Flow cytometry for meningeal infiltration in B acute lymphoblastic leukemia in a low middle income country.

Meningeal infiltration in children with B acute lymphoblastic leukemia is one of the most serious complications. Timely diagnosis not only significantly enhances treatment efficacy but also leads to improve patient outcome and reduce risk of relapse. This is particularly crucial in low to middle income countries facing health constraints, where optimizing resources is essential. Conventional cytology (CC) study of cerebrospinal fluid (CSF) is considered in different countries to be the Gold-standard despite its low sensitivity (< 50%). The study of CSF by multiparametric flow cytometry (MFC) appears to be an alternative. The aim of our study was to assess MFC analytical performance compared with CC. Our cross sectional study was conducted over a six-month period in the biological hematology department. CSF samples underwent analysis for the presence of blasts using both CC and MFC. Cytological slides of the CSF were prepared by cytocentrifugation in a Shandon Cytospin 4™. Flow cytometric analysis was performed on the BD FACSLyric™ flow cytometer. All statistical analyses were performed using SPSS version 21.0 (SPSS Inc.). Agreement between the two methods was made using the Kappa index and χ2 test. This study was approved by the local ethics committee. Sixty CSF samples from 39 children with B acute lymphoblastic leukemia were analyzed. Meningeal infiltration was detected respectively in 20% of cases by MFC and 5% of cases by CC, with a significant difference p = 0.006. Comparing the two methods, the Kappa coefficient was 0.35, indicating weak agreement between the two methods. Moreover, MFC positivity was higher even for hypocellular samples. Of the 51 hypocellular samples, eight were positive by MFC while they were negative by CC. MFC shows better sensitivity while retaining good specificity for the detection of meningeal involvement. MFC could therefore be a complementary method to CC for detecting blast cells in the central nervous system.

Challenges associated with access to plasma-derived medicinal products in low middle-income and low-income countries.

BACKGROUND AND OBJECTIVES: Plasma-derived medicinal products (PDMPs) are essential to treat many chronic conditions such as haemophilia and primary immunodeficiency. Patients living in low middle-income and low-income countries (LMICs and LICs, respectively) have limited access to PDMPs. The aim of this article is to explore the challenges of accessing PDMPs in LMICs and LICs. MATERIALS AND METHODS: A review of the literature and reports on blood safety, plasma production and its utilization to produce PDMPs in LMICs and LICs was carried out. RESULTS: There is huge wastage of recovered plasma in LMICs and LICs as a result of a lack of good manufacturing practice (GMP) in the production of plasma for fractionation. Together with the high cost of imported PDMP procurement, patients have limited access to such products. CONCLUSION: There is a need to improve the situation by using domestically sourced plasma through the initiation of local plasma programmes through a stepwise approach to improve access to PDMPs in LMICs and LICs.

Clot waveform analysis in acute promyelocytic leukemia.

The aim of this study was to evaluate the activated partial thromboplastin time (APTT) and prothrombin time (PT)-based clot waveform analysis (CWA) in patients diagnosed with acute promyelocytic leukemia (APL). APTT-based and PT-based CWA parameters of patients diagnosed with APL were analyzed and compared with healthy volunteers. Four APTT-CWA parameters were noted, maximum velocity corresponding to the first peak of the first derivative (max1), maximum acceleration corresponding to the first peak of the second derivative (max2) and the corresponding peak times of max1 and max2 (Tmax1, Tmax2). For the PT-CWA, two PT-CWA parameters were noted, maximum velocity (max1') and the corresponding timing (Tmax1'). The results were expressed in medians. Mann-Whitney U test was used to compare the CWA parameters. Correlations were examined using the Spearman correlation test. Tmax1 and Tmax2 were significantly prolonged in patients with APL in comparison with healthy volunteers. Although max1 and max2 were lower in APL patients compared with healthy volunteers, no significant difference was noted. There was a strong and significant correlation between the DIC score and the parameters max1, max2 and max1' and a very strong and significant correlation between fibrinogen levels and max1, max2 and max1'. When comparing DIC patients with hypofibrinogenemia and DIC without hypofibrinogenemia, a significant difference was noted in max1, max2, Tmax1 and Tmax2. The APTT and PT-based CWA analysis is a good tool to evaluate the bleeding tendency in APL, as it offers a novel approach for evaluating global hemostasis, predicting the bleeding risk and delivering improvements to APL patients management.

