Zsolt Oláh

Stem Cell Therapy: A Promising and Prospective Approach in the Treatment of Patients With Severe Buerger’s Disease

No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow—derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. A total of 5 patients with SPAD were treated by ABMSCT using isolated CD34+ cells with characterized phenotype administered by intramuscular injections. The follow-up before and 1, 3, 6, 9, and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, nonhealing ulcers, ankle-brachial index [ABI]) and laboratory (angiography, duplex and laser ultrasonography, TcPO2) parameters. Significant improvement of pain and walking distance was observed in all patients. Nonhealing ulcers disappeared in 3 patients and became smaller and thinner in 1 patient. The average of ABI improved significantly on the treated limb but did not change on the contralateral limb. New collaterals were detected by angiography in 3 patients, but duplex ultrasonography detected improvement in one patient only. Laser ultrasonography showed a mild significant change, TcPO2 values improved mainly on the foot. Severe adverse events were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, and results in sustained clinical benefit for patients with SPAD.

Founder effect is responsible for the p.Leu131Phe heparin-binding-site antithrombin mutation common in Hungary: phenotype analysis in a large cohort

Essentials Antithrombin Budapest3 (ATBp3; p.Leu131Phe) causing heparin‐binding‐site defect is common. We studied the clinical and laboratory phenotype of a large Hungarian ATBp3 cohort (n = 102). Founder effect of ATBp3 was confirmed by 12 genetic markers; anti‐FXa AT assay was 100% sensitive. The spectrum of thrombotic symptoms was wide in ATBp3 patients including arterial thrombosis.

The superiority of anti-FXa assay over anti-FIIa assay in detecting heparin-binding site antithrombin deficiency.

OBJECTIVES Antithrombin is a progressive inhibitor of active factor X (FXa) and thrombin (FIIa). Its effect is 500- to 1,000-fold accelerated by heparin or heparan sulfate. Heterozygous type I (quantitative) and most type II (qualitative) antithrombin deficiencies highly increase the risk of venous thromboembolism (VTE), while homozygous mutations are lethal. The functional defect affecting the heparin-binding site confers moderate risk of VTE to heterozygous and high risk of VTE to homozygous individuals. METHODS Antithrombin activity assays based on the inhibition of FIIa and FXa were compared for their efficiency in detecting heparin-binding site defects. RESULTS With a single exception, in heterozygotes for heparin-binding site defects (n = 20), anti-FIIa activities remained in the reference interval, while anti-FXa activities were uniformly decreased. In individuals who were homozygous for heparin-binding site mutations (n = 9), anti-FIIa activities were in the range of 48% to 80%; the range of anti-FXa activities was 9% to 25%. Anti-FIIa and anti-FXa activities in type I deficiencies and type II pleiotropic deficiency did not differ significantly. CONCLUSIONS Anti-FXa antithrombin assay is recommended as a first-line test to detect type II heparin-binding site antithrombin deficiency.

Rapid-onset heparin-induced thrombocytopenia without previous heparin exposure.

Heparin-induced thrombocytopenia (HIT) is one of the most common immune-mediated drug reactions. Immunoglobulin G-type antibodies against platelet factor 4(PF4)/heparin complexes are known to play a key role in the pathogenesis of HIT. Rapid-onset HIT is caused by the presence of circulating HIT antibodies at the time of heparin readministration. These antibodies are generally resulted from a recent immunizing exposure to heparin. Here we report a case of rapid-onset HIT developed after a septicemia without previous heparin exposure. The diagnosis of HIT as well as the presence of platelet activating and heparin-dependent antibodies was confirmed by ELISA and flow cytometric functional assays. Our case report reinforces that rapid-onset HIT cannot be excluded only based on the absence of previous heparin exposure. In addition, it may support the new theory of pre-immunization by PF4-coated bacteria in the pathomechanism of HIT. We also call the attention that venous limb gangrene can be rarely associated with HIT and thrombosis even in the absence of coumarin therapy. Furthermore, transient presence of anti-phospholipid antibodies can cause a differential diagnostic problem in the cases of HIT.

Management and outcome of pregnancies in women with antithrombin deficiency: a single-center experience and review of literature.

