Lab assays

Question 1

DRVVT

Answer 1

Direct FX activator, dependent on PL
Measure ratio patient to normal APTT
The measure amount of correction with addition of PL
Long with no correction = F def
Long with correction = LA

Question 2

PT

Answer 2

Prep PPP
Mix with TF (contains phospholipid)
Incubate immediateley 37deg with addition calcium 25mM
Measure time to clot

Question 3

APTT

Answer 3

Prep PPP
Incubate 2-10 min with PL and contact activator (e.g. Silica microbeads)
Add calcium
Measure time to clot

Question 4

X-match

Answer 4

Serum or plasma
Incubate with donor red cells and AHG
Spin
Look for agglutination

Question 5

G&S

Answer 5

Card for group (room temp, no AHG)
IAT based panel for ab screen

Question 6

PIFT

Answer 6

ELISA style assay
Panel platelets + serum
Wash
Secondary antibody + detection (flow)

Question 7

MAIPA

Answer 7

ELISA style
Glycoproteins with know binding sites immobilised on a plate
Incubate serum
Wash
2nd antibody + detection

Question 8

DAT

Answer 8

Column based
With AHG

Question 9

FBC

Question 10

F8 inhib screen

Answer 10

Test normal, patient and mixed plasma (incubated 50:50)
Incubate at 37deg for 120min
Quench in ice
Prepare a 2nd mix sample (immediate mix)
Do APTT on all 4
F8 def normal in incubated mix
Fast inhibitor prolonged in all tubes
Slow inhibitor prolonged in test and incubated mix

Question 11

Bethesda

Answer 11

1 BU neutralises 50% of F8 after 2 hour incubation at 37deg

Make serial doubling dilutions of patient plasma in buffer
Make control dilutions of reference plasma in same buffer
Incubate all for 2 hours at 37deg
Measure F8 activity with 1-stage APTT
Dilution that reduces FVIII to 50% gives 1xBUxtitre

Nijmegen modification - improves stability of F8
Protein concentration - increases sensitivity
Heat treatment - removes porcine F8

Question 12

TT

Answer 12

Prep PPP
Add low conc thrombin
Measure clot time
Measures time for fibrinogen to generate fibrin
Sensitive to e.g. heparin

Question 13

Clauss Fn

Answer 13

Prep PPP
Add high conc thrombin
Measure clot time
Measures time for fibrinogen to generate fibrin
Insensitive to e.g. heparin

Question 14

APTT F assays

Answer 14

Dilute patient and ref plasma in F def plasma
Do APTT on each
Compare dilutions with same APTT to calculate F level

2-stage = incubate first with XIa then measure activity of Xa wg chromogenic substrate