sample and confirm Lot No and Expiry Date with reference values specific to the
machine.
 Make a note of the storage temperature.
 Controls are stored at 2-8oC in specified Biochemistry Refrigerator
 Vials are kept at room temperature for a maximum period of 1 hour while running tests.
 Record sample validity period after opening or reconstitution.
 Prepare aliquots (if applicable) and store in recommended temperature.
 Internal Controls are run on a daily basis immediately after the specific machines are switched
on and in perfect running mode. Only authorized personnel in each department run controls.
 Values observed are recorded and plotted in L-J charts.
 Observe for any violation of Multi QC Deviation Rule and proceed accordingly.

INTERPRETATION OF QUALITY CONTROL DATA

QC Results are reviewed & if any of the parameters run out of 2SD or do not follow any of the MULTI
QC RULES described below, then a QC Level is run for the second time. If the results still do not fall
within the expected range, then the test parameter is calibrated, QC checked & samples run. All the
QC values are checked and filed and reviewed by the Consultant Pathologist.

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

The rules to follow when 1 level QC material are used:

Reject QC if:

13S: One control observation exceeding the mean ± 3s; primarily sensitive to random error.
22S: Two consecutive control observation exceeding the same mean plus 2 s or mean minus 2 s
limit; sensitive to systematic error.
10X: 10 consecutive control observations falling on one side of the mean (above or below, with no
other requirement on size of the deviations); sensitive to systematic error.
41s: Four consecutive values on either side of mean are within 1SD to 2SD (41S).

The rules to follow when 2 level QC material are used:

Reject QC if:
1.
Either QC value is outside 3 SD (1 3 S)
2.
Both QC value are outside 2 SD on the same side, but within 3 SD (2 2 S)
3.
Different between both QC value is > 4 SD i.e. one level QC is > 2 SD and other level QC is
<2SD (R 4 S)
4.
Ten consecutive value of the same level QC are >/< the mean, but within 2 SD (10x)
5.
Five consecutive value of one level QC and five consecutive value of another level QC are
>/< the mean but within 2 SD (10x).
6.
Four consecutive values on either side of mean are within 1SD to 2SD

Procedure to be followed when QC results are “out of control” (> ± 2 SD).

FOLLOW STEP WISE TILL QC RESULTS ARE “IN CONTROL”

1. Repeat with same aliquot of QC.
2. Repeat with new aliquot of QC.
3. Reconstitute new QC.
4. Calibrate with current calibration pack and same reagent.
5. Calibrate with new calibration pack and same reagent.
6. Check generation no. of slide/ lot no. Cartridge/ reagent expiry / on board reagent expiry.
7. Use new reagent.
8. Calibrate with new calibration pack and new reagent
9. Contact respective Application Specialist.

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

B. Participation in EQAS / Inter-Laboratory Comparison:

The laboratory participates in EQAS with Bio-Rad. Before testing of EQAS sample daily QC results
are reviewed, if results are found satisfactory then EQAS sample is tested. ILC is done with NABL
accredited labs once in a month.

C. Retesting of retained sample:

Retesting of the sample is done daily. Primary sample is chosen at a random and tested within 24
hrs. Consultant Pathologist reviews retesting results and record is maintained.

D. Correlations of results:
Different Tests when results indicate the same pathology are used to confirm test results when
necessary. Both inter and intradepartmental correlation is done. Pathologist reviews the record

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

CALIBRATION PROCEDURE

Diasys Respons-910
 Machine is calibrated by the manufacturer as and when required
 Individual parameter calibration is done in-house by calibrators having
international traceability.

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

SAFETY AND PRECAUTIONS PROCEDURE:

 Safety instructions of different instruments and chemicals given by manufacturers

are followed strictly.
 General safety precautions of biohazard are observed.
 Samples are handled with utmost care.
 Samples are handled wearing gloves, facemask and apron.
 Reagents and chemicals are kept according to the company instructions

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

List of Critical Alert Value

SL NO TEST DISCIPLINE TEST PARAMETRE CRITICAL VALUE

1. Biochemistry Amylase > 700 U/L

Ref: Wallsch. J interpretation of diagnostic test 7th edition page 29-31. Lippincort

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