Use of Viper Venom

8/14/2019 Use of Viper Venom

1/7

1986 68: 869-874

P Thiagarajan, V Pengo and SS Shapiro

anticoagulantsThe use of the dilute Russell viper venom time for the diagnosis of lupus

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Blood, Vol 68. N o 4 (O ctober), 1986: pp 869-874 86 9

T he U se of the D ilute R ussell V iper V enom T im e

for the D iagnosis of L upus A nticoagu lants

B y P erum al Th iagarajan, V ittorio P engo , and SandorS. Shapiro

W e desc ribe he re a te st fo r lupus anticoagulants ba sed on

a m odified Russe ll v ipe r veno m tim e (RV V T). u sing lim iting

am ou nts o f phosph olipid an d venom . W e have s tud ied 29

p atien ts w ith a prolong ed dilu te RV VT. F iv e of the 29 had an orm a l activa ted p artia l th rom boplas tin tim e and th ree of

1 4 tested by the tis su e throm bo pla stin inh ibition te st w ere

n orm a l. In 1 7 of 1 9 p atients te s ted. the d ilu te RV VT was

com p letely no rm al w hen iono phore -trea ted pla telets we re

L U PU S A N TIC O A G U LA N TS are antibodies reactiveagainst anionic phospholip ids , thereby prolonging

phospholipid-dependent coagulation tests.5 Thoughm i-tia lly r ec og niz ed in patients w ith system ic lupus erythem ato-

sus, sim ilar inhibitors have been described ina variety ofdisorders a nd , o cc as io na ll y, in appa ren tly norm a l individu-

als.29 In recent years it has becom e clear thatth e p re se nce

of a lupu s an ticoagulant is arisk factor for throm bosis#{176}8

and recu rrent spontaneous abo rtions.928 Neverthe less, th e

prevalence of lupus an tico ag ulants ha s been difficult to

determ ine, sincec riteria for the d iag nosis oft his c oa gu la ti oninh ib itor have not been clearly estab lished. Suspicion of a

lu pu s a ntic oa gu la nt is u su ally aroused by the finding ofa

prolonged activated partial throm boplastin tim e (aPTT) that

is no t c orrected by addition of an equal volum e of norm alp la sm a . C o rr ob or at io n is freq uently soug ht by performance

of the tissue throm bop la stin inhib itio n test (TTI).9 29 H ow -

ever, the TTI can be negative in the presence of som e lupus

an ticoagulants303 and has been reported to be positive in the

presence of som e factor V III or factor IX antibodies, as w ell

as in the presence of heparin.32 Inorder to avo id som e ofthese

problem s, w e haveem plo yed a m odified R ussell vipervenom

tim e, using diluted venom and a lim iting concentration of the

phospholipid reagen t.33 W e describe here details of the

m ethod, as w ell as som e studies of its sensitivity and specific-

ity com pared tothe TTI and the aP TT in the diagnosis ofl up u s a n ti co a gu la n ts .

M ATER IA LS AN D M E TH OD S

C oagu lation te sts . Blood was collected in one-tenth volume of

3.8% trisodium citra te. P late let-poor plasm a w as obtained by cen-

trifugation at 2 ,500 g for 15 m inutes at room tem perature . T he

prothrom bin tim e, activated partial throm boplastin tim e and throm -

bin tim e, using tissue throm boplastin (G eneral D iagnostics, M orris

Plains, N J), Throm bofax (O rtho D iagnostic System s, Raritan, N J),

and T hrom bin (U pjohn, K alam azoo, M ich), respectively , w ere

performed as desribed previously .34 T he norm al ranges for these tests

in our laboratory are I 2 to I5 seconds, 23 to 36 seconds, and 20 to 26seconds, respectively . The tissue throm boplastin inhibition test

(TTI) w as perform ed according to the m ethod of Schleider et al,9

using a 1:100 dilu tion of tissue throm boplastin. T he titers ofan tibodies to specific coagulation factors w ere determ ined by the

Bethesda A ssay.35 A ll test resu lts described here w ere perform ed in

ou r laboratory.

