From www.bloodjournal.org by on July 21, 2009. For personal use only.

1986 68: 869-874

The use of the dilute Russell viper venom time for the diagnosis of lupus
anticoagulants
P Thiagarajan, V Pengo and SS Shapiro

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Copyright 2007 by The American Society of Hematology; all rights reserved.
 From www.bloodjournal.org by on July 21, 2009. For personal use only.

The Use of the Dilute Russell Viper Venom Time

for the Diagnosis of Lupus Anticoagulants

By Perumal Thiagarajan, Vittorio Pengo, and Sandor S. Shapiro

We describe here a test for lupus anticoagulants based on substituted for phospholipid; the remaining two patients.
a modified Russell viper venom time (RVVT). using limiting both with very long phospholipid-dependent dilute RVVTs.
amounts of phospholipid and venom. We have studied 29 were nearly completely normalized. The dilute RVVT is not
patients with a prolonged dilute RVVT. Five of the 29 had a prolonged in the presence of antibodies to factors VIII. IX.
normal activated partial thromboplastin time and three of or Xl. Thus. the dilute RVVT appears to be a simple.
1 4 tested by the tissue thromboplastin inhibition test were reproducible, sensitive. and relatively specific method for
normal. In 1 7 of 1 9 patients tested. the dilute RVVT was the detection of lupus anticoagulants.
completely normal when ionophore-treated platelets were a 1986 by Grune & Stratton, Inc.

L UPUS ANTICOAGULANTS are antibodies reactive Thrombofax

thromboplastins
was diluted
can
1:8 in Tris-buffered
be substituted for Thrombofax;
saline. Other
however,
partial
the
against anionic phospholipids, thereby prolonging
phospholipid-dependent coagulation tests.’5 Though mi- dilution of each reagent needs to be determined in the manner
described for Thrombofax (see Results). The dilute RVVT was
tially recognized in patients with systemic lupus erythemato-
performed by incubating 0.1 mL of plasma, 0.1 mL of diluted
sus, similar inhibitors have been described in a variety of
Russell viper venom, and 0. 1 mL diluted phospholipid for 30 seconds
disorders and, occasionally, in apparently normal individu-
at 37 #{176}C,
after which 0.1 mL of 0.03 mol/L calcium chloride was
als.29 In recent years it has become clear that the presence added and the clotting time recorded. In experiments using platelets,
of a lupus anticoagulant is a risk factor for thrombosis’#{176}’8 0. 1 mL of calcium ionophore-treated platelets was substituted for
and recurrent spontaneous abortions.’928 Nevertheless, the phospholipid.
prevalence of lupus anticoagulants has been difficult to Preparation of washed. ionophore-treated platelets. Citrated
determine, since criteria for the diagnosis of this coagulation blood was centrifuged at 1 50 g for I 5 minutes at room temperature.
inhibitor have not been clearly established. Suspicion of a The platelet-rich plasma supernatant was centrifuged at I ,500 g for
lupus anticoagulant is usually aroused by the finding of a ten minutes at room temperature and the platelets were washed
twice by suspension and centrifugation in a buffer consisting of 0.15
prolonged activated partial thromboplastin time (aPTT) that
mol/L NaCI, 0.02 mol/L Tris, 0.001 mol/L EDTA, 0.005 mol/L
is not corrected by addition of an equal volume of normal
glucose, pH 7.5. The twice-washed platelets were resuspended at a
plasma. Corroboration is frequently sought by performance
concentration of 8 x I 08/mL in the same buffer, but without EDTA.
of the tissue thromboplastin inhibition test (TTI).9’29 How- Activation was achieved by adding to 2 mL of platelet suspension I
ever, the TTI can be negative in the presence of some lupus zL of a 5 mmol/L solution of calcium ionophore A23 1 87 (Calbio-
anticoagulants30’3’ and has been reported to be positive in the chem, San Diego, Calif) in absolute ethanol (giving a final concen-
presence of some factor
as in the presence
problems,
time, using
VIII or factor
of heparin.32
we have employed
IX antibodies, as well
In order to avoid some of these
a modified
diluted
Russell
venom
viper venom
and a limiting concentration of the
tration
temperature.
or frozen
of 2.5
The
in aliquots
tmol/L,