Efficacy and safety of von Willebrand factor concentrate almost devoid of factor VIII (Wilfactin®) in paediatric patients under 6 years of age with severe von Willebrand disease.

BACKGROUND: Plasma-derived von Willebrand factor (VWF) (Wilfactin®, LFB, France) was developed for prophylaxis and treatment of haemorrhages in both adults and adolescents with von Willebrand disease (VWD). Replacement therapy in paediatric patients is a key element of the clinical trial programme. MATERIAL AND METHODS: Patients aged <6 years with severe VWD were enrolled in a multinational, open-label study to evaluate the in vivo recovery for Wilfactin®, and its efficacy in preventing and treating bleeding episodes and during surgery. Overall haemostatic efficacy based on a 4-point scale was assessed by investigators. The treatment period ≥18 months investigated the long-term safety. RESULTS: Nine patients, including 7 with type 3 VWD were exposed to treatment with Wilfactin® for up to 4.2 years. Recovery of VWF in 7 patients (n=5 type 3, n=1 type 2, n=1 type 1) was 1.8±0.4 IU/dL per IU/kg. Of the 62 bleeds, 89% were controlled with one (73%) or two (16%) infusions of Wilfactin®. The median dose per infusion was 54 IU/kg. A factor VIII dose was co-administered in 1.6% of bleeds. "Excellent"/"Good" haemostatic efficacy was achieved in 90.3% of episodes. Six patients underwent 11 minor surgical interventions. Treatment duration was 1 day (range: 1-6 days) with a dose administered 30-60 minutes before procedure of 56 IU/kg (range: 41-106 IU/kg). Haemostasis was rated as "Excellent" in all surgeries. During 4-year prophylactic treatment in one patient, breakthrough bleeds were reported in 2.2% of infusions. No VWF inhibitors, thromboembolic events or allergic/anaphylactic-type reactions were observed following a total exposure of 770 days. DISCUSSION: The results show that Wilfactin® provides a safe and effective treatment in patients <6 years of age with severe VWD.

Molecular heterogeneity of factor XI deficiency in Tunisia.

Factor XI (FXI) deficiency is a rare inherited bleeding disorder that is highly prevalent in Ashkenazi Jewish ancestry but sporadically observed in most ethnic groups. It is heterogeneous both in clinical presentation and in genetic causality. Although a large spectrum of mutations associated with this disorder has been reported in several populations, genetic data of FXI deficiency in Tunisia are poorly described. The purpose of this study was to determine the molecular basis of FXI deficiency among Tunisian patients. Fourteen index cases from nine unrelated families with FXI deficiency, referred to Hemophilia Treatment Center of Aziza Othmana Hospital, were included in this study. The patients' F11 genes were amplified by PCR and subjected to direct DNA sequencing analysis. Sequencing analysis of F11 genes identified three distinct mutations; the Jewish type II nonsense mutation E117X, one previously reported missense mutation E602Q and one novel missense mutation V271M, which led to the disruption of the third apple domain structure of FXI. Furthermore, seven polymorphisms previously described, were also detected: C321F, c. 294A>G, -138 A>C, p.D125D, p.T249T, p.G379G, p.D551D. This report represents the first genetic study analyzing the molecular characteristics of factor XI deficiency within Tunisian population. Identification of the Jewish type II mutation in two families, as well as one missense previously reported mutation and one novel mutation confirmed the genetic heterogeneity of this disorder. Screening a large number of Tunisian factor XI deficient would reveal the spectrum mutations causing factor XI deficiency in Tunisia.

Factor XI deficiency: About 20 cases and literature review.

