quality assurance activities in coagulation_ippt_97

8/12/2019 Quality Assurance Activities in Coagulation_IPPT_97

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Quality Activities inCoagulation Laboratory

Kiu Ling Ling

Application Manager

All Eights (M) SDN.BHD.


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Outline

1. Establish Control Range with commercial QC

material

2. Transference & Validation of reference interval

3. Calibration of PT-INR system with certified

plasma

4. Factor assay: constructing calibration curve

for accurate result


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Establish Control Range

with Commercial QCMaterial


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What

Why

When

How


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WHATis Internal Quality Control ?

Set of procedures undertaken by laboratory

staffs for the continuous monitoring of operationsand the results of the measurement in order to

decide whether the results are reliable enough to

be released.


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IQC

To monitor the

accuracy &precision of the

completeanalyticalprocess

To detectimmediateanalytical

errorsDay to Day

monitoring oftestperformance

quality

( long term

constancy )

WHYwe need IQC ?


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Control Material- Important

CharacteristicsSimilar to test specimens ( Matrix )

Can be liquid or freeze-dried ( lyophilized )

known concentration of analyte 2 or 3 levels of control ( normal & abnormal )

Values cover medical decision points

Stability up to at least 8 hoursQC material must be tested in the same

manners as patient sample


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To Establish Control Range

Collecting Control data

Perform control repeatedly (minimum 20times) with testing spread over at least 10section

Calculate Mean , Standard Deviation (SD)

Determine control limit ( Mean 2SD )

Plotting Levey-Jennings chart

- Manually on graph paper

- MS Excel Software

- Built in quality software in coagulationanalyzer


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How to Create Levey-Jennings Graph


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APTT normal control data

1. 23.0 sec

2. 22.0 sec

3. 22.5 sec

4. 23.2 sec

5. 22.2 sec6. 22.5 sec

7. 22.8 sec

8. 23.3 sec

9. 22.5 sec10. 23.1 sec

11. 22.4 sec

12. 22.6 sec

13. 23.0 sec

14. 23.0 sec

15. 22.8 sec

16. 22.5 sec

17. 23.0 sec

18. 23.0 sec

19. 22.9 sec20. 22.5 sec

Step 1

Calculate MEAN

Mean = Sum ( X1 + X2 +.X20)

Total Number of Data

(N)

MEAN = 454.8 / 20

= 22.7 sec

Step 2

Calculate SD

SD = 0.4 secSum = 454.8 sec


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Step 3

Set control limit In Gaussian Distribution :

68.3% of data is within 1.0 SD

95.5% of data is within 2.0

SD

99.7% of data is within 3.0 SD

Control limit for APTT

= Mean 2SD

= 21.9 23.5 sec


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Step 4: Plotting LJ graph

MEAN

+1SD

+2SD

-1SD

-2SD

-3SD

+3SD

22.7

23.1

23.5

23.9

22.3

21.9

21.5


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CLSI C28-A3:Define, Establishing, and

Verifying ReferenceIntervals in the Clinical

Laboratory

3rd

Edition


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Reference Interval Minimum 120 healthy donors ( CLSI 28-A3)

Criteria of donor selection

equal number of sex

preferred 1650 years old no under any anticoagulant therapy and

medication

collect sample over few daysday-to-day variation

Normal Range = Mean 2SD


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Reference Interval Establishment


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Local PT/INR System Calibration

Norma

lRange:Exam

ple

ID PT ( sec) APTT (sec) Fib (g/l) TT (Sec)