Women with antithrombin (AT) deficiency have an increased risk for pregnancy-associated venous thromboembolism (VTE) and adverse pregnancy outcome. AT deficiency is a rare thrombophilia with heterogeneous genetic background. Owing to the few cases reported in the literature, management strategies of pregnancy with AT deficiency are inconsistent. Our aim was to examine the type of the genetic defect, management, maternal, and pregnancy outcome in patients with hereditary AT deficiency. Five expectant mothers with AT deficiency were followed in our center to evaluate thrombotic events, and maternal and pregnancy outcomes. AT gene sequencing was performed in all cases, and levels of AT and anti-activated factor X were regularly measured to guide the risk-adopted anticoagulant prophylaxis. Three mothers had homozygous type II heparin-binding site mutations and two had heterozygous type I mutations of the gene encoding AT. Two women had additional factor V Leiden heterozygous mutations. Three maternal VTEs – four healthy newborns and five pregnancy losses – were observed. The risk of patients to VTE and adverse pregnancy outcome was found to associate with the homozygous type II heparin-binding site mutation of the AT gene. High risk of maternal VTE and frequent pregnancy complications were observed to associate with AT deficiency. Our results support the need of individualized, risk-adopted anticoagulant therapy in patients with AT deficiency.

Direct Thrombin Inhibitors and Factor Xa Inhibitors Can Influence the Diluted Prothrombin Time Used as the Initial Screen for Lupus Anticoagulant

CONTEXT Lupus anticoagulant (LA) is a heterogeneous group of antiphospholipid antibodies. Among others, diluted prothrombin time (dPT) is a sensitive screening test for LA; however, the interpretation of LA tests is difficult in patients treated with anticoagulants. The effect of different types of anticoagulants on the result of LA tests, particularly on dPT, has not been studied extensively. OBJECTIVE To determine whether the direct thrombin inhibitors lepirudin and argatroban and the predominantly factor Xa inhibitors enoxaparin, danaparoid, and fondaparinux could interfere with LA screening based on dPT. DESIGN Each drug was added to normal and LA-positive plasmas in clinically relevant concentrations. Each sample was tested for dPT. Samples with factor Xa inhibitors were investigated before and after addition of heparinase. Mixing and confirmatory tests for LA were not performed. RESULTS In the presence of lepirudin or argatroban, dPT increased notably and the dPT ratio exceeded the cutoff value even at subtherapeutic concentrations resulting in false positivity. With increasing factor Xa inhibitor concentrations, a linear increase of dPT ratios and false-positive results were also demonstrated. Although heparinase could almost completely neutralize the anti-Xa effect of all investigated factor Xa inhibitors, dPT ratio returned to the basal level only in case of enoxaparin. CONCLUSIONS Here we provide evidence that both the direct thrombin and indirect factor Xa inhibitors influence dPT assay for LA, causing false positivity. This should be considered when interpreting LA results during anticoagulant therapy. However, dPT seems to be a reliable test for LA screening under enoxaparin therapy after neutralization by heparinase.

Autológ csontveloi eredetu ossejtterápia eredmńye elorehaladott perifériás artériás érbetegségben

BACKGROUND AND AIMS Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease. Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. PATIENTS AND METHODS Five patients with severe peripheral arterial disorder were treated by autologous bone marrow-derived stem cell therapy. CD34+, CD133+ and CD45+/- cell number and ratio were determined. CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The cell suspension was administered by local intramuscular injections (0.5-1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1, 3, 6, 9 and 12 months after the autologous bone marrow-derived stem cell therapy) based on clinical (rest pain, walking distance without pain, changes of non-healing ischaemic ulcers, ankle-brachial index) and laboratory (angiography, Color- and Laser-Doppler scan, measurement of transcutaneous oxygen tension and endothelial function test) parameters was documented and analyzed. RESULTS Improvement of pain and walking distance was observed in all five cases. In three cases the non-healing ischaemic ulcers disappeared, in one other case they became smaller and thinner, and in one case no change was realized. The average of ankle-brachial index improved significantly (before: 0.41, twelve months after: 0.83). New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in one patient only. Before and 1, 6 and 12 months after stem cell therapy the transcutaneous oxygen tension changed on the foot from 18.80/16.78/23.83/37.50 mmHg, and on the calf from 36.66/31.25/45.00/37.30 mmHg. The macro- and microcirculation parameters did not show improvement after 1 month, however, after 3, 6, 9 and 12 months improved parameters were recorded. Severe adverse events were not observed. In one case elevated level of serum creatinine phosphokinase, and in another case a mild form of vasculitis were detected. CONCLUSION autologous bone marrow-derived stem cell therapy with isolated CD34+ cells is effective, safe and results in sustained clinical benefit for patients with severe peripheral arterial disease.