INTRODUCTION: Factor XI deficiency is a rare coagulation disorder with variable bleeding manifestations. AIM: To evaluate the correlation between the degree of factorXI deficiency and the clinical expression of the disease. METHODS: Retrospective study, spanning 10 years from January 1, 2010 to December 31, 2019, concerning patients followed at the Hemophilia Center at Aziza Othmana Hospital in Tunis. The data were collected from the medical records. The determination of PT, APTT, fibrinogen level and coagulation factors are performed by coagulometric technique on STA® compact / ACL TOP®. FactorXI deficiency was confirmed on two different samples. Statistical analysis of the clinical-biological correlation was performed using the chi-square test. The significance level was 0.05. RESULTS: Twenty patients were collected. The mean age of discovery was 25 years with a sex ratio (M/F) =0.33. The circumstances of discovery were incidental in 14 patients. A family history of bleeding was reported in 30% of cases. Eight patients underwent surgery, six of whom had a simple postoperative course. The APTT was prolonged and isolated in 75% of cases. The hemostasis test was normal in 5 cases. The average FactorXI level was 24%. The tendency to bleed did not seem to be correlated with FactorXI levels. CONCLUSION: Prospective multicenter studies including molecular study would be necessary to better elucidate this rare disorder.

Clinical phenotype and F7 gene genotype in 40 Tunisian patients with congenital factor VII deficiency.

Congenital factor VII (FVII) deficiency is an autosomal recessive bleeding disorder characterized by a weak phenotypic and genotypic correlation. This study aimed to determine the genetic alterations of 40 Tunisian patients and to evaluate their relationships with the collected clinical and biological data. Forty FVII-deficient Tunisian patients have been included in this study. First, diagnosis of the FVII deficiency was made on the basis of FVII coagulant activity (FVII:c) levels performed using the prothrombin time assay. Then, clinical and anamnesis data were set up and filed out from the regional registry of bleeding disorders and the medical file of each patient. Finally, genetic alterations were determined by direct sequencing of the coding regions, intron/exons boundaries of the F7 gene. Clinical heterogeneity was noticed, and the direct sequencing allowed the identification of 13 F7 gene mutations of which one was a novel mutation. The clinical manifestations are variably associated with FVII activity FVII:c levels. Lack of relations between severity of clinical manifestations and genotypes was observed; however, a relationship between the nonpathogeneous mutations and clinical phenotypes was noticed. A wide phenotypic inter-individual variability was detected, which suggests the presence of other extra-genetic components influencing the expressivity of the deficiency.

Low dose prophylaxis and antifibrinolytics: Options to consider with proven benefits for persons with haemophilia.

INTRODUCTION: Prophylaxis has become standard of care for persons with severe phenotype haemophilia (PWsH). However, 'standard prophylaxis' with either factor or non-factor therapies (emicizumab) is prohibitively expensive for much of the world. We sought to evaluate whether haemophilia care can be provided at a lower cost yet achieve good results using Lower dose/Lower frequency prophylaxis (LDP) and with increasing use of antifibrinolytics (Tranexamic acid and Epsilon amino caproic acid). METHODS: We identified 12 studies that collectively included 335 PWsH using LDP. Additionally, we undertook a literature search regarding the benefits of antifibrinolytics in haemophilia care. RESULTS: Identified studies show that LDP is far superior to no prophylaxis (On demand [OD] therapy) resulting in significant patient benefits. Patients on LDP showed (in comparison to patients OD) on average: 72% less total bleeds; 75% less joint bleeds; 91% less days lost from school; 77% less hospital admission days; and improved quality of life measures. These benefits come at similar or only slightly higher (< 2-fold greater) costs than OD therapy. Antifibrinolytics are effective adjunctive agents in managing bleeds (oral, nasal, intracranial, possibly other) and providing haemostasis for surgeries (particularly oral surgeries). Antifibrinolytics can substitute for more expensive factor concentrates or can reduce the use of such concentrates. There is evidence to show that antifibrinolytics may be used in conjunction with factor concentrates/emicizumab for more effective/less costly prophylaxis. CONCLUSIONS: The use of LDP along with appropriate and increased use of antifibrinolytics offers less resourced countries good options for managing patients with haemophilia.

