Munira Borhany

Bleeding disorders in the tribe: result of consanguineous in breeding

ObjectiveTo determine the frequency and clinical features of bleeding disorders in the tribe as a result of consanguineous marriages.DesignCross Sectional StudyIntroductionCountries in which consanguinity is a normal practice, these rare autosomal recessive disorders run in close families and tribes. Here we describe a family, living in village Ali Murad Chandio, District Badin, labeled as haemophilia.Patients & MethodsOur team visited the village & developed the pedigree of the whole extended family, up to seven generations. Performa was filled by incorporating patients, family history of bleeding, signs & symptoms, and bleeding from any site. From them 144 individuals were screened with CBC, bleeding time, platelet aggregation studies & RiCoF. While for PT, APTT, VWF assay and Factor VIII assay, samples were kept frozen at -70 degrees C until tested.ResultsThe family tree of the seven generations comprises of 533 individuals, 63 subjects died over a period of 20 years and 470 were alive. Out of all those 144 subjects were selected on the basis of the bleeding history. Among them 98(68.1%) were diagnosed to have a bleeding disorder; 44.9% patients were male and 55.1% patients were female. Median age of all the patients was 20.81, range (4 months- 80 yrs). The results of bleeding have shown that majority had gum bleeding, epistaxis and menorrhagia. Most common bleeding disorder was Von Willebrand disease and Platelet functional disorders.ConclusionConsanguineous marriages keep all the beneficial and adversely affecting recessive genes within the family; in homozygous states. These genes express themselves and result in life threatening diseases. Awareness, education & genetic counseling will be needed to prevent the spread of such common occurrence of these bleeding disorders in the community.

Transfusion Transmitted Infections in Patients with Hemophilia of Karachi, Pakistan

The aim of the study was to assess the prevalence of HCV, HBV, and HIV infections among the patients with hemophilia. Patients with Hemophilia A and B were evaluated who visited hospital for factor replacement therapy. The viral markers tested in these patients included anti-HCV-Ab, HBsAg, and anti-HIV-Ab. Seroprevalence was compared from 5717 exchange healthy blood donors for same markers. A total of 173 multitransfused male hemophiliacs showed prevalence of 51.4% for HCV, 1.73% for HBV, and nil for HIV. In blood donors seroprevalence was 1.9% for HCV, 1.81% for HBV, while no HIV-positive case was detected. Prevalence of anti-HCV-Ab was significantly high in patients with hemophilia than normal donors (P = .0005). This study showed that HCV infection was more frequently identified than HBV and HIV infections in multitransfused hemophiliacs. The frequency of hepatitis C among blood donors is also higher than that of hepatitis B which is showing downward trend.

Clinical and molecular characterisation of 21 patients affected by quantitative fibrinogen deficiency

Fibrinogen is a plasma glycoprotein mainly synthesised by hepatocytes and circulating as a 340-kDa hexamer consisting of two sets of three different polypeptide chains (Aα, Bβ, and γ, encoded by the FGA, FGB, and FGG gene, respectively). Congenital afibrinogenaemia and hypofibrinogenaemia are rare bleeding disorders characterised by abnormally low levels of functional and immunoreactive fibrinogen in plasma, associated with haemorrhagic manifestations of variable severity. While afibrinogenaemia is caused by mutations in the homozygous or compound heterozygous state in one of the three fibrinogen genes, hypofibrinogenaemia is generally due to heterozygous mutations, and is usually characterised by a milder phenotype. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations often affecting fibrinogen assembly and/or secretion. Here we report the clinical and molecular characterisation of 13 unrelated afibrinogenaemic and eight hypofibrinogenaemic patients, leading to the identification of 17 different mutations (10 hitherto unknown). All the newly-identified missense and splicing mutations werein vitro expressed to verify their pathogenic role. Our data increase the number of mutations causing quantitative fibrinogen deficiencies by about 7 %. The high number of private mutations identified in the analysed probands indicates that the full mutational screening of the three fibrinogen genes is still required for molecular diagnosis.