at -
ionophore-treated
and

70 #{176}C
incubating

for future

RESULTS
platelets
for

use.
five
were
minutes
used immediately
at room

phospholipid reagent.33 We describe here details of the

method, as well as some studies of its sensitivity and specific- Effect ofphospholipid concentration on the RVVT. Rus-
ity compared the TTI to and the aPTT in the diagnosis of sell viper venom was diluted to give a clotting time in normal
lupus anticoagulants. plasma of approximately 25 seconds. At this dilution, slightly
better separation of lupus anticoagulant and normal plasma
MATERIALS AND METHODS was achieved than at higher venom concentrations. Since

Coagulation tests. Blood was collected in one-tenth volume of lupus anticoagulants are antibodies with immunologic reactiv-
3.8% trisodium citrate. Platelet-poor plasma was obtained by cen-
trifugation at 2,500 g for 15 minutes at room temperature. The
prothrombin time, activated partial thromboplastin time and throm- From the Cardeza Foundation for Hematologic Research.
bin time, using tissue thromboplastin (General Diagnostics, Morris Department of Medicine. Jefferson Medical College of Thomas
Plains, NJ), Thrombofax (Ortho Diagnostic Systems, Raritan, NJ), Jefferson University. Philadelphia.
and Thrombin (Upjohn, Kalamazoo, Mich), respectively, were Supported in part by NIH Grants HL-09163 (555). HL-27278
performed as desribed previously.34 The normal ranges for these tests (PT), a Grant-in-Aid and an Established Investigator Award from
in our laboratory are I 2 to I 5 seconds, 23 to 36 seconds, and 20 to 26 the American Heart Association (PT) and the Sheryl N. Hirsch
seconds, respectively. The tissue thromboplastin inhibition test Award of The Lupus Foundation ofPhiladelphia (555).
(TTI) was performed according to the method of Schleider et al,9 Submitted March 6. 1986; accepted May 12, 1986.
using a 1:100 dilution of tissue thromboplastin. The titers of Address reprint requests to Dr Sandor S. Shapiro. Cardeza
antibodies to specific coagulation factors were determined by the Foundation for Hematologic Research. Jefferson Medical College.
Bethesda Assay.35 All test results described here were performed in 1015 Walnut Street. Philadelphia. PA 19107.
our laboratory. The publication costs ofthis article were defrayed in part by page
The dilute Russell viper venom time (RVVT) was performed as charge payment. This article must therefore be hereby marked
described previously.33 Briefly, Russell viper venom (Burroughs “advertisement” in accordance with 18 U.S.C. § 1734 solely to
Wellcome, Raleigh, NC) was reconstituted as suggested by the indicate this fact.
manufacturer and further diluted I :200 in Tris-buffered saline (0.15 cc 1986 by Grune & Stratton, Inc.
mol/L NaC1, 0.02 mol/L Tris, pH 7.5). The phospholipid reagent 0006-4971/86/6804-O0I0$03.00/0

Blood, Vol 68. No 4 (October), 1986: pp 869-874 869

 From www.bloodjournal.org by on July 21, 2009. For personal use only.