Acquired FVII Deficiency and Acute Myeloid Leukemia: A Case Report and Literature Review.

Factor VII (FVII) deficiency is the most common among all rare inherited bleeding disorders. However, acquired FVII deficiency (aFVIID) is uncommon. Only few cases in the literature have been reported. Herein, we present a case of an aFVIID associated with acute myeloid leukemia (AML), along with a literature review regarding this condition. A 50 year old Arab male patient was diagnosed with AML at the hematology department of our institution. At admission, coagulation tests showed a prolonged prothrombin time (PT) with a normal activated partial thromboplastin time (aPTT) and a slightly elevated fibrinogen level. Prothrombin complex coagulation factors dosing (PCCFD) revealed a decrease only in FVII levels. The patient, however, did not experience any bleeding. The evolution of the health of the patient was marked by a normalization of PT and FVII levels and complete remission.

[Contribution and limits of CD157 in the detection of the paroxysmal nocturnal hemoglobinuria clone: a case report]. / Apport et limites du CD157 pour la recherche d'un clone d'hémoglobinurie paroxystique nocturne Illustration par un cas.

Flow cytometry is the gold standard for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones. Several antibody panels have been used with varying sensitivities and specificities. The CD157 is one of the glycosylphosphatidylinositol-anchored molecules tested and widely used. The CD157 deficiency is rare. We report a case of an isolated CD157 deficiency discovered during the search for the PNH clone in a patient with a plastic anemia. The interpretation of the results in this case poses a problem of false positive. We discuss how to deal with these difficulties encountered by the biologist, detailing the various possible causes. This observation also underlines the importance of following international guidelines before making the diagnosis of the PNH clone which has significant implications.

Show me the evidence: Effectiveness of low-dose prophylaxis.

Prophylaxis is the gold standard treatment for haemophilia but requires more amount of clotting factor concentrates, than on demand therapy. Low dose prophylaxis is an alternative for countries with limited resources. There are data of evidence showing the superiority of low-dose prophylaxis than episodic treatment. Studies from China, India, Tunisia, Thailand and Indonesia reported experiences with low dose prophylaxis using outcome assessment. These studies have shown the effectiveness of various protocols regimen with once, twice or thrice injection of 10-15 UI Kg-1 per injection. These protocols allow reduction of joint bleeds and at least delay of joint damages. There is not enough long-term data nowadays, but low dose prophylaxis is certainly better than on demand therapy and should be considered as a first step of prophylaxis in some countries but not the final goal.

Heavy menstrual bleeding in women with inherited bleeding disorders.

Heavy menstrual bleeding (HMB) is the commonest bleeding symptom among women with inherited bleeding disorders (IBD). Since HMB starts at the very onset of menarche and continues throughout the reproductive life, the health related quality of life of these women is affected and they are at an increased risk of developing iron-deficiency anemia. Because of the entrenched stigma and taboos, women and girls are often reluctant to discuss the problem of HMB within their families and do not seek medical advice. Increased awareness and multidisciplinary management approach for the management of these women are essential in ensuring an optimal outcome. It is important to take a careful history and undertake a thorough gynecological assessment to exclude other underlying/concomitant causes of HMB. Iron supplementation is essential. Strategies for decreasing menstrual blood flow are similar to those used for HMB in general with the addition of desmopressin and replacement therapy and the exclusion of non-steroidal anti-inflammatory drugs. Tranexamic acid and/or hormonal intervention are usually recommended as first-line therapy. Treatment choice should be individualized taking into account whether the woman wishes to preserve her fertility, if she requires contraception, the type of IBD, the severity of bleeding, and her social and religious background as well as acceptability and availability of the treatment options.

