Labs

The following is the complete blood count (CBC) at the time of admission:

WBC (109/L)Hb (g/dL)MCV (fL)PLT (109/L)
7.214.193235

What’s what: WBC, white blood cell count; Hb, hemoglobin; MCV, mean cell volume; MCHC, mean cellular hemoglobin concentration; RDW-SD, red cell distribution width-standard deviation; platelets, PLT; Normal values: WBC 5-10 x 109/L, RBC 4-6 x 1012/L, Hb 12-16 g/dL, Hct 35-47%, MCV 80-100 fL, MCHC 32-36 g/dL, RDW-SD < 45 fL, platelets (PLT) 150-450 x 109/L

TimePT (seconds)INRaPTT (seconds)
Day of admission12.11.166.7
5 years earlier12.31.031.5

What’s what: PT, prothrombin time; INR, international ratio; aPTT, activated partial thromboplastin time; Normal values: PT 9.4-12.5 seconds, aPTT 25-36.5 seconds.

If the patient had presented with a soft tissue hematoma instead of an ischemic stroke, would you have ordered a lupus anticoagulant as the next test?

a
Yes
b
No
In this case, you would be more concerned about deficiency or inhibitor of a specific clotting factor (especially in the intrinsic pathway), so a mixing study followed by specific factor assays would be a more reasonable approach.

Principles behind testing for a lupus anticoagulant:

At this point, given the clinical scenario (thrombosis + elevated aPTT), it is reasonable to order testing for a lupus anticoagulant. Before we look at the results, let’s discuss the principles behind the test:

  • The term lupus anticoagulant (LA) refers to a panel of different functional assays detecting a heterogeneous group of immunoglobulins behaving as acquired in vitro inhibitors of the coagulation.
  • LA is one of the three laboratory criteria for the identification of the antiphospholipid syndrome (APS). The other two are:
    • Anticardiolipin antibody
    • Anti-beta-2-glycoprotein 1 antibody
  • LA detection is based on phospholipid (PL)-dependent coagulation tests.
  • Testing involves three-step procedure with two test systems:
    • Three steps:
      • Screening
      • Mixing study
      • Confirmatory tests
    • Two test systems based on different principles:
      • Diluted Russell’s viper venom time (dRVVT)
      • Activated partial thromboplastin time (aPTT), typically with silica as the activator (silica clotting time)
  • Screening tests:
    • No single test is 100% sensitive to LA.
    • dRVVT should be the first test considered:
      • Based on the activation of factor X by a fraction of the venom derived from the Russell viper in combination with dilute phospholipids.
      • Recommended for its specificity and robustness.
    • The second test should be a sensitive aPTT (suitable PL composition and low concentration) and preferably silica as activator (silica clotting time).
    • Screening tests are performed with dRVVT and aPTT, and regarded to be positive if the normalized clotting time is prolonged beyond the locally established cutoff.
  • Mixing studies:
    • A mixing test (1:1 mix with pooled normal plasma) with screening reagent is performed if the screening test on undiluted sample is prolonged.
    • Consider performing the mixing step and the confirmatory test at the same time in all samples with a prolonged screening test, since a confirmatory test is suggested even if the mixing study is normal.
    • Results of mixing test are suggestive of LA when the normalized clotting time is greater than the local cutoff value.
  • Confirmatory tests:
    • Confirmatory test to be performed if the screening test suggests LA presence, irrespective of the result of the mixing test with screening reagent.
    • Based on rationale that increasing the concentration of phospholipids in the test system neutralizes the effect of LA and shortens the prolonged coagulation time if it is due to the presence of LA:
      • Source of phospholipids may include:
        • Aged platelets
        • Synthetic phospholipids:
          • Bilayer
          • Hexagonal (II) phase
      • Assays may include:
        • dRVVT
        • Silica clotting time

Interpretation of lupus anticoagulant (LA) testing:

  • LA should be considered positive if one of the two test systems gives a positive result in the three steps (screen-mix-confirm)
  • LA is reported with a final conclusion as positive or negative.
  • Results should always be related to the results of anticardiolipin antibodies (aCL) and anti-beta-2-glycoprotein I (β2GPI) to assess the risk profile.
  • Repeat testing is required after an initial positive result on a second occasion after at least 12 weeks to confirm persistent positivity.

