Dilute Russell's Viper Venom Test
dRVVT, DVVtest®, DVVconfirm®
The dilute dRVVT is an in vitro qualitative assay for lupus anticoagulants (LAs), the most common type of nonspecific coagulation inhibitor. LAs are immunoglobulins (IgG, IgM, both) directed against certain phospholipid epitopes, including the phospholipid added as a surrogate source of phospholipid in the aPTT. In addition to SLE (only 20% occur in SLE), LAs have been reported in patients with other autoimmune diseases, malignancies, infections, AIDS, and otherwise normal individuals. In addition, LAs are common in patients receiving chlorpromazine, procainamide, thorazine, and other medications. In children, LAs most frequently occur in association with tonsillitis and infected adenoids.

Paradoxically, clinical bleeding is not associated with the presence of LA alone because the platelet membranes provide an alternative phospholipid surface for in vivo coagulation complexes, which are not inhibited by LA. However, LAs have been associated with a variety of other clinical problems, including thrombosis, recurrent pregnancy loss, thrombocytopenia, cutaneous lesions (livido reticularis, leg ulcers (erythema nodosum-like nodules), neurologic problems, and hemolytic anemia. Thrombosis (venous and/or arterial) occurs in about 25% of patients with LA, irrespective of the presence of SLE.

The venom of Russell's viper (xxxxx), a poisoninous snake common in xxxxxx, causes massive thrombosis when injected in vivo. A potent in vitro clot promoting effect of RVV was demonstrated by Macfarlane and Barnett in 1934, and this substance has since been available as a local hemostatic for hemophiliacs, under the name of "Stypven." The coagulant protein in RVV is an enzyme (serine protease) which directly activates factor X in the presence of Ca++, bypassing the intrinsic and extrinsic pathways. The activated factor X then activates prothrombin (factor II) in the presence of factor V and phospholipid. In the dilute Russell’s viper venom time, phospholipid is diluted to the point that the clotting time becomes very sensitive to the presence of phospholipid.

No specific patient preparation is required. However, since lipemia may interfere with photo-electric measurements of clot formation, specimens should not be obtained after a meal.
Citrated, platelet-poor plasma is used for the dRVVT.
Citrated, platelet-poor plasma is collected as for the aPTT.

The blood must be centrifuged as soon as possible after procurement (a minimum of 5000 x g for 15 minutes) to obtain platelet-poor plasma with a platelet count of < 104/uL. Alternately, acceptable platelet-poor plasma can be prepared by passing plasma through a 0.22 micron filter. Normal and control plasma with a platelet count of < 104/uL must alos be available for the dRVVT. The platelet-poor plasma should be separated and placed into capped plastic tubes within 60 minutes of venipuncture. The DVVtest® and DVVconfirm assays can be performed on plasma stored at 0oC to 8oC within 4 hours of venipuncture or on plasma frozen and stored at -70oC for up to three months. Frozen plasma must be thawed rapidly at 37oC and either tested immediately, or stored for up to two hours at 2oC to 8oC prior to testing .
The DVVtest® is a commercial reagent (American Diagnostica, Inc., Greenwich, CT) developed to standardize the dRVVT. The DVVtest® reagent combines RVV, plant phospholipid, and calcium into a single reagent. A second reagent, DVVconfirm®, contains RVV, extra plant phospholipid, and calcium. The extra phospholipid in the DVVconfirm® reagent corrects a prolonged DVVtest® time in a manner analogous to platelet addition in the APTT-based platelet neutralization procedure (PNP).

The DVVtest® and DVVconfirm® assays are performed at 37oC. Prewarmed (37oC) citrated plasma is added to prewarmed (37oC) DVVtest® or DVVconfirm® reagents and the time required for the formation of fibrin monomers is determined visually, mechanically, or opto-electronically. Each test sample or normal plasma specimen are assayed in duplicate and the average of the duplicate times is taken as the final result. Platelet-poor normal and abnormal LA control plasma should be tested with each group of patient assays, and fall within the laboratory's established control range.

The results are interpreted as follows:

DVVtest® time of test plasma within established normal reference range - Test is negative for LA.

DVVtest® time of test plasma greater than normal reference range - LA or Factor II, V, or X deficiency is present. An LA can be differentiated from a factor deficiency by two different techniques:

(1) The DVVtest® time of a 1:1 mixture of test plasma and pooled normal plasma can be determined and a DVVtest ratio calculated from the following formula:

DVVtest® time of 1:1 plasma mixture
DVVtest Ratio = ---------------------------------------------------------
DVVtest® time of pooled normal plasma

If the DVVtest ratio is within the laboratory normal reference ratio range, the test is negative for LA.