870 THIAGARAJAN ET AL

ity towards anionic phospholipids, we wished to increase the

test sensitivity further by using the minimal concentration of
phospholipid reagent necessary for prothrombin activation. As
can be seen in Fig I , Thrombofax was slightly inhibitory when
used undiluted and was optimal at a dilution of 1:2 to 1:4,
when tested in normal plasma. However, optimal separation of
lupus anticoagulant from normal plasma was achieved at a 50
Thrombofax dilution of I :8 or greater. At this phospholipid
concentration lupus anticoagulant patients were clearly
abnormal, even though some had a normal RVVT with
undiluted phospholipid reagent. The coefficient of variation of
this test, determined by performing 20 replicates on a single
normal plasma, was 0.8%. The normal range, determined on 40-
I 4 fresh normal plasmas, was 26.2 ± 1 .5 sec ( ± 2 SD). The
normal range observed in our laboratory over a 3-year period, (
using fresh and frozen plasmas, was 25.6 ± 2.6 sec ( ± 2 SD).
We consider clotting times of 30 seconds or greater (>3.8 SD -

above the mean) to be abnormal, and make a presumptive F

diagnosis of a lupus anticoagulant when an abnormal test is i..-. 30
not corrected by addition of an equal volume of normal Q
plasma. Using this test, we have diagnosed the presence of a
lupus anticoagulant in 29 patients referred to us because of the
suspicion of such an anticoagulant. Of the 29 patients, only 24
had a prolonged aPTT. Of the remaining five, two patients
were on steroid therapy at the time of our study but had a 20
prolonged aPTT previously; one patient had a chloroproma-
zine-related lupus syndrome with an antinuclear antibody f
titer of 1:320 and a rapid plasma reagin (RPR) titer of 1:16; 0
the fourth patient had a history of recurrent abortions; and the PH OS P HO L I P ID P L AT E L E T S
fifth patient had systemic lupus erythematosus.
Substitution of platelets for phospholipid in the dilute Fig 2. Effect of platelets and phospholipid on the RVVT of
. . . . lupus anticoagulant plasmas. Hatched areas are the 2 SD normal
RVVT. The effect of substituting calcium ionophore-
. . . . . ranges.
activated platelets for the phospholipid reagent was investi-
gated as follows. Ionophore-treated platelets were diluted to 50 l08/mL and 200 x 108/mL. Washed, ionophore-
a concentration giving an RVVT of 23 to 28 seconds. The treated platelets are stable in suspension at least during the
concentration
is variable with
of platelets
the
necessary
platelet preparation,
to give this clotting
ranging between
time day they are prepared,
and
activity.
stored
As can
for I 2 months
be seen
and

in
can be quick-frozen
or more
Fig
at - at least
70

2, in 17 of 19 cases
without
#{176}C

tested
once
loss of
in
I0 0 - this manner the RVVT was completely normal when per-
formed with platelets rather than phospholipid. In the other
. two cases the RVVT was not completely normalized. These
8 0 two patients had very prolonged dilute RVVTs and, in
addition, one of the two was receiving oral anticoagulants.
w The dilute R VVT in coagulation factor deficien-
6 0 . cies. The dilute RVVT is normal in plasma deficient in
I-. . factors VII, VIII, IX, XI, or XII. Plasmas with factor V or
c.: . factor X levels below 0.4 U/mL and plasmas from patients
4 0 . receiving oral anticoagulants give a prolonged dilute RVVT.
:: #{149} However, in these situations the prolonged test is normalized
0 by the addition of an equal volume of normal plasma (Fig 3).
2 0 In contrast, addition of an equal volume of normal plasma
does not correct the dilute RVVT of lupus anticoagulant

O plasma. In fact, an occasional lupus anticoagulant plasma

I :I I:2 I :4 I :8 I:I 6 I:3 2 shows a further prolongation of the RVVT test on admixture
with normal plasma (Fig 3), an effect previously referred to
PHOSPHOLIPID DILUTION as the “lupus cofactor” phenomenon.36’37

Fig 1. Effect of phospholipid concentration on the dilute The dilute R VVT in the presence of heparin or antibodies
RVVT. Bars are normal controls and closed circles are lupus to coagulation factors. The presence of antibodies to fac-
patients. tors VIII, IX, or XI does not prolong the dilute RVVT (Table
 From www.bloodjournal.org by on July 21, 2009. For personal use only.