True of false: False positive LA results may occur in patients treated with warfarin, heparin, or direct oral anticoagulants.

a
True
b
False

Testing for lupus anticoagulant (LA) in a patient on anticoagulation:

One of the main confounding factors in LA testing is anticoagulant therapy, prolonging the clotting times in the phospholipid (PL)-dependent assays used for LA detection. Ideally, LA testing should be deferred until anticoagulation is discontinued, but requests for LA testing during therapy occur frequently in routine clinical practice, resulting in potentially false-positive or false-negative results.

  • Heparins:
    • Interfere with LA clotting assay
    • Unfractionated heparin (UFH) and enoxaparin affect the dRVVT at supra-therapeutic anti-Xa levels.
    • Enoxaparin caused false-positive aPTT-based LA detection only at supra-therapeutic anti-Xa activity levels.
    • Some reagents, such as diluted Russell’s viper venom time (dRVVT) reagents and some LA-specific aPTT reagents contain heparin neutralizers.
    • Checking anti-Xa activity alongside LA testing can ensure that results are reliable if anti-Xa activity levels are within the therapeutic range.
    • Samples should be taken, when feasible, at least 12 hours after the last dose of low molecular weight heparin (LMWH) was administered and as near as possible to the next dose.
    • If anti-Xa levels are supra-therapeutic, positive LA results should be interpreted with care since false positives occur.
  • Vitamin K antagonists (VKAs):
    • May cause false-positive or false-negative results.
    • Taipan snake venom/Ecarin clotting time may help in LA detection in VKA- and rivaroxaban-treated patients.
  • Direct oral anticoagulants (DOACs):
    • DOACs give false-positive results in aPTT and dRVVT test systems, even at low concentrations.
    • Pretreatment of plasma with adsorbents may help.
    • If feasible, LA testing should progress after a brief interruption of DOACs—on a pragmatic, empirical basis at least 48 hours after the last dose, and longer in patients with renal impairment.

Bottom line: the LA test should not be ordered for patients taking anticoagulants, or should be interpreted with caution if performed.

The patient was started on aspirin, but was not anticoagulated. The following are the results from her lupus anticoagulant test:

Notes:

  • The test result is read out as positive or negative (in this case positive).
  • The results include a recommendation to retest > 12 weeks later as per diagnostic criteria.
  • dRVVT screen ratio (patient dRVVT screen result/mean of dRVVT screen normal range (NR). 
  • dRVVT confirm ratio (patient dRVVT confirm/mean of dRVVT confirm NR).
  • Integrated result reported as a normalized ratio (dRVVT screen ratio/dRVVT confirm ratio).
  • In this particular workflow, if dRVVT is positive, there is no need to proceed to second clotting assay (for example, silica clotting test) or perform mixing study.

dRVVT, diluted Russell’s viper venom time

What is the dRVVT?

a
Clotting time using a snake venom that activates tissue factor
b
Clotting time using a snake venom that activates prothrombin
c
Clotting time using a snake venom that activates factor X
The venom obtained from Russell’s viper (Vipera russelli) contains enzymes that directly activate coagulation factors V and X, bypassing the activation of factors VII, VIII, IX, XI, and XII, and therefore, the effect of deficiencies or inhibitors of these factors.
d
Clotting time using scorpion venom that activates factor XII

Dilute Russell viper venom time (dRVVT):

