Lab in APS - Confusion over consensus

Dr Vandana KamathChristian Medical College

Vellore

LA testing

Tests Case 1 Case 2 Case 3 History 34 year old

-H/o recurrent abortions

50 year old gentleman during a routine preoperativework up

24 year young man with history of DVT – now on Warfarin

APTT(reference range: 24-32secs)

60 secs 30 secs 67secs

Actin FS (reference range: 22-32secs)

30 secs - 43 secs

Ratio 2 1.5PT(reference range:9-13.5secs /INR

- 49.4/2.8 32secs/2.8

Results LA detected

? Factor deficiency F II, VII,IX,X – in normal range

LA detected

Tests Case 1

History h/o recurrent abortions

APTT 60 secs

Actin FS 30 secs

Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)

1. Prolongation of phospholipid dependant clotting test time .

3. Correction in the presence of excess phospholipids

Tests Case 1 History h/o recurrent abortions

APTT( highly sensitive) 60 secs

Mix (1/2 pt+1/2 control) 50 secs

Actin FS(less sensitive) 30 secs

Result Markedly positive LA

Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)

2. Failure to correct the prolonged clot time on mixing with normal control plasma and repeating the test.

4. Exclusion of coagulopathies.

Case- 2 Routine pre-operative work up

APTT 30secs

PT/ INR(ISI- 1 )

49.4/2.8

? Factor deficiency

F II, VII,IX,X levels with normal range.

Case- 2 Routine pre-operative work up

APTT (reference range: 24- 30secs)

30secs

PT/ INR(ISI- 1 )Reference range : (9-13.5secs)

49.4/2.8

F II, VII,IX,X levels with normal range.

PT -1 mix with control plasma 46.2secs

? inhibitor

Case- 2 Routine pre-operative work up

APTT 30secs

PT-1/ INR(ISI- 1 )

49.4/2.8

PT -1 mix with control plasma 46.2secs

F II, VII,IX,X levels with normal range.PT -2 (Rabbit brain thromboplastin)

13.4secs

Result LA specific recombinant thromboplastin

Gamma carboxylation of Gla by Vit K --- increasing Negative Charge

VII

IX

X

II

Increased affinity for calcium providing nett strong positive charge..

VII

Ca Ca Ca+IX

Ca Ca Ca+ X

Ca Ca Ca+ II

Ca Ca Ca+

VII

Ca Ca Ca+PE PI PS

IX

Ca Ca Ca+PE PI PS

X

Ca Ca Ca+PE PI PS

II

Ca Ca Ca+PE PI PS

..to bind anionic

phospholipids.

..on the surface of activated platelets

Sensitivity of PT reagentsISI: International sensitivity index.

ISI = 1/ sensitivity

INR: International normalised ratio.

MNPT: Mean normal pooled plasma.

INR = (PT of patient/ MNPT)ISI.

PT- 1 (ISI- 1) more sensitive.

PT-2 (Rabbit brain thromboplastin) (ISI-1.8)- less sensitive.

LA testing

Tests Case 3 History Patient on Warfarin

dRVVT (screen) 67 secs

dRVVT(confirm) 43 secs

Screen/confirm 1.56

PT-1 /INR(ISI -1)

32 secs/ 2.8

PT-2/INR(ISI-1.8)

20.7secs/2.8

PT-1/PT-2 1.56

Result LA detected

LA testing Tests Case 3

History Patient on Warfarin

dRVVT (screen) 67secs

Mixing study 32 secs

dRVVT (confirm) 43secs

Screen/confirm 1.5

PT-1 /INR(ISI -1)

32 secs/ 2.8

PT-2/INR(ISI-1.8)

20.7secs/2.8

Result (referral lab) LA not detected

Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)

1. Prolongation of phospholipid dependant clotting test time .

2. Failure to correct the prolonged clot time on mixing with normal control plasma and repeating the test.

3. Correction in the presence of excess phospholipids

4. Exclusion of coagulopathies.

Mixing studies

Reporting a prolonged or a positive screening tests (either APTT or dRVVT) as lupus anticoagulant without mixing studies which is the only way to demonstrate the inhibition or the anticoagulant effect of Lupus anticoagulant is unethical and against the standard as it reports a lot of false positives consistently.

Indications of LA testingPart of all thrombotic work ups.An unexpectedly prolonged screening APTT

may also trigger an LA investigation.

Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)

1. Prolongation of phospholipid dependant clotting test time .

2. Failure to correct the prolonged clot time on mixing with normal control plasma and repeating the test.

3. Correction in the presence of excess phospholipids

4. Exclusion of coagulopathies.

LA - SSC-ISTH consensus criteriaNo single PL dependent clotting test is 100%

sensitive to detect LARecommended to perform at least one

additional screening clotting test Category 1 Screening test - activation of

Intrinsic pathway – aPTT or KCTCategory 2 Screening test - direct activation

of clotting – dRVVT or dPT (TTI)Confirmatory tests – dRVVT kit comes with

extra phospholipid. Few aPTT reagents – hexagonal PL.

Simple Confirmatory for aPTT, dPT, and KCT make PRP and Freeze thaw to make platelet phospholipid available – then do these tests.

QC – reference range to be established

Laboratory tests should employ A detection or screening stage

(prolongation of the clotting time) – SCREENING TESTS – reduced levels of PL.

• Failure of correction of the prolongation when normal plasma is added in order to exclude factor deficiency as the cause of the prolongation – MIXING STUDIES.

• A confirmation stage showing that the prolongation is phospholipid dependent, for example by showing that addition of excess phospholipid corrects the clotting time – CONFIRMATORY TESTS

Mixing/Correction Studies

Done when PT/APTT are prolongedMixing studies :- Mix equal volumes of test

(abnormal) plasma and control pool plasma (where all factors are present in normal quantity) and repeat the test.

Rationale: - if the prolongation in time is due to deficiency of

factor (s) in the test plasma, normal plasma will provide the deficient factor when they are mixed correcting the prolonged time

- if the prolongation in time is due to an inhibitor (antibody to factor or heparin) the normal plasma will also be inhibited and the prolonged time will remain prolonged

Correction of time: Deficiency of factorNo correction: Inhibitor

No correctionWhatever caused the abnormal

(prolonged) timing is also causing an abnormality to normal plasma after the patients plasma was mixed in it.

- Heparin - Lupus anticoagulant

(antiphospholipid antibody) - Factor Inhibitor ( antibodies to

Clotting factors) - FDP/D-Dimer

Correction StudiesDirect interpretation to therapyPatient who is bleeding with a

prolongation in plasma clotting testsCorrection of the time by mixing studies

in which the normal plasma supplied the deficient factor will directly mean that if you give (transfuse) the patient - normal plasma (FFP) his clotting defect, that lead to the prolongation of the time, will get corrected and the bleeding will stop.

Non Correction of time – No benefit of transfusing FFP. Use safer productsHeparin – use ProtaminInhibitor – other agents

Standard Mixing studies – will miss a FVIII inhibitor

APTT – One Month Statistics

aPTT by SynthASiL (N=7174)

27.5% Prolonged aPTT (n=1964)

2nd line aPTT: Actin FS

80% Normal aPTT (n=1460)

72.5% Normal aPTT (n=5183)

Only 7 % remains to be sorted (n=504)

29

APTT STA – PTT A

Normal (within reference range)

Release report withReference range.

APTT Abnormal (more than upper limit of ref. range)

Normal(within ref. range)

Release APTT FS with its ref. range

Abnormal-do APTT mix with STA – PTT A

Release APTT STA-PTT A result with Mix

Do APTT FS