1820425 13.4 39.7 3.7 15.8

1712056 13.2 41.0 3.0 15.0

1712084 13.8 41.9 2.7 15.9

1820420 13.5 40.5 3.1 15.9

1820421 12.8 33.2 3.0 16.2

1712088 13.4 37.6 3.6 17.2

1820422 13.0 35.6 3.4 16.3

1820369 13.5 37.9 3.1 16.5

1712087 13.5 41.7 3.1 16.6

1712086 13.0 39.1 3.0 16.6

1712085 13.1 33.4 3.3 16.1

1822001 13.0 41.2 3.0 17.5

D14 12.8 37.8 2.9 15.7

D15 13.5 35.6 3.2 16.0

D16 13.1 38.2 2.6 15.8

D17 12.8 40.8 3.3 17.4

D18 13.6 41.0 3.4 15.9

D19 13.0 36.0 2.4 16.4

D20 12.6 31.7 2.3 17.0

D21 13.6 35.6 2.7 15.8

D22 13.0 33.7 2.7 17.4

D23 13.4 33.5 2.3 17.2

D24 13.2 36.9 2.6 17.0

D25 13.3 33.8 3.0 17.3

D26 12.7 35.0 3.5 15.4

Mean 13.7 37.2 3.0 16.4

SD 0.4 3.1 0.4 0.7

Normal Range

Mean2SD 12.513.9 sec 30.943.4 sec 2.23.7g/l 15.017.8 sec


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Challenge in Determination of

Reference IntervalTo determine reliability of reference

interval (RI) costly & heavy task

minimum 120 healthy subjects needed

Difficulty in collecting sufficient number ofqualified reference individuals

Transfer of reference range from manufactureror other laboratory


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Transference of RI

If Laboratory has previously established a

RI for its own population

simple comparison between two analytical system

( new & old ) could be sufficient

Advantages

no need to collect sample from referenceindividuals

can use existing patients samples


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Transference of RI

If completely comparable ( Excellentcorrelation coefficient , small slope bias, smallintercept, comparable range of value )

y = 1.004x0.628, r2 = 0.990 Assume the RI for the current method is 50150

when x = 50, y = 1.004 (50)0.628 = 50

When x = 150, y = 1.004 (150)0.628 = 150

New RI still remain 50150

Values are completely comparable


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Transference of RI

y = 1.57x0.832, r2 = 0.990 Assume the RI for the current method is 50150

when x = 50, y = 1.57 (50)0.832 = 78

When x = 150, y = 1.57 (150)0.832 = 235

New RI transforms to 78 - 235

Highly correlated but one showed higher or lowerbias

result

Strongly encourages to validate the RI with small

sample ( n=20 ) of reference individual


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Transference of RI

same or comparable analytical system

from another lab or manufacturer package

insert

Different population 3 validation method proposed:

(1) Subjective assessment

(2) Validation with small number of reference

individuals ( eg. N = 20 )

(3) Evaluation with large number of reference

individuals ( N=60)


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1. Subjective Assessment

Transfer of RI without validation study

carefully inspection of all the pertinent

factors of original appropriate reference

value study

reference population demographic/ geographic,pre-analytical & analytical procedure, method of

determine RI


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2.Validation with Small No. of Reference Individuals ( n = 20 )


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3. Evaluate with Large No. of Reference

Individuals ( n = 60 )

Methodology similar to validation with

n= 20Advantage :

increasing power for observing statistical

different between two sets reference specimens


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CLSI H54-A:

Procedure for Validation ofINR and Local Calibrationof PT/INR Systems


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PT INR System Verification

PT INR Mathematic conversion of PT result based on

the responsiveness of the thromboplastine

reagent used in the assay

Monitoring of Oral Anticoagulant therapies

INR = Patient PT ( seconds )

GMNPT ( Seconds )

ISI


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ISI

Quantitative measure/ mathematic

indicator of the responsiveness of PT

system to the defect induced by VKA

therapy compare to primary WHO

international Reference Preparation (IRP )

ISI , responsive to VKA induced defect

Thromboplastine / instrument specific ISI

Generic ISI


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IF reagent / instrument specific ISIis

used

ISI verification should be perform

IF ISI verification fail Local ISI calibration

IF generic ISIis used

ISI verification is MANDATORY Local ISI calibration is strongly recommended


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PT INR System Verification Change of reagent & reagent lot number

Change of instrument, following major repair

Unexplained major discrepancy of EQA result

After PT/INR calibration

At least once per year if not major change Mandatory if generic ISI is used

When

Minimum 3 INR certified plasma(INR 1.54.5 ) & 1 NPP

Commercial plasma calibration set( calibrated against WHO standard )

