Practice and performance of lupus anticoagulant tests: A single centre experience

@#Introduction: Lupus anticoagulant (LA) is a well-known risk factor for thrombosis. Correct diagnosis of LA is essential in patient management with anticoagulation. The objectives of this study were to document the clinical and laboratory characteristics of patients tested for LA and to evaluate existing LA testing methods in our laboratory with the aim of improving the performance of LA test interpretation and reporting. Methods: Tests for LA include dilute Russell’s viper venom time (dRVVT) and Kaolin clotting time (KCT). Patients with LA ratio (dRVVT screen ratio/dRVVT confirm ratio) of ≥1.2 were considered as LA positive irrespective of KCT results. KCT was considered positive if there was a prolongation in KCT screening test which was not corrected on mixing with normal plasma. Results: Of 577 patients’ results, 295 were normal, 178 were KCT positive with negative dRVVT and 104 were LA positive. Incidences of thrombosis, connective tissue disease (CTD) and bad obstetric events were noted in 13%, 16% and 44% of normal patients, 9%, 22% and 49% of KCT+ patients and 23%, 37% and 17% of LA+ patients respectively. On further evaluation of dRVVT screen ratios, 431 had a ratio of <1.1, 59 had a ratio between 1.1 and 1.2 and 87 had a ratio of >1.2. Positive LA results were found in 3%, 29% and 87% of patients with dRVVT screen ratios of <1.1, 1.1 - 1.2 and >1.2 respectively. Conclusion: LA+ patients had higher incidences of thrombosis and CTD as compared to normal and KCT only positive patients. There was no significant difference in clinical characteristics between normal and KCT+ patients which suggests the presence of a high rate of false-positive KCT results. Since confirmatory testing for KCT is not widely used, the option of using another LA screening test method should be considered. In regard to dRVVT testing, confirmatory test should only be performed in patients with prolonged dRVVT screening result which was not corrected upon mixing with normal plasma as required by the International Society of Thrombosis and Haemostasis guidelines on LA testing. This practice will not only result in significant cost reduction but also avoid diagnostic confusion.

Laboratory measurement of bleeding risk for patients receiving Dabigatran / 临床检验杂志

Objective To observe the correlation between conventional coagulation tests and Dabigatran concentration in order to find proper test method to predict the bleeding risk of patients receiving Dabigatran.Methods The clinical data of forty-nine non-valvular atrial fibrillation (NVAF) patients who took Dabigatran in Peking University Third Hospital from 2015 to 2017 were analyzed and the bleeding rates were calculated.The plasma samples from twenty healthy volunteers were collected and mixed up into normal pooled plasma (NPP).Different doses of active Dabigatran were mixed with NPP,making Dabigatran concentrations from 0 to 1 000 ng/mL.Prothrombin time (PT),activated partial thromboplastin time (APTT),thrombin time (TT),fibrinogen (Fib) and diluted Russell viper venom time (dRVVT) were determined.Fresh whole blood samples from three normal volunteers were collected and mixed with Dabigatran in the same way to perform thromboelastogram (TEG).Results The total bleeding rate of NVAF patients receiving Dabigatran was 28.6％,among which gastrointestinal bleeding accounts for the most.APTT,dRVVT,R time (R) and clot index (CI) showed a good linear correlation with Dabigatran concentration.The sensitivity of dRVVT was the best,followed by APTT.R and CI showed low sensitivity compared with APTT and dRVVT.Conclusion APTT and dRVVT should be good parameters for monitoring the bleeding risk of Dabigatran and helpful for physicians to choose proper point-in-time for withdrawal of Dabigatran and reduce bleeding events.