Congenital Bleeding Disorders in Karachi, Pakistan

Objective: To determine the frequency of inherited bleeding disorders, its complications, and treatment modalities available for its treatment. Design: Cross-sectional study. Patients and Methods: Patients with a history of bleeding tendency were tested for confirmation of the diagnosis. History and clinical findings were recorded. Laboratory analysis included prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time (BT), and fibrinogen assay. Patients with prolonged APTT were tested for factors VIII (FVIII) and IX (FIX). If FVIII was low, von Willebrand factor: antigen (vWF:Ag) and von Willebrand factor:ristocetin cofactor (vWF:RCo) were performed. When PT and APTT both were prolonged, FV, FX, and FII were tested. Platelet aggregation studies were done when there was isolated prolonged BT. Urea clot solubility test was done when all coagulation tests were normal. All patients with hemophilia A and B were evaluated for inhibitors. Results: Of the 376 patients, inherited bleeding disorder was diagnosed in 318 (85%) cases. Median age of patients was 16.4years. Hemophilia A was the commonest inherited bleeding disorder that was observed in 140 (37.2%) followed by vWD 68 (18.0%), platelet function disorders 48 (12.8%), and hemophilia B in 33 (8.8%) cases. We also found rare congenital factor deficiencies in 13 (3.4%), low VWF in 11 (3.0%) participants and 5 (1.3%) in female hemophilia carriers. Hemarthrosis was the most frequent symptom in hemophilia A and B (79.7%) involving knee joint. Inhibitor was detected in 21 (15%) cases. Fresh frozen plasma/cryoprecipitate were the most common modality of treatment. In 58 patients, no abnormality was detected in coagulation profile. Conclusion: Hemophilia A and vWD are the most common congenital bleeding disorders in this study. Hemarthrosis involving knee joint was the most common complication. Inhibitor was detected in a significant number of patients. Plasma is still the most common modality of treatment.

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A‐ and B‐subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT‐PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.

The utility of International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool and other bleeding questionnaires in assessing the bleeding phenotype in two platelet function defects.

The main objective of this study is to investigate the utility of International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT) in comparison with the condensed form of Molecular and Clinical Markers for the Diagnosis and Management of type 1 and WHO BATs, in assessing bleeding in two well known and clinically significant platelet function defects. Thirty-eight patients previously diagnosed with Glanzmanns thrombasthenia and 10 with Bernard–Soulier syndrome (BSS) were analyzed. Bleeding scores were significantly higher than that of controls using both electronic bleeding questionnaire (eBQ) and ISTH-BAT with no significant difference between both tools. ISTH-BAT had a sensitivity, specificity, positive predictive value and negative predictive value of 100%, 76.2%, 0.9 and 1. This was closely similar to eBQ. Both ISTH-BAT and eBQ are efficient in BSS and Glanzmanns thrombasthenia. However, given the ISTH recommendation, ISTH-BAT should be adopted. Larger study including other platelet defects will enhance its utility and support the integration of bleeding scores with standardized laboratory testing to allow for a universal diagnostic approach to patients with suspected bleeding disorders.