Haemodialysis Tunisian patient with acquired factor V inhibitor associated to arteriovenous shunt thrombosis.

Factor V deficiency is a rare hemostatic disorder. It may present with a diverse spectrum of symptoms due to a variety of mechanisms including development of autoantibodies associated with a number of conditions. We report a first case of factor V deficiency in Tunisian hemodialysis patient due to an autoantibody most likely secondary to antibiotic exposure responsible for an arteriovenous shunt thrombosis rather than bleeding. We discuss here the clinical and biological features of acquired factor V inhibitor and provide a short review of the current literature.

The Glanzmann's Thrombasthenia in Tunisia: A Cohort Study.

BACKGROUND: The Glanzmann's thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder with uncommon neonatal revelation. It is due to abnormalities of quantitative and/or qualitative αIIbß3 integrin. This cell adhesion receptor is essential for platelet aggregation and allows the formation of a hemostatic plug if the vessel is damaged by injury. The clinical picture of GT is variable, with mucocutaneous bleeding due to non-functional platelets. Management requires a good expertise in bleeding disorders. We describe the clinical and the epidemiological data of GT in Aziza Othmana Hospital Hemophilia Center. METHODS: This was a retrospective study of all patients with GT monitored and treated in our hemophilia center during the period of 2011 - 2015. RESULTS: Twenty-seven patients among the 35 patients included in our hemophilia center registry were studied. The most common sign encountered is the gingival bleeding. In our women cohort, one completed her pregnancy. The consanguinity is present with a frequency of 62%. Treatments used depending on the case are tranexamic acid, platelet transfusion, packed red blood cells and rFVIIa, respectively. CONCLUSION: GT is relatively frequent in Tunisia and especially in the North of the country which can be explained by the high consanguinity in our population.

[Evaluation of non-compliance of transfusion requests of packed red blood cells]. / Evaluation des non-conformités des demandes de transfusion des concentres de globules rouges.

BACKGROUND: Despite legislative acts develloped, many deficiencies were identified in blood requests at the National Blood TransfusionCenter impedding board and blood safety. AIM: to evaluate the conformity of the different topics of packed red blood cells requests to the legislation. METHODS: Our study was prospective descriptive lasting six months (March-August 2011). It assessed all packed red blood cells requests which reached the national blood transfusion center. RESULTS: 16064 packed red blood cells requests from 21 public institutions and 28 private institutions were studied. There was different deficiencies in each item.The absence of birth date in 67.18% of request represented the largest non-compliance within administrative information. A predominance of shortcomings related to transfusion and obstetric history was recorded for clinical information with absence of date of the last transfusion in 91.72% cases, lack of accuracy of any previous transfusion reactions in 88.63% cases and absence of the number of previous pregnancies in 93.15% of transfusion requests prescribed to women. Non-conformities related to the prescribing physician concerned mainly the phone number which was absent in 55.82% of cases. CONCLUSIONS: This study revealed a significant lack of awareness of physicians in relation to the law governing transfusion. It is therefore essential to develop training for prescribers to improve transfusion safety.

Hepatitis E virus seroprevalence among hemodialysis and hemophiliac patients in Tunisia (North Africa).

The aims of this study are to determine seroprevalence of Hepatitis E virus (HEV) in Tunisian blood donors and to evaluate its risk of parenteral transmission. Sera collected from 426 blood donors were tested for HEV IgG by indirect ELISA. Individuals were recruited from two national transfusion centers, in the North and the South of the country. Seroprevalence of HEV IgG was then compared with two other groups with increased risk of exposure to parenterally transmitted agents: 80 hemophiliac and 286 hemodialysis patients. Among blood donors, the seroprevalence was estimated to be 4.5%. It was significantly higher in the hemophiliac and hemodialysis groups with 7.5% and 10.2%, respectively, (P = 0.002). No significant correlation was observed for this IgG 1 seroprevalence between age and sex among three studied groups. These results suggest that HEV has a high risk of parenteral transmission and confirm that the low endemicity of hepatitis E in Tunisia was observed.