Dabigatran en plasma: tiempos de trombina diluido, Ecarin y veneno de víbora de Russell diluido / Plasma dabigatran: diluted thrombin time, Ecarin and diluted Russell Viper Venom Time / Dabigatrana em plasma: tempos de trombina diluído, Ecarin e veneno de víbora de Russell diluído

Los objetivos del trabajo fueron verificar la calidad analítica del ensayo tiempo de trombina diluido (DTI) para medición de la concentración plasmática (cc) de dabigatran comparando dos coagulómetros de detección foto-óptica, comparar los resultados con el tiempo de Ecarin (ECT) y correlacionar las cc con las pruebas básicas de coagulación Tiempo de protrombina (TP), APTT y Tiempo de trombina (TT), y tiempo de veneno de víbora de Russell con fosfolípidos concentrados (DRVVTC). Se tomaron 43 muestras de plasma en el valle (10-14 h de la última toma) de 40 pacientes que recibían dabigatran. DTI y ECT presentaron (%) repetitividad <5,4% y <7,5%, CV interensayo <6% y <9%, respectivamente, en el protocolo EP15A2, aceptables para un Error Total permitido (TEa) <15%. Las cc medidas en pacientes fueron: mediana 83 (4-945) ng/mL. La comparación de equipos ACL TOP 300 y 500 dio resultados equivalentes por procedimiento alternativo de comparación de métodos. La comparación ECT vs. DTI fue satisfactoria por regresión de Deming (pendiente 1,143, ordenada al origen -19,33). Las correlaciones de cc vs. APTT, TP y DRVVTC fueron moderadas y no lineales tendiendo a plateau a cc>350 ng/mL, r2 0,59, 0,66 y 0,59, respectivamente. El TT fue extremadamente sensible: >120 s a cc 50 ng/mL. DTI presentó un buen desempeño analítico y permitió cuantificar dabigatran plasmático a cc bajas y altas en ambos equipos utilizados. ECT presentó resultados comparables a DTI. Se verifica una correlación moderada entre cc de dabigatran y las pruebas clásicas y DRVVTC, pudiendo ser estimadores de cc a partir de los 50 ng/mL.

The aims of the study were to verify the analytical performance of Dilute Thrombin Time (DTI) test to measure plasma dabigatran concentration (cc) in two photo-optical coagulometers, compare Ecarin clotting Time (ECT) and DTI results, and correlate cc with classical coagulation tests: prothrombin time (PT), APTT, thrombin time (TT) and diluted Russell Viper Venom Time tests with high phospholipid concentration (DRVVTC). Forty three plasma samples from 40 patients taking dabigatran were drown at through (10-14 hs.since last dose). DTI and ECT showed repetitivity (%) <5.4% and <7.5%, interassay CV <6% and <9%, respectively, following EP15A2 protocol, acceptable considering a Allowed Total Error (TEa)<15%. Patients` cc: median 83 (4-945) ng/mL. The comparison between ACL TOP 300 and 500 coagulometers showed equivalent results by using the alternative method comparison test. ECT vs. DTI: acceptable by Deming`s regression (slope 1.143, Y insert -19.33). cc vs. APTT, TP and DRVVTC: nonlinear and moderate correlations with plateau reached at cc >350 ng/mL, r2 0.59, 0.66 y 0.59, respectively. TT is extremely prolonged at cc >50 ng/mL. In conclusion: DTI showed a good analytical performance in both coagulometers. ECT showed comparable results to DTI. We verified that dabigatran cc presented moderate correlations with PT, APTT and DRVVTC, and that these tests could only qualitative estimate cc >50 ng/mL.

