Haemophilia care in India: innovations and integrations by various chapters of Haemophilia Federation of India (HFI)

Summary.  Care of persons with haemophilia (PWH) in western countries is the responsibility of the government of those countries with or without funding from health insurers. Haemophilia societies in western countries work as pressure groups to ensure better care, and they disseminate information on the disease and some of the societies even support medical research for haemophilia care. In India, Haemophilia Federation of India (HFI) was established in 1982 with few haemophilia families and sympathizers of their cause; subsequently more than 65 chapters involving more than 12 500 PWH came up under HFI. HFI and its constituent chapters are unique in the world in the sense that they are not only trying to involve state and federal government to take responsibility for delivering haemophilia care, but they are also using various innovative and integrative techniques to deliver haemophilia care to PWH themselves, till the time federal and state governments of the country make suitable arrangement for their care. In this study, several of these approaches are discussed with the understanding that 80% of worlds' haemophilia population needs similar help, and the national haemophilia organizations (NMO) of various developing countries will find some of the approaches useful and adaptable to their own circumstances.     

Menorrhagia and reproductive health in rare bleeding disorders: a study from the Indian subcontinent

Abstract.  At this centre, 130 women with rare bleeding disorders (RBD) were investigated over the past 15 years. Fifty patients were above the age of menarche (age of menarche in India is 10 years). Of these 44 presented with menorrhagia. Other complications in these patients involved bleeding because of ruptured graffian follicle (1), severe haemorrhage following caesarean section (1), recurrent pregnancy losses (3), hysterectomy to control menorrhagia (2), laser ablation of endometrium (1) and irradiation of ovary (1). Three patients voluntarily chose to remain unmarried because of the problems associated with menorrhagia which they assumed will interfere with married life. All the 45 patients had iron deficiency anaemia. The spectrum of RBD in these patients comprised Glanzmann's thrombasthenia (17), Bernard-Soulier syndrome (2), storage pool disorder (2), factor V (FV) deficiency (3), combined FV and factor VIII deficiency (5), factor XI deficiency (3), factor XIII deficiency (1), factor X deficiency (5), factor VII deficiency (2), α₂-antiplasmin deficiency (1) and afibrinogenemia (3). RBD in women is diagnosed late and often they are not optimally managed hence suffer both iatrogenic and non-iatrogenic complications in this country.     

Cloning, expression and immunoprotective efficacy of rHaa86, the homologue of the Bm86 tick vaccine antigen, from Hyalomma anatolicum anatolicum

The Bm86 homologue of Hyalomma anatolicum anatolicum Izatnagar isolate was cloned and expressed in methylotropic yeast Pichia pastoris as intracellular, glycosylated and particulated form. It was named as rHaa86, the first recombinant protein of H. a. anatolicum . Seven epidermal growth factor-like domains predicted in Haa86 were structurally similar with that of its Bm86 counterpart. The identity between the corresponding EGF like domains of Bm86 and Haa86 were ranging from 51·3% to 78·3%. The molecular weight of the rHaa86 was 120–140 kDa, with possible 50–70 kDa glycosylation. The purified rHaa86 was characterized immunologically and evaluated for its immunoprotective potential against homologous challenge infestation in three groups of cross-bred calves. The immediate rejection percentage of females of H. a. anatolicum was 36 5%, 12·4% and 10·1% fed on immunized (group 1), adjuvant control (group 2) and untreated control (group 3) calves, respectively. The percent rejection of female ticks fed on immunized calves was 24·1% and 26·4% higher than for the ticks fed on control groups 2 and 3, respectively ( P <  0·05). The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 58·0%, 9·0%, 5·0% and 61·6%, respectively. The mean reproductive index of females fed on group 1 calves was significantly lower ( P <  0·05) than the females fed on the control groups and 44% reduction in the number of engorged larvae was recorded from the group 1 calves. The data demonstrated that rHaa86 antigen based vaccine could serve as one of the effective components in the integrated control of H. a. anatolicum.     

Cancers in patients with hemophilia: a retrospective study from the Italian Association of Hemophilia Centers: a rebuttal

See also Tagliaferri A, Franchini M. Cancers in patients with hemophilia: a retrospective study from the Italian Association of Hemophilia Centers: a reply to a rebuttal. This issue, pp 1201–2. Tagliaferri A, Di Perna C, Santoro C, Schinco P, Santoro R, Rossetti G, Coppola A, Morfini M, Franchini M, on behalf of the Italian Association of Hemophilia Centers. Cancers in patients with hemophilia: a retrospective study from the Italian Association of Hemophilia Centers. J Thromb Haemost 2012; 10 : 90–9.     