Evaluation of flow cytometric HIT assays in relation to an IgG-specific immunoassay and clinical outcome.

Heparin‐induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)–heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT.

Thrombin generation and low-molecular-weight heparin prophylaxis in pregnant women with thrombophilia

Pregnancy is associated with increased risk of venous thromboembolism, especially in the presence of thrombophilia. However, there is no consensus on the optimal approach for thromboprophylaxis in this population. Recent evidence suggests that thrombin generation correlates with the overall procoagulant state of the plasma. Our aim was to evaluate thrombin generation in a prospective cohort of thrombophilic pregnant women, and investigate the effectiveness of low-molecular-weight heparin (LMWH) prophylaxis in pregnancy. Women with severe (n=8), mild (n=47) and no (n=15) thrombophilia were followed throughout their pregnancies. Thrombin generation was evaluated in each trimester as well as five days and eight weeks postpartum (as a reference category). In women undergoing LMWH prophylaxis, thrombin generation and anti-Factor-Xa activity were measured just before and 4 hours after administration (peak effect). Thrombin generation was determined using Technothrombin TGA assay system. For the analysis, median peak thrombin and endogenous thrombin potential were used. Peak thrombin and endogenous thrombin potential were increased during pregnancy compared to the non-pregnant state with the highest results in the severe thrombophilia group. In women receiving LMWH prophylaxis a decrease was observed in thrombin generation at peak effect but over the progression of pregnancy the extent of this decrease reduced in a stepwise fashion. Our results show that thrombin generation demonstrates the hypercoagulable state in thrombophilic pregnancies. In addition, we found the effect of LMWH prophylaxis to progressively decrease with advancing stages of pregnancy.

Progressive chromogenic anti-factor Xa assay and its use in the classification of antithrombin deficiencies.

Abstract Background: Antithrombin (AT) is a slow-acting progressive inhibitor of activated clotting factors, particularly thrombin and activated factor X (FXa). However, the presence of heparin or heparan sulfate accelerates its effect by several magnitudes. AT deficiency, a severe thrombophilia, is classified as type I (quantitative) and type II (qualitative) deficiency. In the latter case mutations may influence the reactive site, the heparin binding-site (HBS) and exert pleiotropic effect. Heterozygous type II-HBS deficiency is a less severe thrombophilia than other heterozygous subtypes. However, as opposed to other subtypes, it also exists in homozygous form which represents a very high risk of venous thromboembolism. Methods: A modified anti-FXa chromogenic AT assay was developed which determines both the progressive (p) and the heparin cofactor (hc) activities, in parallel. The method was evaluated and reference intervals were established. The usefulness of the assay in detecting type II-HBS AT deficiency was tested on 78 AT deficient patients including 51 type II-HBS heterozygotes and 18 homozygotes. Results: Both p-anti-FXa and hc-anti-FXa assays showed excellent reproducibility and were not influenced by high concentrations of triglyceride, bilirubin and hemoglobin. Reference intervals for p-anti-FXa and hc-anti-FXa AT activities were 84%–117% and 81%–117%, respectively. Type II-HBS deficient patients demonstrated low (heterozygotes) or very low (homozygotes) hc-anti-FXa activity with normal or slightly decreased p-anti-FXa activity. The p/hc ratio clearly distinguished wild type controls, type II-HBS heterozygotes and homozygotes. Conclusions: Concomitant determination of p-anti-FXa and hc-anti-FXa activities provides a reliable, clinically important diagnosis of type II-HBS AT deficiency and distinguishes between homozygotes and heterozygotes.