The dilute Russell viper venom tim e (RV V T) w as perform ed as

described previously .33 B riefly, Russell viper venom (Burroughs

W ellcom e, Raleigh, N C ) w as reconstituted as suggested by the

m anufacturer and further diluted I :200 in Tris-buffered saline(0.15

m ol/L N aC1, 0.02 m ol/L Tris, pH7.5) . The phospholipid reagent

substitu ted for phospho lip id; the rem a in ing tw o patien ts .

bo th w ith ve ry long phospholipid -dependen t dilute RV VTs.

w ere nea rly com pletely norm a lized . The dilute R VV T is no t

pro longed in the p re sence o f an tibod ies to fac tors V III. IX .or X l. Thus. the dilute RV V T appears to be a sim ple.

rep roducib le , s ensitive . and rela tively spec ific m ethod for

the detection of lupus anticoagulants .

a 1986 by G rune & Stratton , Inc.

Throm bofax w as dilu ted 1:8in Tris-buffered saline. O ther partial

thromboplast ins can be substituted for T hrom bofax; how ever, the

dilution of each reagent needs to be determ ined in the m annerdescr ibed for Throm bofax (see Results). T he dilute RV V T w as

performed by incubating 0.1 m L of plasm a, 0 .1 m L of diluted

R ussell viper venom , and 0. 1 m L dilu ted phospholipid for 30 seconds

at 37 #{176}C,fter w hich 0.1 m L of 0.03 m ol/L calcium chloride w as

added an d th e clotting tim e recorded. In experim ents us ing platelets ,

0. 1 m L of calcium ionophore-trea ted pla tele ts w as substituted for

phosphol ipid.P repa ration of w ashed. ionopho re-tre ated pla te lets .Citrated

blood wa s centrifuged at 1 50 g for I 5 m inutes at room tem perature.

The platelet-rich p lasm a supernatan t w as cen trifuged at I ,500 g for

ten m inutes at room tem perature and the plate lets w ere w ashed

tw ice by suspension and centrifugation in a buffer consisting of 0.15

m ol/L N aCI, 0.02 m ol/L Tris, 0.001 m ol/L ED TA , 0.005 m ol/L

glucose, pH 7.5. T he tw ice-w ashed platelets w ere resuspended at a

concentra tion of 8 x I 08/m L in the sam e buffer, but w ithout ED TA .

Activation w as ach ieved by adding to 2 m L of platelet suspension I

zL of a 5 m m ol/L solution of calcium ionophore A 23 1 87 (Calbio-

chem , S an D iego, Calif) in absolute e thanol (giving a final concen-

tration of 2.5 m ol/L, and incubating for five m inutes at room

tem perature. T he ionophore-trea ted platelets w ere used im m ediately

or frozen in aliquots at-0 # {1 76 }Cor future use.R E S U LT S

Effect ofphospho lipid concen tra tion on the RV VT.Rus -

sell viper venom w as diluted to give a clotting tim e in norm al

plasma o f a p pr ox im a te ly 2 5 s ec on ds . A t this dilution, sligh tly

better separation of lupus anticoagulant and norm al plasm a

w as achieved than at higher venom concentrations. S ince

l up u s a nt ic o ag u la n ts are an tibodies w ith im m unologic reactiv-

From the Cardeza Founda tion fo r H em atologic R esea rch .

Depa rtm en t o f M edicine. Jefferson M ed ical Co llege of Thom as

J ef fe r so n U n iv er si ty . P hi la de l ph ia .

S upported in part by NIH Grants HL-09163 (555). HL-27278(P T ), a G rant-in-A id and an E stablished Investigator A w ard from

th e A m erican H ea rt Associa tion (P T) and the S he ryl N . H irsch

Aw ard o f The Lupus Founda tion ofP hiladelphia (555 ).

S ubm itted M arch 6. 1986; accepted M ay 12, 1986 .

Address reprint requests to Dr S ando r S . S hapiro. C ardeza

Founda tion fo r Hem a tologic R esearch. Jeffe rson M ed ica l Co llege.