Os objetivos do trabalho foram verificar a qualidade analítica do ensaio tempo de trombina diluído (DTI) para medição da concentração plasmática (cc) de dabigatrana, comparando dois coagulômetros de detecção foto-óptica, comparar os resultados com o tempo de Ecarina (ECT) e correlacionar as cc com os testes básicos de coagulação Tempo de protrombina (TP), APTT e Tempo de trombina (TT), e tempo de veneno de víbora de Russell com fosfolipídios concentrados (DRVVTC). Foram tomadas 43 amostras de plasma no vale (10-14 h. da última toma) de 40 pacientes que recebiam dabigatrana. DTI e ECT apresentaram (%) repetitividade <5,4% e <7,5%, CV interensaio <6% e <9%, respectivamente, no protocolo EP15A2, aceitáveis para um Erro Total permitido (TEa) <15%. Cc medidas em pacientes: mediana 83 (4-945) ng/mL. Comparação de equipamentos ACL TOP 300 e 500: resultados equivalentes por procedimento alternativo de comparação de métodos. Comparação ECT vs. DTI: satisfatória por regressão de Deming (pendente 1,143, ordenada à origem -19,33). Correlações cc vs. APTT, TP e DRVVTC: moderadas e não lineares tendendo a plateau a cc>350 ng/mL, r2 0,59; 0,66 e 0,59, respectivamente. O TT é extremamente sensível: >120 s a cc 50 ng/mL. DTI apresentou um bom desempenho analítico e permitiu quantificar dabigatrana plasmática a cc baixas e altas em ambos os equipamentos utilizados. ECT apresentou resultados comparáveis com DTI. Verifica-se uma correlação moderada entre cc de dabigatrana e os testes clássicos e DRVVTC, podendo ser estimadores de cc a partir dos 50 ng/mL.

<b>Two Cases of Viper Bite Envenomation Treated with Saireito </b> / 日本東洋医学雑誌

Inflammatory swelling after viper bite envenomation has the potential for causing multiple organ dysfunctions. We report 2 cases of viper bite that were successfully treated with saireito. A 79-year-old man suffered viper bite envenomation. Saireito was administered to reduce the swelling of the right lower leg. The patient's symptom was improved without any complications. A 68-year-old man suffered the same condition. The swelling spread over the chest and tako-tsubo-like left ventricular (= apical ballooning cardiomyopathy) dysfunction developed. Saireito was administered to reduce the swelling of the left upper limb. The patient was discharged without any severe complications. Thus, saireito may be useful for the treatment of swelling due to viper bite envenomation.

Recombinant Russell’s viper venom-factor X activator (RVV-X)-specific antibody: neutralization and crossreactivity with Cryptelytrops albolabris and Calloselasma rhodostoma venoms.

Background: Russell’s viper venom-factor X activator (RVV-X) is a major procoagulant in Russell’s viper venom, and is composed of a heavy chain (RVV-XH) and two light chains (RVV-XL). It directly activates factor X in the final common coagulation pathway, which leads to rapid formation of blood clots. Objective: Produce rabbit anti-recombinant protein antibodies and identify their cross-reactivity with two viperine snake venoms. Methods: cDNA clones encoding RVV-XH and one of the light chains (RVV-XL; LC1) were recombinantly expressed in E. coli BL21 and used as antigens for rabbit immunization. The cross-reactivity of these anti-recombinant protein antibodies with two viperine snake venoms was determined using Western blot analysis. Results: rRVV-XH was more immunogenic than rRVV-XL. Rabbit anti-rRVV-XH and rRVV-XL IgG antibodies bind specifically to RVV-X, but they do not neutralize purified RVV-X. In addition, rabbit anti-rRVV-XH IgG antibody also bind to an 18-kDa protein in C. rhodostoma venom, and many proteins in C. albolabris venom. Rabbit antirRVV- XL IgG antibody recognized protein bands of crude venoms of C. rhodostoma and C. albolabris at about 25-kDa and 23-kDa, respectively. Conclusion: Rabbit anti-rRVV-XH and rRVV-XL IgG antibodies cross-reacted with molecules in other viperine venoms, which could have molecules with similar antigenic determinants. These antibodies could be useful to purify snake venom molecules by affinity chromatography as the first step in purification of factor X activator and other cross reacting molecules.

