[Middle meningeal artery embolization to treat acute epidural haematoma, case report and literature review]. / Akut epiduralis haematoma kezelése arteria meningea media embolisatióval, esetismertetés és irodalmi áttekintés.

The treatment of acute epidural haematoma is surgery as soon as possible, elimination of the source of bleeding and evacuation of the haematoma. In case of small epidural haematoma, strict neurological and radiological follow-up is necessary. In a significant percentage of cases, open surgery must also be performed within a few days. In case of small epidural haematomas, embolization of the middle meningeal artery is considered as an alternative solution. We review the literature on middle meningeal artery embolization and present our first treatment. Our case report is the first European report about an acute epidural haematoma which was treated by embolization of middle meningeal artery. Our case study is the first report in which a patient was treated with both open surgery and endovascular treatment for acute epidural haematoma within a year.

Clinical and Molecular Characterization of Nine Novel Antithrombin Mutations.

Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.

High penetrance of inferior vena cava system atresia in severe thrombophilia caused by homozygous antithrombin Budapest 3 variant: Description of a new syndrome.

Atresia of inferior vena cava (IVC) is a rare congenital malformation associated with high risk of venous thrombosis that still has unknown etiology, although intrauterine IVC thrombosis has been suggested to be involved. The identification of IVC atresia in a case with early idiopathic venous thrombosis and antithrombin deficiency caused by the homozygous SERPINC1 c.391C > T variant (p.Leu131Phe; antithrombin Budapest 3) encouraged us to evaluate the role of this severe thrombophilia in this vascular abnormality. We have done a cross-sectional study in previously identified cohorts of patients homozygous for the Budapest 3 variant (N = 61) selected from 1118 patients with congenital antithrombin deficiency identified in two different populations: Spain (N = 692) and Hungary (N = 426). Image analysis included computed tomography and phlebography. Atresia of the IVC system was observed in 17/24 cases (70.8%, 95% confidence interval [CI]: 48.9%-87.3%) homozygous for antithrombin Budapest 3 with available computed tomography (5/8 and 12/16 in the Spanish and Hungarian cohorts, respectively), 16 had an absence of infrarenal IVC and one had atresia of the left common iliac vein. All cases with vascular defects had compensatory mechanisms, azygos-hemiazygos continuation or double IVC, and seven also had other congenital anomalies. Short tandem repeat analysis supported the specific association of the IVC system atresia with SERPINC1. We show the first evidence of the association of a severe thrombophilia with IVC system atresia, supporting the possibility that a thrombosis in the developing fetal vessels is the reason for this anomaly. Our hypothesis-generating results encourage further studies to investigate severe thrombophilic states in patients with atresia of IVC.

Principles of the perioperative Patient Blood Management / A perioperatív vérgazdálkodási program alapelvei.

The perioperative Patient Blood Management (in Hungary National Blood Donation and Blood Saving Program) is an individualized clinical practice based on a multidisciplinary consensus with a comprehensive and complex approach. It supports the rational and judicious utilization of blood products and abolishes irrational transfusion policy. Its practical implementation is based upon three pillars: 1. anemia management without transfusion, if possible; restrictive transfusion strategy; 2. minimization of blood loss; 3. enhancement of anemia tolerance. Early detection, clarification of etiology and appropriate treatment are the most important tools for the management of preoperative anemia before surgeries with a high risk of bleeding. Minimization of blood loss can be achieved by identifying patients with congenital or acquired bleeding disorders, preparing them appropriately for surgery, discontinuing anticoagulants and antiplatelet drugs for a sufficient time in the preoperative phase of surgery and reversing their effects to comply with current guidelines. Minimal-invasive approaches are preferable. Intraoperatively, atraumatic technique and accurate topical haemostasis should be provided by surgeons. Autologous blood salvage techniques and controlled hypotension in lack of contraindications can also reduce the amount of blood loss. In cases of perioperative bleeding, protocols based on international guidelines but adapted to local circumstances must be used. Ideally, it should be managed by viscoelastic test-guided, goal-directed, individualized and factor concentrate-based algorithm. Perioperatively, an ideal oxygen demand/supply ratio must be ensured to avoid oxygen debt. Restoration and maintenance of homeostasis are essential for both the effectively functioning haemostatic system and the avoidance of oxygen deficit. Implementation of the Patient Blood Management improves patient safety, reduces the cost of medical care and facilitates the national blood product supply. Its successful introduction is our common interest. Orv Hetil. 2020; 161(37): 1554-1568.