LUPUS ANTICOAGULANTS 871

70 Table 2. Effect of Factor V Antibody on the Dilute RVVT

Dilute RVVT
Factor V Antibody .
. Phospholupid Platelets
Concentration
60 lBethesda U/mL) (seconds)

663.0 68.9 59.2

U
66.3 66.9 57.8
a)
6.63 61.9 56.7
0.663 38.5 40.1
0.066 32.3 29.2
0 25.4 26.9
50

40 was also normal in the TTI test. Thus, although the correla-
I- tion between results of all three tests was good, the dilute
I-
0 RVVT appears to be the most sensitive of the three.
.J 30
U DISCUSSION

A number of different tests have been proposed and used

for the diagnosis of lupus anticoagulants, reflecting the
20
difficulty in reaching a consensus concerning their definition
0 and mechanism of action.9’38’39’#{176} It is now clear that lupus
P P+N P P+N
anticoagulants have immunologic reactivity towards anionic
Fig 3. (A) Effect of normal plasma on the dilute RVVT of phospholipids and thereby prolong phospholipid-dependent
factor-deficient plasmas. 0. factor X-deficient plasma; 0. factor coagulation tests. Nevertheless, phospholipid-dependent
V-deficient plasma; #{149}.plasma of patients on oral anticoagu- tests may show variable sensitivity and/or specificity toward
lants. (B) Effect of normal plasma on the dilute RVVT of lupus
lupus anticoagulants for several reasons. First, there are no
anticoagulant plasma. Equal volumes of patient (P) and normal (N)
standards for phospholipid reagents used in coagulation
plasma were mixed and the dilute RVVT recorded. Hatched areas
are the 2 SD normal range. tests, and the quantity of phosphatidylserine present in such
reagents, for example, has been shown to affect their sensitiv-
1 ). The presence of an antibody to factor V does prolong the ity toward lupus anticoagulants.4”42 One test38 does not
test. However, in contrast to the lupus anticoagulant, the utilize any added phospholipid, presumably relying on phos-
prolonged dilute RVVT of factor V antibody plasma does not pholipid originating from the plasma or the “platelet
correct when ionophore-activated platelets are used (Table dust.”43’ Second, the physical state of the phospholipid
2). differs in different tests: the aPTT and the RVVT utilize
As shown in Fig 4, the dilute RVVT, like the aPTT, is
prolonged by therapeutic levels of heparin. The dilute RVVT 90

appears to be less sensitive than the aPTT in this regard.

Below 0.2 U/mL of heparin, the prolongation of the dilute
RVVT is corrected when ionophore-treated platelets are
I
substituted for phospholipid (data not shown).
Comparison with the aPTT and the tissue thromboplastin I V

inhibition test. As can be seen in Fig 5A, 25 of 29 patients I

had prolongations of the aPTT and the dilute RVVT, while 0/
Li
four patients with an abnormal dilute RVVT had a normal
/5
aPTT. As shown in Fig 5B, I I of 14 patients studied were
abnormal in both dilute RVVT and TTI tests. However, two I V

S
z
patients with potent 1gM lupus anticoagulants had a normal I V

I-.
TTI while a third patient, with a weak lupus anticoagulant, 0 9/
-A
0
‘V
Table 1 . Effect of Antibodies to Coagulation Factors 0/0
on the Dilute RVVT
‘-, 0 .
Titer Dilute RVVT 3 V
Antibody (Bethesda U/mL) (seconds)

FactorVlll 38 25.9
FactorVlll 87 26.3
OOl 0.02 0.05 Ol 02 05 10
FactorVIll 7 26.8
FactorVIll 23 25.7 HEPARIN (units/mI)
FactorVlll 14 26.7
FactorlX
Fig 4. Effect of heparin on the aPTT and the dilute RVVT of
85 27.1
three normal plasmas. Open symbols. aPTT; closed symbols. dilute
FactorXl 415 26.4
RVVT.
 From www.bloodjournal.org by on July 21, 2009. For personal use only.