Effects of Venom Nerve Growth Factor on GAP-43 of Nerve Cell Following Cerebal Ischemia/reperfusion in Rat / 中国康复理论与实践

@# Objective To investigate the cerebral ischemia/reperfusion protection mechanism of viper venom nerve growth factor(vNGF) by the change of expression of growth associated protein-43 (GAP-43) and neurological function.Methods 45 adult male Wistar rats (weight 220～280 g) were divided randomly into 3 groups: sham group(S, n=9), balanced salt solution group (BSS, n=9) and venom nerve growth factor group (vNGF, n=27). Each group was observed for 7 days. vNGF group was divided into 25 U, 50 U and 100 U subgroups respectively. The following indexes in 3 groups were observed respectively: neurologic deficits and the expression of GAP-43 (immunohistochemistry method).Results Neurological function: The scores of neurological function was 0 in S group. The neurological deficits score was lower at the same time in vNGF group than that in BSS group (P<0.05). Immunohistochemistry: GAP-43 expressed in both BSS group and vNGF group. The expression of GAP-43 in vNGF group increased in 25 U, and to maximum in 100 U. The expression of GAP-43 in BSS group was significantly lower than in vNGF group (P<0.05). Conclusion vNGF can effectively enhance and prolong the expression of GAP-43, increase the survival rats of nerve cells, and has the protection effect on nerve cells after cerebral ischemia injured.

Preparation anti biological activity of bivalent IgY against cobra anti viper venom / 中华微生物学和免疫学杂志

Objective To prepare the bivalent immunoglobulin yolk (lgY) against eobra and viper venom and to detect its activities as the foundation for production and application of polyvalent . Methods The venom of Naja atra Cantor and Daboia russellii siamensis injected alternately into the leghorn hen. Biva-lent lgY was extraeted by water dilution. The biological activity of bivalent lgY were deteeted in several as-pects, sueh as the potency ( by indireet ELISA assay), the cross immunity ( by double immunodiffusion), the membrane lysis activity ( by experiments of vitelline membrane lysis) and 50% lethal activity ( LD50 ). Results Bivalent IgY was extracted from eggs yolk in 28-42 days after the first immunization. The titers of bivalent lgY against cobra and viper venom were 1:12 800 and 1: 6400. The cross immunologic reactions of bivalent IgY were found obviously with six kinds of snake venoms from Elapinae and Viperinae. There were not immunologic precipitation lines between bivalent IgY and four kinds of snake venoms from Crotalinae. Bi- valent lgY obviously deereased the vitelline membrane lysis activity of cobra and viper venom and prolonged the average survival time of mice with cobra or viper envenomation (P < 0.05). Moreover, with the same dose of bivalent IgY, the survival rate of mice with cobra venom envenomation was higher than those with vi-per venom envenomation. Conclusion Bivalent lgY could signifieantly neutralize biologieal activities of co-bra and viper venom, protect animals with cobra or viper envenomation.

Usefulness of Silica Clotting Time for Detection of Lupus Anticoagulants / 대한진단검사의학회지

BACKGROUND: The presence of lupus anticoagulants (LA) is a strong risk factor for thrombosis in antiphospholipid syndrome. We investigated the usefulness of addition of silica clotting time (SCT) to the pre-existing dilute Russell's viper venom test (dRVVT) for detection of LA. Also, we analyzed differences in the thrombotic features and the characteristics of antiphospholipid antibodies between dRVVT and SCT. METHODS: A total of 167 patients positive for LA or anti-cardiolipin (anti-CL) antibody and 76 healthy controls were enrolled. The dRVVT and SCT were used for detection of LA. Anti-CL, anti-beta2-glycoprotein I (anti-beta2 GPI) and anti-prothrombin (anti-PT) antibodies were measured using commercial ELISA kits. RESULTS: In detection of thrombosis, the sensitivity of the combined test of SCT and dRVVT was 56.4%, which was higher than that of dRVVT alone (46.2%) or SCT alone (23.1%). The specificity of the combined test (80.9%) was comparable to that of dRVVT (81.9%). Also, odds ratio for predicting thrombosis was higher in the combined test than in dRVVT or SCT alone. When normalized LA ratio of the two tests was compared, the group of patients with higher ratio of SCT showed significantly higher prevalence of recurrent abortion and higher positivity of IgG types of anti-CL, anti-beta2 GPI and anti-PT than the group with higher ratio of dRVVT. CONCLUSIONS: Addition of SCT to dRVVT can improve the detection sensitivity of thrombosis in LA test. And the high normalized LA ratio of SCT may be a useful parameter for detection of recurrent abortion.