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Factor assay and inhibitor testing

Definition
The ability of various dilutions of the test plasma to correct the APTT/PT of a severely factor deficient plasma and compare with similar dilutions of control plasma.

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.

Reagents
Imidazole buffer (BBS/ Owrens veronal buffer may be used; pH: 7.35-7.4). CaCl2. APTT reagent.

Manual factor assay


Step 1: Prepare dilutions of calibrator, test & control plasma in duplicate. 3 plastic tubes: 1/10, 1/20, 1/40. 1st tube 100 uL test plasma + 900 uL buffer. 2nd & 3rd tubes- add 500 uL buffer. Make doubling dilutions 1 in 20, 1 in 40 by transferring 500 uL from each tube to the next. Take 100 uL from each tube, transfer to glass tubes, add 100 uL factor def plasma to eachincubate at 37 deg for 5 min, perform APTTs.

Plot values on a semi log paper control and test. Log scale reqd- biologic assays do not follow linear pattern.

90

80
70

60
50

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%

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 controls N & Abn are run once. MDA low and high are possible.

Inhibitor screen
Qualitative detection of inhibitors. Factor VIII inhibitors are time dependant. Principle test and control plasma are mixed and APTT is checked immediately and after 1 & 2 hours of incubation. FIX inhibitors are immediate acting, so 15 min incubation sufficient.

Method
3 plastic tubes 500 ul PNP, 500 ul test plasma, 250 PNP +250 ul test in 3rd.

APTTs of :
PNP Test Test + PNP

1 hour incubation
Incubated mix

PNP

Test

Fresh mix 2 hour incubation

APTT (sec) of Incubated fresh mix > 5 sec: Inhibitor +

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

Bethesda assay
Bethesda unit = amount of inhibitor that would inactivate half the FVIII activity in the incubation mixture under specified test conditions.

Procedure
Take 10 plastic tubes. Make serial dilutions of test plasma in imidazole buffer from 1:2 to 1:1024.
Take 12 glass tubes.

Make doubling dilutions. Incubate at 37 deg for 2 hours. Perform factor VIII assays on all.

PNP+Buffer

PNP+Undil test plasma

1:2

1:4

1:8

1:16

1:32

1:64

1:128

1:256

1:512

1:1024

Note the dilution that gave a residual factor VIII assay closest to 50% eg. C:B 95.8% 1:2 2.7% 1:4 3.7% 1:8 8.5% 1:32 17.1% 1:64 33.9% 1:128 52.8% 1:256 67.1% 1:512 72.2%

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.

Nijmegen modification
Factor VIII deficient plasma as diluent in place of buffer higher protein media for inhibitor to act. Other factors are provided undiluted. Buffered PNP is used- with pH adjusted to 7.4. Imidazole buffer 6.8g + 2ml PNP, adjust pH by adding 5N HCl. More sensitive than Bethesda for low titer inh. Done in lab when screen is neg or <10 s diff in screen.

Acquired inhibitors
Allo antibodies, few auto antibodies follow simple order kinetics. Majority of autoantibodies follow complex kinetics.