Pattern of bleeding and response to therapy in Glanzmann thrombasthenia

Glanzmann Thrombasthenia (GT) is a rare autosomal recessive bleeding disorder affecting the megakaryocyte lineage. The incidence of this disorder is higher in communities where consanguinity is prevalent like Jordan, Iran, South India and Iraqi Jews [1,2]. The molecular basis shows the quantitative and/or qualitative abnormalities of the platelet fibrinogen receptor, the aIIbb3 integrin (glycoprotein (GP) IIb/IIIa, CD41/CD61) which mediates the incorporation of platelets into an aggregate or thrombus at the sites of vessel injury [3]. Clinical manifestations in GT usually start in childhood and have clinical variability; ranging from minimal bruising to severe and potentially fatal haemorrhages. Diagnosis is associated with mucocutaneous bleeding with no platelet aggregation in response to all physiologic stimuli, a normal platelet count and morphology. Deficient or non-functional platelet aIIbb3 should be confirmed using flow cytometry. Genetic studies reveal multiple mutations that can affect the GPIIb/IIIa complex assembly on the platelet membrane. Standard treatment includes platelet transfusion to stop bleeding when conservative measures fail, but repeated transfusions may result in the development of antibodies (alloimmunization) against GPIIb/IIIa and/or human leucocyte antigen (HLA), resulting in refractoriness to further platelet transfusion [4]. In Pakistan, consanguineous unions continue to be extremely common as in SouthWest Asia. The Pakistan Demographic and Health Survey (DHS) shows that two-thirds of marriages in Pakistan are consanguineous. As a result of consanguineous marriages, rare autosomal recessive disorders run in close families and tribes [5]. Therefore, the specific aim of this study was to assess the frequency of GT, to see the pattern of bleeding and their response to therapy in our patients by comparing them over worldwide presentation. This is a retrospective study of GT patients who were diagnosed and followed at the National Institute of Blood Disease & Bone Transplantation (NIBD), Karachi, Pakistan from October 2008 to November 2011. The study was approved by the Institutional ethics committee, informed consent was obtained from all adult subjects, parents or legal guardians. The data were taken from hospital medical records which included outpatient files, emergency department notes, operative reports and inpatient charts. Information for each clinical encounter was recorded and tabulated. Patients were diagnosed on the basis of clinical suspicion of the disease, normal platelet count on complete blood counts (CBC), prolonged bleeding time (BT), normal prothrombin time (PT), activated partial thromboplastin time (APTT), normal to suboptimal platelet aggregation in the presence of ristocetin and defective aggregation with collagen, ADP and epinephrine. Patients with other coagulation and platelet function disorders were excluded from this study. Platelet aggregation studies were done on platelet-rich plasma (PRP) with a count adjusted to 200–250 9 10 L 1 by Helena Agg RAM using ristocetin, adenosine diphosphate (ADP), epinephrine and collagen. The following demographic and clinical data were obtained: age, gender and type of bleeding symptoms i.e. epistaxis, bruising, gum bleeding, bleeding at circumcision (in males), bleeding at dental extraction, muscle bleed, haematoma, subcutaneous bleed, haematuria, haematemesis, gastrointestinal(GI) bleeding, ear bleeding and menorrhagia (in females). Information was also obtained on the number of bleeding episodes, hospital visits, requiring or not requiring admission, duration of bleeds between two consecutive episodes were recorded and responses of the symptoms to different treatment modalities were observed. The initial treatment offered for minor bleeds was local haemostatic measures such as pressure bandage, ice therapy and antifibrinolytic agents such as tranexamic acid. Platelet transfusions were used if bleeding failed to stop and in the presence of severe bleeding, dental procedures and surgery. Response was assessed and when no response was observed another treatment modality such as DDAVP or rFVIIa was added to the treatment regimen and the response assessed. Female patients presented with episodes of menorrhagia were treated with oral contraceptives, tranexamic acid, iron supplements and if needed with platelet transfusions. If a patient required any other treatment modality, they were considered as non-responders for the previously offered treatments. The response to treatment was measured from the initiation of treatment until complete cessation of bleeding in less than 6 h, in more than 6 h, less than 24 h and more than 24 h and labelled as responders. Patients were considered as non-responders when they continued to bleed for more than 36 h from the start of the initial treatment. The statistical package SPSS-13 was used to analyse the data. Frequency and percentage were computed for categorical variables and mean and standard deviation (SD) were estimated for quantitative variables. A total of 43 GT patients were diagnosed at NIBD hospital of the 123 congenital platelet function disorders (35%). These patients were followed in OPD, inpatient and emergency department. There were 20 males with a mean age of 12.1 years (range, 6 months– 30 years) and 23 females with a mean age of 10.5 years (range, 1–17 years). Parents of 34 (79%) patients were first or second cousins. Positive family history of bleeding was noted in 26 (60%) patients, no history of bleeding in 7 (16.2%) patients and unknown in 10 (23%) patients. A total of 245 bleeding episodes were identified during different time periods requiring visit to hospital, but only 173 bleeding episodes could be evaluated because of the availability of complete details. The average number of medical visits per patient was 12 (range, 2–66). Hospitalization was required in 147/173 (85%) of these visits for a mean of 31 days spent as an inpatient Correspondence: Dr. Munira Borhany, National Institute of Blood Disease and Bone Marrow Transplantation (NIBD), Karachi, Pakistan,: ST 2/A Block 17, Gulshan-e-Iqbal KDA Scheme 24 Karachi, Pakistan 75300. Tel.: +0092-21-34821502-3; fax: +0092-21-34821504; e-mail: drmunirashoaib@yahoo.com