Russell`s Viper Daboia Russelii on Branch of Tree. Stock Photo - Image of  outdoor, wild: 149767700
Vipera russelli
American Society for Clinical Laboratory Science January 2017, 30 (1) 7-14
  • One of several available in vitro tests that may be used to screen and confirm for presence of lupus anticoagulant.
  • Uses venom obtained from Russell’s viper (Vipera russelli) which contains enzymes that directly activate coagulation factors V and X, bypassing the activation of factors VII, VIII, IX, XI, and XII, and therefore, the effect of deficiencies or inhibitors of these factors.
  • Diluting the phospholipid necessary for the clotting factor interactions increases the sensitivity to LA and the likelihood of identifying a phospholipid-dependent inhibitor that may not be detected by other coagulation tests with higher phospholipid content (eg, LA-insensitive aPTT reagents).
  • Patient plasma is incubated for a specified time then combined with a dRVVT screening reagent containing Russell’s viper venom, phospholipids, heparin neutralizing agents, calcium, buffers and stabilizers to trigger the coagulation process. Time to clot formation is measured optically using a wavelength of 671 nm.
  • The patient dRVVT screening clotting time is normalized by dividing the patient result by the mean DRVVT screening clotting time of normal pooled plasma to yield a ratio dRVVT screen ratio).
    • A normal dRVVT screen ratio (<1.20) indicates that lupus anticoagulant (LA) is not present or not detectable by this method (but might be detected with other methods). 
    • An abnormal dRVVT screen ratio (dRVVT screen ratio > or =1.20) may suggest presence of LA as well as coagulation factor deficiencies, anticoagulant effects, or other types of coagulation factor inhibitors.
  • Patient samples with a prolonged dRVVT (dRVVT screen ratio > or =1.20) are further studied (reflexive testing) by:
    • Adding an equal volume of normal pooled plasma (platelet-depleted) and repeating the DRVVT test procedure, with mathematical normalization, to yield the dRVVT mix (1:1) ratio
    • Repeating dRVVT after addition of excess phospholipid (DRVVT confirmatory reagent) to obtain quotient obtained from dividing the patient dRVVT screening clotting time by the patient dRVVT confirmatory clotting time (dRVVT confirm ratio).

Regarding the term lupus anticoagulant, which statement(s) is/are correct:

a
LA is most commonly seen in patients with lupus erythematosus (SLE)
Lupus anticoagulants were so-named because they were first found among patients with SLE, but they are more frequently seen in patients without SLE.
b
The term “anticoagulant” is a misnomer when considering the effects of LA in vivo
The term “anticoagulant” is part of the name because lupus anticoagulants prolong clotting time in laboratory tests that are used to evaluate coagulation.
c
The term “anticoagulant” refers to the ability of LA to prolong clotting assays in the test tube

Our patient underwent additional work-up to rule out other causes for her stroke:

  • Transthoracic echocardiogram to evaluate for right to left shunt (patent foramen ovale or atrial septal defect) – normal
  • Holter monitor to evaluate for paroxysmal atrial fibrillation – normal
  • Serum LDL – elevated. She was started on atorvastatin.

In addition to LA, there are two other types of antiphospholipid antibodies:

  • Anticardiolipin antibodies
  • Anti-beta-2-glycopetein I antibodies

These are most commonly detected by:

a
Clotting assays
b
FACS
c
ELISA
d
Cell culture

Would you test this patient for anticardiolipin (aCL) and anti-beta-2-glycopetein I antibodies?

a
Yes
It is important that all three aPL tests (LA, aCL and anti-beta-2-GP1) are performed as the aPL phenotype influences thrombotic risk. Triple aPL-positivity (i.e. the presence of LA, IgG and/or IgM anti-beta-2-GP1 and IgG and/or IgM aCL positivity) is correlated with the highest risk of thrombosis.
b
No

Here are the results of the additional antibody testing:

Based on these criteria, this patient has a high-risk profile.

The risk may be further increased by virtue of her being “triple positive”:

  • Positive for LA, anticardiolipin [IgG or IgM], and anti-beta-2 glycoprotein I [IgG or IgM].
  • Triple positivity in asymptomatic patients reported to be associated with first thrombosis.
  • Triple positivity in patients with APS reported to be associated with recurrence of thrombosis.

APL antiphospholipid antibodies; GPI, glycoprotein I

True of false: The LA test correlates better with clinical events than do aCL and anti-beta-2 glycoprotein I tests

a
True
b
False

Should all patients with ischemic stroke be tested for aPL?

a
Yes
b
No
While routine screening for aPL in patients with ischemic stroke is not warranted, it is recommended by the British Society of Hematology in young adults < 50 years.
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