If the DVVtest ratio is greater than the laboratory normal reference ratio range, the test is positive for LA.

(2) The DVVconfirm test can be performed on the test plasma. DVVconfirm contains a high concentration of phospholipid, which corrects for the presence of LA by providing alternative phospholoipid binding sites. A DVVtest/DVVconfirm ratio is calculated from the DVVtest and DVVconfirm times of the test plasma from the following formula:

DVVtest® time of plasma
Ratio = --------------------------------------------
DVVconfirm® time of plasma

If the DVVtest/DVVconfirm ratio is within the laboratory normal reference ratio range, the test is negative for LA. The plasma should be tested further for the presence of a factor II, V, or X deficiency.

If the DVVtest/DVVconfirm ratio is greater than the laboratory normal reference ratio range, the test is positive for LA.

Normal Values
Critical Limits
The DVVtest time and DVVconfirm time are instrument and laboratory dependent. Therefore, each laboratory must establish normal reference ranges (mean +/- 2 S.D., in seconds) for the DVVtest® and DVVconfirm®. The normal reference ranges should be derived from a minimum of 20 healthy blood donors, spanning the adult age range and including both men and women. Expected values obtained on different commercial coagulation analyzers in quality control studies are provided by American Diagnostica, Inc.
The blood:anticoagulant ratio must be adjusted during sample collection in patients with hematocrits < 20% or > 60%. Adequate centrifugation of the specimen must be achieved to produce platelet-poor plasma (platelet count < 104/uL). Deficiencies of Factors II, V, and X or anticoagulant therapy with warfarin may prolong the DVVtest® and DVVconfirm®. However, these assays are not affected by the presence of heparin in concentrations < 1.0 U/mL, deficiencies of Factors VIII, IX or XI, and antibodies against Factors XI or XII. Very strong Factor VIII antibodies (> 1000 Bethesda Units) may increase the DVVtest® by about 10%.

Icterus, lipemia, or hemolysis will interfere with the DVVtest® and DVVconfirm® assays if a photoelectric instrument is used for endpoint detection. Specimens containing heparin levels greater than 1.0 U/mL may give incorrect results and should not be evaluated by this assay. Inhibitors to factors II, V, or X may interfere with the detection of LAs. The dRVVT can be used to detect LA in patient samples with a normal aPTT since phospholipid dilution increases the sensitivity of the assay.

Clinical Utilization
The dRVVT is used clinically to detect the presence of lupus anticoagulants (LA). LA are autoantibodies of the IgG and IgM classes which interfere with the function of anionic phospholipids and prolong phospholipid-dependent clotting tests such as the aPTT and dRVVT. LA bind the available phospholipid, block the activation of factor II, and lead to a prolongation of the clotting time. The dRVVT is more specific for LA than the aPTT since it is not influenced by deficiencies of the contact or intrinsic pathway factors or antibodies to Factors VIII, IX, or XI.

The finding of a prolonged dRVVT with patient plasma is presumptive evidence for the presence of a lupus anticoagulant. This presumption is "confirmed" if the dRVVT is not corrected with a mixture of normal and platelet plasma, but is corrected by the substitution of platelets for phospholipid. With the DVVtest® and DVVconfirm® reagents, a DVVtest®/DVVconfirm® ratio >1.2 is confirmatory for the presence of LA.

Clinical correlation is absolutely essential in the interpretation of laboratory assays of lupus anticoagulants, since no single assay is reliable in every plasma specimen.

Exner, T., Papadopoulos, G., and Koutts, J. Use of a simplified dilute Russell viper venom time (dRVVT) confirms heterogeneity among "Lupus Anticoagulants." Blood Coagulation and Fibrinolysis. 1:259-266, 1990.

Harris, E.N., Asherson, R.A., and Hughes, G.R.V. Antiphospholipid antibodies - autoantibodies with a difference. Annu. Rev. Med. 39:261-271, 1988.

Lazarchick, J. and Kizer, J. The laboratory diagnosis of lupus anticoagulants. Arch. Pathol. Lab. Med. 113:177-180, 1989.

Saxena, R. Evaluation of four coagulation tests to detect plasma lupus anticoagulants. Am. J. Clin. Pathol.96:755-758, 1991.

Thiagarajan, P., Pengo, V., Shapiro, S.S. Use of a simplified dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood 68:869-874, 1986.
Web Links
http://www.coulter.com/Coulter/Viewpoint-Online/Coagulation/lupus.asp - Coulter