Thromboplastine reagent

What


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Reconstitute set of calibration

plasmas

( 4 vialsA : INR 1.0 ; B: INR 2.0C: INR 3.0 ; D: INR 4.0 )

Aliquot each vial of calibration plasma into 3 microtubes

for day to day variation assessment

A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3


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A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3

DAY 3

Frozen at -70 CPerform PT assay

(Duplicate)

DAY 2

Frozen at -70 CPerform PT assay

(Duplicate)

DAY 1Perform PT assay

(Duplicate)

Calculate the Mean of PT seconds ( 3 days duplicate ) for

each calibration level ( A, B, C & D)Calculate the INR of each calibration plasma

( reagent /instrument specific ISI & local GMNPT )

Compare INR obtained with INR value from product insert


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Compare INR obtained with INR value from product

insert

Different 15 %

( one level of calibration

plasma )

FAILLocal PT/INR calibrationDifferent certified plasma used

for verification

Preferably from different

manufacturer

if from same manufacturer,

different

lot must be use

Verification of local ISI

( obtained from calibration graph

)

Test system with

different thromboplastin

local PT/INR system

calibration with diff set of

CP

Contact manufacturer

for assistance

FAIL

AGAIN

Different < 15 %

( each calibration plasma )

Manufacturer assigned ISI

is valid for local PT INR

reporting


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ExampleThromboplastin Certified Calibration

Plasma

Name STA Neoplastine Cl Plus AK-Calibrant

Lot Number 105513 0U03B00

Expiry Date 2011-10 2011-12-31

ISI 1.33 -

AK

Calibrant

Day 1 Day 2 Day 3 Mean

A 13.1 13.3 13.5 13.7 13.3 13.0 13.3

B 23.5 24.0 25.1 24.6 24.0 23.7 24.2

C 32.4 33.4 34.4 34.4 34.1 35.1 34.0

D 45.9 46.7 50.0 49.9 46.7 46.5 47.6


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AK Calibrant INR target

value

( from flyer )

INR acceptable

range

( 15 % from

target value )

Mean PT

( seconds )

Local

generated INR

A 1.03 0.881.18 13.3 1.06

B 2.29 1.952.63 24.2 2.35

C 3.43 2.92

3.94 34.0 3.71

D 5.31 4.516.11 47.6 5.80

ISI = 1.33

GMNPT = 12.7 seconds Manufacturer assigned ISI is

valid for local PT INR

reporting


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IF ISI Verification FAIL

Local ISI Calibration is

MANDATORY!!


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Local ISI Calibration

Reference method:

20 normal plasma + 60 stabilized warfarinised

samples

Assay for PT

Plot Log PT ( y-axis ) versus Log assigned INR ( x-

axis )

Determine local ISI ( 1/Slope )

Only if manufacturer do

not provide local ISI

calculation service

i d


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Assigned INR

( From flyer )

Mean PT

( from 3 days

replicate )

1.03 13.3

2.29 24.23.43 34.0

5.31 5.31

Log INR Log PT

0.013 1.124

0.360 1.384

0.535 1.531

0.725 1.678

y = 0.779x + 1.110R = 0.999

0.600

0.800

1.000

1.200

1.400

1.600

1.800

0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800

Y = 0.4

Local MNPT = y intercept at X = 0

= 1.110

= anti-log ( 1.110 )

= 12.9 seconds

INR cal plasma Method:

1. measure PT

2. calculate log10 of PT and INR

3. Plot the values

4. Draw line of best fit

5. Calculate Slope & ISI

Slope =change in Y/change in X

= 0.4/0.51

= 0.784 ( 0.779 frm formula equation)

Local ISI = 1 / Slope

= 1 / 0.784 ( 1/0.779 )

= 1.275 ( 1.28 )

Log PT

Example:


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Local MNPT &

ISI ( frommanufacturer )

Mean PT from

3 daysreplicate

PT INR

(reagent/

instrumentspecific ) from

flyer

No Manual

calculation of

local ISI & MNPT

Example:


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After Local ISI Calibration..