Obstetrical aspects of the National Blood Donation and Blood Saving Program / A Nemzeti Véradó és Vérmento Program szülészeti vonatkozásai.

The aims of the National Blood Donation and Blood Saving Program are to support the rational and judicious utilization of blood products and abolish irrational transfusion policy to improve patient safety. In addition to the general principles, this program has got some special obstetrical aspects. Obstetrical, especially the postpartum haemorrhages belong to the leading causes of maternal mortality worldwide. In developed countries, a trend in increasing incidence can be observed. Preparing for delivery includes some important elements such as optimization of hemoglobin level, routinely applied prophylactic or therapeutic iron supplementation and early screening and comprehensive care of patients with high risk of obstetrical bleeding. The main causes of peripartum bleeding are abruptio placentae, placenta praevia, uterine atony, retained tissue in the uterus, trauma during delivery, and haemostatic disorders or their combinations. To prevent postpartum bleeding, it is important to use the active management of the third stage of labour including prophylactic utilization of uterotonics as an essential element. Utilization of blood salvage techniques with adequate indications may be considered in cases of cesarean section or postpartum haemorhage. In cases of obstetrical haemorrhage, management of surgical bleeding has the main priority by the obstetrician. Secondary coagulopathy associated with massive bleeding should be managed by viscoelastic test-guided, individualized and factor concentrate-based algorithm, however, pregnancy-specific reference and target ranges must be used that are different from the non-pregnancy values. Obstetrical bleedings belong to the potentially preventable causes of death. Hopefully, the implementation of the National Blood Donation and Blood Saving Program in the field of obstetrics can decrease the associated morbidity and mortality further. Orv Hetil. 2020; 161(37): 1588-1598.

A decade-long clinical experience on the prophylactic use of activated prothrombin complex concentrate in acquired haemophilia A: a case series from a tertiary care centre.

: In acquired haemophilia A (AHA), risk for recurrent bleeding exists until the inhibitor is detectable. Thus, patients with persisting inhibitor may benefit from prophylaxis with activated prothrombin complex concentrate (aPCC). Potential thromboembolic complications and cost are also factors to consider. Today, no high level evidence or clear recommendations are available on aPCC prophylaxis in AHA. Recently, a small prospective study demonstrated a favourable outcome with short-term, daily administered aPCC infusion. Here we report a retrospective case series of 19 patients with AHA to demonstrate our practice on aPCC prophylaxis. In our practice, clinical bleeding tendency guided our decision on the initiation of aPCC prophylaxis. In patients with serious bleeding tendency, aPCC infusion was prolonged beyond bleeding resolution in a twice-weekly or thrice-weekly regimen. Serious bleeding phenotype included a single episode of life-threatening bleeding or recurrent, severe haemorrhages. Patients who did not present such events were treated on-demand. The preventive dose of aPCC was equal with the lowest effective therapeutic dose. Prophylaxis was continued until the inhibitor disappeared. Eleven patients received aPCC prophylaxis. In nine cases, prophylaxis lasted beyond two months. No severe bleeding developed spontaneously and no thromboembolic complication occurred in the median 16 weeks (interquartile range 9-34) duration of prophylaxis. Eight patients of the nonprophylaxis group did not present any severe haemorrhage. According to our experience, we consider prophylaxis with aPCC effective and well tolerated for patients with AHA and serious bleeding tendency, until the acquired inhibitor persists.

[Laboratory aspects of novel oral anticoagulant treatment]. / Az új orális antikoagulánsokkal történo kezelés laboratóriumi vonatkozásai.