1015 W alnut S tree t. Ph iladelph ia . P A 19107 .

T he publication cos ts ofth is article w ere defrayed in part by pagecharge paym en t. Th is article m ust the refore be hereby m arked

advertisement in acco rdance w ith 18 U .S .C . 1734 sole ly toind ica te this fact.

cc 1986 by G rune & Stratton , Inc.

0006-4971/86/6804-O0I0$03.00/0

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3/7

50

40-

3 0

20

ity tow ards anionic phospholipids, w e w ished toincrease th etest sensitiv ity f urther by using the m in im al concentration of

p h o s p h o lip id re a g e n t n e c e s sa ryfor prothrom bin activ ation . A s

ca n be seen in Fi g I , Thrombofax w as sligh tly inhibitory w henused undiluted and w as op tim al at a dilution of 1:2 to 1:4,

w hen tested in norm al plasm a. H ow ev er, optim al separation of

lupus anticoagulant from norm a l pla sm a w as achiev ed at aThrom bofax dilution ofI :8 or greater. A t th is phospholipid

concen tration lupus anticoagulant patients w ere clearly

abnorm al, ev en though som e had a norm al R V V T w ith

undiluted phospholipid reagent. T he coef f ic ient of v ariation of

t h i s t es t, d et erm i ne d b y p er fo rm i ng 2 0 re pl ic at es on a single

norm al plasm a, w as 0 .8% . T he norm al range, determ ined on

I 4 fre sh n orm al plasm as, w as 26.2 1 .5 sec ( 2 S D ). T he

norm al range observed in o ur laborato ry over a 3 -year period, (using fresh and frozen p lasm as, w as 25.6 2.6 sec ( 2 S D).

W e consider clotting tim es of 30 seconds or greater (>3 .8 S D-

abov e the m ean) to be abnorm al, and m ak e a presum ptiv e Fd i a g n o s i s of a lupus an ticoagu lant w hen an abnorm al test isi..-.not corrected by addition of an equal v olum e of norm al Qp l a s m a . U sing this test, w e hav e diagnosed the presence of a

lupus anticoagulant in 29 patients referred to us because of the

s us pic io n o f s uc h a n a n t ic o a g u la n t . O f th e 29 patien ts, only 24had a prolonged aPT T . O f the rem aining f iv e, tw o patients

w ere on steroid therapy at the tim e ofou r stud y but had a

prolonged aPT T prev iously ; one patientha d a c hl oro pro m a -

zine-related lupus syndrom e w ith an antinuclear antibody ftiter of 1:320 and a rapid p lasm a reag in (R PR ) titer of 1:16; 0th e fo ur th patient had a his tory of recurrent abortions; and the PH O S P H OL I P ID P L AT E L E T Sf if th patient had sy stem ic lupus ery them atosus.

Substitution of plate le ts for phospholipid in the dilute F ig 2 . E ffe c t o f p la te le ts a n d p h o s p h o lip id o n th e R VVT o f. . . . lu p u s a n t ic o a g u la n t p la s m a s . H a tc h e d a re a s a re th e 2 S D n o rm a l

RVVT. T he ef f ect of substitu ting calcium ionophore-. . . . . r a n g e s .

activ ated platelets for the phospholipid reagent w as inv esti-

gated as follows. Io noph ore -treated plate letsw ere dilu ted to 50 l08/m L and 200 x 108/m L . W ashed , ionophore-

a concentration giv ing an R V V T of 23 to 28 seconds. T he treated platelets are stable in suspension at least during

concentration of platelets necessary to giv e this clotting tim e day they are prepared, and can be qu ick -f roz en at least

is v ariable w ith the p latelet preparation, ranging betw een and stored for I 2 m onths or m ore at-70 #{176}Cithou t loss ofa c t i v i t y. A s can be seen in Fig 2 , in 17 of 19 cases tested in

I 0 0 - this m anner the RVV T was com pletely norm al whenper-form ed w ith platelets rather than phospholip id . In the o ther

. tw o cases the R V V T w as no t com pletely norm alized . T hese

8 0 tw o patients had v ery prolonged d ilu te R V V T sand, inaddition , one of the tw o w as receiv ing oral anticoagulants .

w T he dilute R V V T in coagulation factor deficien-

6 0 . cies. The d ilu te R VV T is no rm al in p la sm a deficient inI- . . fac tors V II, V III, IX , X I, orX I I . Plasm as w ith factor V or

c.: . f actor X levels below 0.4 U /m L and plasm as f rompatients

4 0 . receiv ing oral anticoagulants giv e a pro longed dilute R V V T .:: #{149} However, in these situations the prolonged test is norm aliz ed0 by the a ddition of an equal v olum e of norm al plasm a (Fig 3).