Verification should be carry out on local

calculated ISI

IF verification pass

Local calculated ISI is valid for PT INRreporting

IF verification FAIL again

Try with different thromboplastine ( reagent )

Repeat the ISI calibration with another set of

CP

Call manufacturer for assisstance


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Factor Assay:

Constructing CalibrationCurve for accurate result


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Factor Assay: Test Principle

Factor activities of plasma sample is

measured by its ability to correct the

prolonged clotting time of factor deficient

plasma

The APTTs or PTs mixtures of diluted test

plasma and factor deficient plasma areinversely proportional to the concentration

of the factor in the test plasma mixtures


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Factor Assay Principle

Diluted sample 100l (calibrator, control or patient )

Factor deficient plasma 100l

Test reagent (PT 200l or APTT 100l and CaCl2 100l)

Measure Clotting of PT or APTT

Wh d d ?


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What do you need ?

PT or APTT reagents

- PT ( Extrinsic : FVII )

- APTT ( Intrinsic : FVIII, FIX,FXI,

FXII )

Factor Deficient Plasma

- < 1 % of particular coagulation

factor- >50 % of all other coagulationfactors

- from individual with severecongenital factor deficiency ( factor

inhibitor & LA free )- May also prepared by in vitroimmunodepletion

Reference Plasma

- At least 20 healthy donors

- Should have 100 20 % coagulantactivities for all coagulation factors

- Calibrated against WHO standardor a secondary standard

- Commercial

Quality Control

- Normal control plasma- Abnormal control plasma

- Should be assayed witheach batch of test


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Preparation of Calibration curve

Two types of reference curve:

1. Extended curve To establish the range of linearity for the labs specific

analytical system

Change of reagent lot, reference plasma or instrument

2. Working curve to validate the method of that run

Used as analytic reference against which test sample aremeasured

Each run of assay

Fewer dilutions

( limited within linear portion of extended curve )


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Extended Standard Curve

Reconstitute Reference Plasma ( follow product insert

)

Prepare dilution of reference plasma

Test each dilution standard for PT or

APTT

Add Factor deficient plasma to each diluted standard ( 1 :

1 )

At least 7 dilution ( 1:5 , 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 )

Plot PT or APTT results on Y-axis against dilution on X-

axislo -lo a er


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Reference Plasma Dilution

Linear range

( 1:5 to 1:160 )

100 %50 %25 %12.5%6.25

%

3.13

%


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Working Standard CurveReconstitute Reference Plasma

(Same lot as used in extended reference curve )

Prepare dilution of reference plasma

Test each dilution standard for PT or

APTT

Add Factor deficient plasma to each diluted standard ( 1 :

1 )

At least 3 dilutions which fall within the linear range

Plot PT or APTT results on Y-axis against Factor activity

on X-axis ( log-log paper )


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79.0

70.3

63.5

57.4

13 25 50 100

Concentration %

Clotting Time, sec

Log( c) = ( -6.531* Log(t) ) + 13.478

r = - 0.999


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79.0

70.3

63.5

57.4

13 25 50 100

Concentration %

Clotting Time, sec

Log( c) = ( -6.531* Log(t) ) + 13.478

r = - 0.999

Reference Curve: Validation Criteria

R > 0.99

Interpolate Vs Standardvalues < 10 % different

Within control range

Duplicate, 10%

different tolerance


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Raw Data Target Range

** Based on 100% unicalibrator value


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How to look at raw data ?

1.000


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How to look at raw data ?

Good curve

Bad curve ( short raw data)Bad curve ( long raw data)


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Optimization of Standard Curve

Calibrator & reagent preparation

Fresh dH2O or diluents

Stabilize time as recommended by

manufacturer

Do not use beyond stability calibrator or

reagent

Automation

Perfect washing system

Syringe is free from bubble


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Optimization of Standard Curve

Manual

standard plasma dilution preparation

Plotting standard curve:

reference plasma values

data points plotting


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Thank You !

quality assurance activities in coagulation_ippt_97

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