The introduction of novel oral anticoagulants (NOAC) have long been expected drugs and they quickly became used widespread as their clinical effectiveness was as good as, or even better than the previously used only oral anticoagulant drug, the coumarins. Thus, the direct thrombin inhibitor dabigatran and the activated factor X inhibitors (rivaroxaban, apixaban, edoxaban) have become the part of daily therapeutic practice. Their permeation was facilitated by the guideline which suggested that no laboratory monitoring was required during NOAC treatment and this was very convenient for both patients and doctors. The clinical experience obtained in the past years, however have proved that the 'one size fits all' view is oversimplified and there are numerous situations when the determination NOAC levels is unavoidable or highly recommended. This review discusses the laboratory aspects of NOAC treatment, primarily summarizing their effect on the screening tests and special assays of hemostasis and we also describe the correct methods to determine their plasma concentrations. Orv Hetil. 2017; 158(49): 1930-1945.

Clinical and laboratory characteristics of antithrombin deficiencies: A large cohort study from a single diagnostic center.

INTRODUCTION: Inherited antithrombin (AT) deficiency is a heterogeneous disease. Due to low prevalence, only a few studies are available concerning genotype-phenotype associations. The aim was to describe the clinical, laboratory and genetic characteristics of AT deficiency in a large cohort including children and to add further laboratory data on the different sensitivity of functional AT assays. PATIENTS AND METHODS: Non-related AT deficient patients (n=156) and their family members (total n=246) were recruited. Clinical and laboratory data were collected, the mutation spectrum of SERPINC1 was described. Three different AT functional assays were explored. RESULTS: Thirty-one SERPINC1 mutations including 11 novel ones and high mutation detection rate (98%) were detected. Heparin binding site deficiency (type IIHBS) was the most frequent (75.6%) including AT Budapest3 (ATBp3), AT Padua I and AT Basel (86%, 9% and 4% of type IIHBS, respectively). Clinical and laboratory phenotypes of IIHBS were heterogeneous and dependent on the specific mutation. Arterial thrombosis and pregnancy complications were the most frequent in AT Basel and AT Padua I, respectively. Median age at the time of thrombosis was the lowest in ATBp3 homozygotes. The functional assay with high heparin concentration and pH7.4 as assay conditions had low (44%) sensitivity for ATBp3 and it was insensitive for AT Basel and Padua I. CONCLUSION: Type IIHBS deficiencies behave differently in clinical and laboratory phenotypes from each other and from other AT deficiencies. Heparin concentration and pH seem to be the key factors influencing the sensitivity of AT functional assays to IIHBS.

Antithrombin Debrecen (p.Leu205Pro) - Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency.

INTRODUCTION: Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS: Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS: Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans­Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS: AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

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

BACKGROUND: 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. METHODS: The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. RESULTS: EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. CONCLUSIONS: EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society.

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.

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.

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.

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.

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.

Acquired haemophilia: an often overlooked cause of bleeding - experience from a Hungarian tertiary care centre.

Acquired haemophilia is a potentially life-threatening bleeding disorder. Its early diagnosis and treatment is of major importance. We evaluated the elapsed time between the first presentation of the bleeding symptoms and the correct diagnosis in the cases of the acquired haemophilia patients referred to our centre between 1999 and 2011. The causes and consequences of the often delayed diagnosis were also examined. The clinical and laboratory data of 13 patients with acquired haemophilia were analysed. Eleven patients had inhibitors to factor VIII (FVIII), in one case the autoantibody developed to factor XIII (FXIII) and in one to factor V (FV). The median period between the onset of the bleeding symptoms and the correct diagnosis was 1.5 months (3.0 days-9.0 months). In four cases 4.0-9.0 months were needed to establish the diagnosis. The main reason of this delay was that either the prothrombin time was used exclusively to evaluate haemostasis in primary care and also in some secondary care centres, or the prolonged activated partial thromboplastin time went unnoticed despite the obvious bleeding symptoms. Our observation underlines the importance of early referral of patients with unexplained bleeding symptoms to centres with appropriate laboratory facilities and experience in the diagnosis of bleeding disorders.

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.

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, TcPO(2)) 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, TcPO(2) 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.