2 0 In con trast, add ition of an equal v olum e of norm al p lasm a

does no t co rrect the dilute RV VT of lupus anticoagulant

O plasm a. In fact, an occasional lupus anticoagulant p lasm aI : I I : 2 I : 4 I :8 I : I 6 I : 3 2 show s a further prolongation of the R V V T test on adm ix ture

w ith norm al plasm a (Fig 3), an ef fect prev iously referred toPH O SPH O LIP ID DILU TIO N as the lupus cofactor phenom enon.3637

Fig 1. E ffe c t o f p h o s p h o lip id c o n c e n tra t io n o n t h e d ilu te T he dilute R V V T in the presence of heparin or antibodiesR VVT . B a rs a re n o r m a l c o n tro ls a n d c lo s e d c irc le s a re lu p u sto coagula tion factors. The pre sence of antibodie s to fac-p a t i e n t s . tors VIII, IX , or XI does not prolong the dilute R VVT (Table

8 7 0 THIA G AR AJAN E T A L



4/7

T a b le 2 . E ffe c t o f F a c to r V A n tib o d y o n th e D ilu te R V V T

F ac to r V A ntib od y.

Concentration

lB ethes da U /m L)

D ilute R VV T.

Phospholupid

(seconds)

Platelets

663.0

66.3

6.63

0.663

0.066

0

68.9

66.9

61.9

38.5

32.3

25.4

59.2

57.8

56.7

40.1

29.2

26.9

P P +N P P+N

70

60

U

a)

50

40I-I-0.J 30U

20

0

Fig 3 . (A ) E f fe c t o f n o rm a l p la s m a o n th e d ilu t e R V V T o f

fa c to r-d e f ic ie n t p la s m as . 0 . f a c to r X -d e f ic ie n t p la s m a; 0 . fa c to r

V -d e f ic ie n t p la s m a ; # {1 4 9 }. la s m a o f p a t ie n t s o n o ra l a n t ic o a g u -

la n ts . (B ) E ffe c t o f n o rm a l p la s m a o n t h e d ilu te R V V T o f lu p u s

a n t ic o a g u la n t p la s m a. E q u a l v o lu m es o f p a t ie n t (P ) a n d n o rm al (N )

p la s m a w e re m ix e d a n d th e d ilu te R V V T re c o rd e d . H a t c h e d a re a s

a re th e 2 S D n o rm al ra n g e .

1 ). The pres enc e of an antibo dy to factor V does prolong the

test. How ever, in contrast to the lupus anticoagulant, the

prolonged dilute RV V T of factor V antibody plasma does not

correct w hen ionophore-activated platelets are used (Table

2 ) .

A s show n in Fig 4, the dilute RV V T, like the aPTT, isprolonged by therapeutic levels of heparin. The dilute RV V T

appears to be less sensitive than the aPTT in this regard.Below 0.2 U /mL of heparin, the prolongation of the dilute

RV V T is corrected wh en ion opho re-trea ted p latele ts a re

substituted for phospholipid (data not show n).

C om parison with the aP T T and the tissue throm boplastin

inhibition test. A s can be seen in Fig 5A, 25 of 29 patientshad pro longations of the aPTT and the dilute RV V T, w hile

four patients w ith an abnormal dilute RV V T had a normal

aPTT. A s show n in Fig 5B ,I I o f 14 patients studied w ere

abnorm a l in both dilute R VV T and TT I tests. How ever, tw o

patients w ith potent 1gM lupus anticoagulants had a normal

T T I w hile a third patient, w ith a w eak lupus anticoagulant,

w as also norm al in the TTI test. Thus, although the corre la-

tion betw een results of all three tests w as good, the dilute

RV V T appears to be the most sensitive of the three.

