factor assay and inhibitor testing

8/13/2019 Factor Assay and Inhibitor Testing

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Factor assay and inhibitor

testing


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Definition

The ability of various dilutions of the test

plasma to correct the APTT/PT of a

severely factor deficient plasma andcompare with similar dilutions of control

plasma.


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Reagents

Normal reference plasma : to draw std graph -

commercial (IL cal)/ in house (PNP, traceable to a

calibrator). Control plasma: Normal (IL control) & abnormal

(Dade P).

Test plasma.

Factor deficient plasma: in house/ commercial

immunodepleted.


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Reagents

Imidazole buffer (BBS/ Owrens veronal buffer

may be used; pH: 7.35-7.4).

CaCl2. APTT reagent.


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Manual factor assay

Step 1: Prepare dilutions of calibrator, test & controlplasma in duplicate.

3 plastic tubes: 1/10, 1/20, 1/40.

1sttube100 uL test plasma + 900 uL buffer.

2nd& 3rdtubes- add 500 uL buffer. Make doubling

dilutions1 in 20, 1 in 40 by transferring 500 uL

from each tube to the next.

Take 100 uL from each tube, transfer to glasstubes, add 100 uL factor def plasma to each-

incubate at 37 deg for 5 min, perform APTTs.


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Plot values on a semi log papercontrol and test.

Log scale reqd- biologic assays do not follow

linear pattern.


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50

60

70

80

90


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Extrapolate test values from control graph.

Multiply by dilution and calibration factor.

eg. In this case : 29% x 1 = 29 x 0.89 = 25.8%


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Automated assays

Machine reproduces manual technique, variables

involved in manual method are eliminated.

Calibrator is run only after any major servicing,change of reagent lot, buffer, def plasma.

Daily controlsN & Abn are run once.

MDAlow and high are possible.


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Inhibitor screen

Qualitative detection of inhibitors.

Factor VIII inhibitors are time dependant.

Principletest and control plasma are mixed andAPTT is checked immediately and after 1 & 2

hours of incubation.

FIX inhibitors are immediate acting, so 15 minincubation sufficient.


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Method

3 plastic tubes500 ul PNP, 500 ul test plasma,

250 PNP +250 ul test in 3rd.


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PNP TestTest +

PNP

1 hour

incubation

Incubated

mix

Fresh

mixPNP Test

2 hour

incubation

APTT (sec) of Incubatedfresh mix > 5 sec: Inhibitor +

APTTs of:


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Inhibitor assay

Quantitation based on amount of FVIII inactivated

by patients plasma in an incubation mixture under

specified conditions. Bethesda assay

New Oxford method

Nijmegen modification


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Bethesda assay

Bethesda unit = amount of inhibitor that would

inactivate half the FVIII activity in the incubation

mixture under specified test conditions.


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Procedure

Take 10 plastic tubes. Make serial dilutions of test

plasma in imidazole buffer from 1:2 to 1:1024.

Take 12glass tubes. Make doubling dilutions.

Incubate at 37 deg for 2 hours.

Perform factor VIII assays on all.


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1:2 1:4 1:8 1:16 1:32

1:64 1:128 1:256 1:512 1:1024

PNP+Buffer PNP+Undil test plasma


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Note the dilution that gave a residual factor VIII

assay closest to 50% eg.C:B95.8%

1:22.7%

1:43.7%

1:88.5%1:3217.1%

1:6433.9%

1:12852.8%

1:25667.1%1:51272.2%


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Correction for PNP value of FVIII :.

Residual FVIII activity = FVIII(pt) x 100

FVIII(PNP)52.8/95.8 x100 = 55.1

Correction for difference from 50% residual FVIII

is done and conversion to Bethesda unit factor.

128 x 0.85 = 108.8 BU.


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Acquired inhibitors

Allo antibodies, few auto antibodies follow simple

order kinetics.

Majority of autoantibodies follow complexkinetics.

factor assay and inhibitor testing

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