872 THIAGARAJAN ET AL

50 : since it is unaffected by the presence of antibodies to factors

. I VIII, IX, or XI, and have further increased the sensitivity of
0 the test by using limiting concentrations of both phospholipid
a)
40
. :: #{149}#{149}
#{149}
#{149}#{149} and venom. This test is easy to perform and highly reproduc-
. . . !#{149}#{149}
#{149}.#{149} ible. Nevertheless, it is not entirely specific for lupus antico-
I-
> 30 0 #{149}L#{149} agulants. Coagulation factor deficiencies can prolong the
> dilute RVVT, but these can be easily excluded by repeating
Li the test on a mixture of normal and patient plasma. The
I-. 20
presence of an antibody to factor V. an extremely rare event,
-I
also prolongs this test. However, in one factor V antibody
0 patient whom we had the opportunity to study, substitution
of ionophore-treated platelets for the phospholipid reagent
did not correct the test, unlike the findings with lupus
lO 2’0 30 50 60 anticoagulants. Furthermore, the presence of a factor V
A
p T T ( ) antibody is associated with a marked prolongation of the PT,
a sec a rare with lupus
occurrence anticoagulants. Nevertheless,
50 definitive exclusion of a factor V antibody requires specific
measurement of factor V inhibition in a mixture of patient
. C and normal plasma.
40 0 Finally, therapeutic concentrations of heparin prolong the
U
a) C #{149}
#{149} #{149} dilute RVVT, although to a somewhat lesser extent than the
in

.c:. aPTT (Fig 4). 1-leparin effects can be ruled out by measure-
!;; 3#{176} ment ofthe thrombin time, since this test is normal in the
>
a: presence of a lupus anticoagulant.
Li The significance of the presence of a lupus anticoagulant
20
does not lie in its association with bleeding, which rarely, if
: ever, occurs as a result of the lupus anticoagulant alone,37’8”5
IO but rather in the fact that approximately 25% to 30% of
: patients with this antibody have a history of thromboembolic
phenomena,’#{176}8 or repeated spontaneous abortions)28 The

B 05 I 0 I 20 25 presence of anticardiolipin antibodies, as measured by any

TTI RATIO one ofseveral immunologic techniques, has also been identi-
fled as a risk factor for thrombosis.5’2447 However, the
Fig 5. Comparison of the dilute RVVT with the aPTT (A) and relationship between these two measurements has not been
the TTI (B). Open circles are patients with 1gM lupus anticoagu- evaluated adequately. It is clear, for example, that some
lants. Closed circles are patients with IgG or a mixture of lgG and types of “anticardiolipin” antibodies are not associated with
gM lupus anticoagulants. Dotted lines indicate upper limits of . . . . .

normal for the tests. an increased thrombotic risk. For example, positive serologic
tests in patients with syphilis are due to reactivity of patient
serum with cardiolipin, yet patients with syphilis do not have
phospholipid in micellar form, whereas the TTI and the an increased prevalence of lupus anticoagulants or of throm-
prothrombin time (PT) make use oftissue thromboplastin, in boembolic disease.48 For an adequate understanding of the
which the phospholipid is protein-bound. Third, the tests biologic role of antibodies reactive with anionic phospho-
vary in their response to deficiencies of clotting factors and to lipids, it will be necessary to standardize both types of tests.
antibodies to clotting factors. For instance, antibodies to The dilute RVVT which we describe here is simple and
factor VIII may prolong the aPTT, but not the RVVT. reproducible. Moreover, as we have demonstrated, it is more
Finally, there appear to be variations in test sensitivity sensitive than the aPTT and the TTI, and thus should be
related to the immunoglobulin class of the lupus anticoagu- highly useful as a screening diagnostic test for further
lant; for example, as we have shown, some 1gM anticoagu- studies.
lants do not prolong the TTI, although they do prolong other
tests. As a result, the incidence of lupus anticoagulants has ACKNOWLEDGMENT
not been clearly established, since patients may be positive in
one test and not another. We wish to thank E. BatIle for expert secretarial assistance.
Strategies to increase the sensitivity of tests have taken
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