DISCUSSION

A number of different tests have been proposed and used

for the diagnosis of lupus anticoagulants, reflecting the

difficulty in reaching a consensus concerning their definition

and mechanism of action.93839#{ 176} It is now clear that lupus

anticoagulants have immunolog ic reactiv ity tow ards anionicphospholipids and thereby prolong phospholipid-dependent

coagulation tests. N evertheless, phospholipid-dependent

tests may show variable sensitivity and/or specificity tow ard

lupus anticoagulants for several reasons. First, there are no

standards for phospholipid reagents used in coagulation

t e s t s , and the quantity of phosphatidylserine present in such

reagents, fo r e xa mp le , has been show n to affect their sensitiv-

ity tow ard lupus anticoagulants.442 One test38 does not

utilize any added phospholipid, presumably rely ing on phos-

pholipid o r i g i n a t i n g from the plasma or the plateletdust.43 Second, the physical state of the phospholipid

d i f f e r s in different tests : the aPTT and the RV V T utilize

90

II V

I0/

Li

z

I-.0-A

0

/5

I VI V

S

9/V

0/0

3Antibody

Titer

(B eth es da U /m L)D ilute R VV T

(seconds)

FactorVlll 38 25.9

FactorVlll 87 26.3

F a c t o r V I l l 7 2 6 . 8

FactorVIll 23 25.7

FactorVll l 14 26.7

FactorlX 85 27.1

FactorXl 4 1 5 2 6 . 4

-, 0

V

.

05 10

F ig 4 . E ff e c t o f h e p a r in o n t h e a P T T a n d th e d ilu t e R V V T o f

t h re e n o rm al p la s m as . O p e n s y m bo ls . a P T T ; c lo s e d s y m bo ls . d ilu te

RV V T.

L UP US A NT IC OA GU LA NT S 87 1

T a b le 1. E f fe c t o f A n t ib o d ie s to C o a g u la t io n F a c t o rs

o n th e D ilu te R V V T

O O l 0.02 0.05 O l 02

HE PA RIN (un its /m I)



5/7

a)

A

50

: since it i s u naf fected by the presence of antibodies to facto rs

. I V III, IX , or X I, and hav e further increased the sensitiv ity of

0 the test by using lim iting concentrations of bo th phospholip id

. : :#{149}{149}{149}{149}{ 149} and venom . T his test is easy to perfo rm and highly reproduc-. . . !#{ 149} #{ 149}{149}.#{149} ib le. N ev ertheless, it is no t en tirely specif ic for lupus antico-

0 #{149}L#{149} agulants. C oagulation factor def iciencies can prolong thedilute R V V T , but these can be easily ex cluded by repeating

the test on a m ix ture of norm al and patient p lasm a. T he

presence of an antibody to factor V . an ex trem ely rare ev ent,

also pro longs this test. H ow ev er, in one f actor V antibody

patient w hom w e had the opportunity to study , substitution

of ionophore-treated platelets fo r the phospholipid reagen t

did no t correct the test, un lik e the f indings w ith lupus

lO 2 0 30 50 60 an ticoagulan ts. Furtherm ore, the presence of a f actor V

p T T ( ) an tibody isassociated w ith a m ark ed prolongation of the PT ,a sec a ra re occu rrence w ith lupus anticoagulants. N ev ertheless,

def initiv e ex clusion of a factor V antibody requires specif ic

m easurem ent of facto r V inhibition in a m ix ture of patient

. C an d norm al p lasm a.

0 Finally , therapeu tic concentrations of heparin prolong the

C #{149} #{149}{ 149} dilute R V V T , although to a s om ew hat lesser ex tent than the

c: . aPT T (Fig 4). 1 -leparin ef f ects can be ruled out by m easure-

m ent of the throm bin tim e, since this test is norm al in the

presence of a lupus an ticoagu lant.