Clinical features and types of von Willebrand disease in Karachi.

This study was conducted on patients with a history of congenital bleeding disorders or with suspected bleeding tendencies. Laboratory analysis revealed Von Willebrand disease (VWD) in 68 (21.3%) of 318 participants with male to female ratio of 0.8: 1 (31 to 37) and median age 17 years (range 2-45 years). Type 3 being the most frequent, 35 (51.4%) of 68, type 2, 20 (29.4%) of 68, and lastly type 1, 13 (19.1%) of 68. A total of 55.8% patients with VWD presented with mucocutaneous bleeding. Menorrhagia was the most common presentation of female patients. Von Willebrand disease (21.3%) was the second common bleeding disorder and the most common coagulation defect among females with menorrhagia. However, the frequency in the study was quite low when compared to the western world. Similarly, low frequency of VWD type 1 might be due to the fact that only symptomatic patients visited us. Further studies are needed as there is limited information on VWD in the developing countries. This will help in the development of expertise for the accurate diagnosis & proper management.

Prevalence of transfusion transmissible infections in blood donors of Pakistan

BackgroundTransfusion-transmitted infections threaten the safety of patients requiring blood transfusion, which in turn imposes serious challenges for the availability of safe blood products that are still affordable in health care systems with limited resources. The aim of the study was to determine the prevalence of transfusion-transmitted infections in blood donors and to evaluate the demographic characteristics of reactive and non-reactive blood donors.MethodsA prospective cohort study was conducted at our institute in Karachi, Pakistan. Donors were required to fill a detailed questionnaire and were screened for Hepatitis B, Hepatitis C, Human immunodeficiency viruses, Syphilis and Malaria by ELISA and thick film (malaria).ResultsOf the 16,602 blood donors, 16,557 were males and 45 females (mean age 28.6 ± 2). Nine hundred and seventy three (5.8%) donations were reactive in any screening assay, with 58 (0.35%) donations reacting in more than one assay. The prevalence of Hepatitis B, Hepatitis C, Human immunodeficiency viruses, Syphilis and Malaria was found to be 1.84, 1.7, 0.04, 2.1 and 0.07% respectively. Characteristics among the infections were evaluated and it was found that unmarried donors had a higher chance to be infected by Hepatitis B virus and Syphilis as compared to the other infections. On the other hand, construction workers and married donors were at more risk to be infected by Syphilis rather than the other infections. In case of co-infections, personnel with different occupations and marital status were infected by more than one pathogen.ConclusionA substantial percentage of the blood donor’s harbored transfusion-transmitted infections. Prevention of TTIs should be the main goal right now. There is a need for stringent selection of blood donors with the emphasis on getting voluntary donations and comprehensive screening of donor’s blood for TTIs using standard methods to ensure the safety of blood recipient.

A new report of FVII‐inhibitor in a patient suffering from severe congenital FVII deficiency

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