Li T he sign if icance of the presence of a lupus anticoagulant

does not lie in its association w ith bleeding, w hich rare ly , if

: ev er, occurs as a result of the lupus anticoagulant alone,3785

but rather in the f act that approx im ately 25% to 30% of

: pa tients w ith t his a nt ib od y have a history of throm boem bolicphenom ena,#{176}8 o r repea ted spontaneous abortions)28 T he

B 05 I 0 I 20 25 presence ofan ticardiolipin antibodies, as m easured by anyTTI RA TIO oneofsev eral im m unologic techniques, has also been identi-

f led as a risk factor for throm bosis.5 2447 H ow ev er, the

Fig 5. Com parison o f th e d ilu te R VVT w ith th e a P TT (A ) a n d relationship betw een the se tw o m easurem ents h as no t beenth e TTI (B). O pen circle s are patients w ith 1gM lupus anticoagu - eva lua ted adequa te ly.It is clea r, for exam ple , tha t som ela n ts . C lo s e d c irc le s a re p a t ie n t s w ith Ig G o r a m ix tu r e o f lg G a n d t y p e s o f a n t ic a rd io lip in a n t ib o d ie s a re n o t a s s o c ia t e d

g M lu p u s a n t ic o a g u la n t s . D o t te d lin e s in d ic a te u p p e r lim its o f . . . . .

n o rm a l fo r t h e te s ts . a n in c r e a s e d th ro m b o t ic r is k . F o r e x a m p le , p o s it iv e s e ro lo g ictests in patien ts w ith sy philisar e due to reactiv ity of patient

serum w ith cardiolipin, y et patients w ith sy ph ilis do not hav e

phospholipid in m icellar form , w hereas the T T I and the an increased prev alence of lupus anticoagulants or of th rom

prothrom bin tim e (PT ) m ak e use of tissue throm boplastin, in boem bolic disease.48 For an adequate understanding

w hich the phospho lipid is p rotein -bound. T hird, the tests b io logic role of antibodies reactiv e w ith anion ic phosp

vary in their response to def iciencies of clo tting facto rs and to lipids, it w ill be necessary to standard iz e bo th ty pes of

antibodies to clo tting factors. For instance, antibod ies to T he dilute R V V T w hich w e describe here is sim ple

f actor V III m ay prolong the aPT T , but not the R V V T . reproducible. M oreov er, as w e hav e dem onstrated , it is m o

Finally , there appear to be v ariations in test sensitiv ity sensitiv e than the aPT T and the T T I, and thus shou ld

related to the im m unoglobu lin class of the lupus an ticoagu- highly useful as a screen ing diagnostic test fo r fu r

lan t; for ex am ple, as w e hav e show n, som e 1gM anticoagu- stud ies.

lan ts do not prolong the T T I, although they do prolong other

tests. A s a result, the incidence of lupus anticoagulants has A C K N O W L E D G M E N T

not been clearly established, since patients m ay be positiv e in

o ne te st an d no t ano ther. W e w ish to thank E . B atIle fo r e x pert secretarial assistance.

S trategies to increase the sensitiv ity of tests hav e tak en

tw o form s. S ince lupus an ticoagu lan t activ ity is less ev iden t R E FE R E N C E S

in the presence of platelets 1.3,45,46 the use of p latelet-f ree . .. 1. T h iagarajan P. S hapiro 55 , D eM arco L : A m onoclonal 1gM

plasma has been advocated. Second, asin o ur p rocedure , lambda coagulation inhibitor w ith phospholip id specif icity : M echa-seve ral te sts m ak e use of lim iting concentrations of phospho- nism of a lupus anticoagulant. J C lin Inv est 66:397, 1980

l ip ids to increase their sensitiv ity . W e hav e chosen the R V V T 2. S hapiro 55, T hiagarajan P. D eM arco L : M echanism of

as our prim ary test f or the diagnosis o f lupus anticoagulants, of the lupus anticoagulant. A nn N Y A cad S ci 370 :359, 1981

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3. Shapiro 55, Thiagarajan P: L upus anti coagul ants. Prog

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anticoagul ants. J L ab Clin M ed 62:416, 1963

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