Effect of glycoPEGylation on factor VIIa binding and internalization

Summary. Recombinant coagulation factor VIIa (rFVIIa), which is widely used for treatment of bleeding episodes in haemophilia patients with inhibitors, is cleared from the circulation relatively fast with a plasma half‐life of 2–4 h. PEGylation is an established and clinically proven strategy for prolonging the circulatory life‐time of bio‐therapeutic proteins. The aim of this study was to investigate the effect of glycoPEGylation of rFVIIa on rFVIIa binding to its cellular receptors and its subsequent internalization. rFVIIa and glycoPEGylated rFVIIa were labeled with ¹²⁵I and the radio‐iodinated proteins were used to monitor rFVIIa binding and uptake in endothelial cells and fibroblasts. FVIIa‐TF activity at the cell surface was analyzed by a factor X activation assay. Modification of rFVIIa with PEG impaired rFVIIa binding to both endothelial cell protein C receptor and tissue factor (TF) on cell surfaces. The internalization of PEGylated rFVIIa in endothelial cells and fibroblasts was markedly lower compared to the internalization of rFVIIa in these cells. PEGylated rFVIIa was able to activate factor X on TF expressing cell surfaces at a rate similar to that of unmodified rFVIIa when the cells were not subjected to multiple washings to remove the free ligand. General effects such as steric hindrance or changes in electrostatic binding properties of the modified rFVIIa to its receptors are probably responsible for this impairment rather than a loss of specific recognition of the receptors, which could explain near normal activation of factor X by glycoPEGylated rFVIIa on TF expressing cells while its uptake is reduced.     

Syringocystadenoma Papilliferum: An Unusual Presentation

Abstract: An 8‐year‐old boy presented with a rapidly growing, unusually large, fleshy, lobulated, cauliflower‐like mass on the lower back. Incisional biopsy revealed the histologic picture of syringocystadenoma papilliferum. The case is reported in this study for its unusual site, very large size, and peculiar morphology.     

Subpopulations of T Lymphocytes in the Peripheral Blood, Dermal Lesions and Lymph Nodes of Post Kala-azar Dermal Leishmaniasis Patients

Distribution of different subpopulations of T cells in the dermal lesions, lymph nodes and peripheral blood of post kala-azar dermal leishmaniasis (PKADL) patients was studied by using appropriate phenotypic markers for CD2⁺, CD4⁺ and CD8⁺ cells. Histopathological studies of skin lesions showed marginal to massive infiltration of mononuclear cells depending upon the duration of illness and type of lesions. Thus, while the hypopigmented patches were represented by small focal collections of lymphocytes with scanty parasites in the dermis, these were replaced at the nodular stage with massive granulomas consisting of lymphocytes, plasma cells and histiocytes with numerous amastigotes. The involvement of CD4⁺ and CD8⁺ cell types in these lesions also showed a gradual change from the appearance of a few cells of both the phenotypes in early hypopigmented type to massive accumulation of cells, primarily of CD8⁺ phenotype, in the granuloma of nodular type. However, the observed preponderance of CD8⁺ cells at the lesion site of chronic PKADL patients is in contrast to their peripheral blood CD4⁺/CD8⁺ cell ratio (1.9:1) which remained within the normal limits. Similar studies of lymph nodes from PKADL patients with lymphadenopathy revealed infiltration of the cortical areas by T cells which were more of CD8⁺ than CD4⁺ phenotypes. All these results document the involvement of CD8⁺ cells in leishmanoid lesions. Thus, it is likely that these cells, in association with appropriate subpopulations of CD4⁺ cells, play a profound role in the evolution of dermal pathology in PKADL.     

Recurrent Pyogenic Meningitis -- A Retrospective Study

Records of 17 patients who had two or more attacks of pyogenicmeningitis were collected from eight centres in the United Kingdomfor retrospective analysis. Thirteen patients had intracranial abnormality; of seven withhead injury five produced cerebrospinal fluid rhinorrhoea. Thefirst of the 28 attacks seen in these occurred between a fewweeks and 12 years of the head injury. Pneumococci were identifiedin 25 episodes in cerebrospinal fluid. Of six patients withouta history of head injury, one had ‘spontaneous cerebrospinalfluid rhinorrhoea’ and five had pathological changes ofthe ear. Various organisms were found in the cerebrospinal fluidduring the 12 attacks in these five. Four of the 17 patientshad primary complement deficiency (C7, C5, C4 and C3b inhibitor);10 (possibly 11) of 16 attacks in these cases were due to Neisseriameningitidis. Routine radiological investigations includingcomputerized tomography did not always identify the abnormality;radioactive cisternography can help to establish cerebrospinalfluid leak. All 58 episodes of pyogenic meningitis in these 17 patientswith different underlying disease responded to conventionaltreatment with antibiotics without mortality and without unduemorbidity. Surgical procedures in intracranial disease had variablesuccess. Correction of complement deficiency is not practicalat present. In some patients prophylaxis with antimicrobialdrugs is the only method of preventing future attacks.     

Mutations in GPIIIa molecule as a cause for Glanzmann thrombasthenia in Indian patients

BACKGROUND: Glanzmann thrombasthenia (GT) results from a quantitative or qualitative defect of GPIIb-IIIa complex, the fibrinogen receptor on platelets, which plays a very important role in platelet aggregation. In this report we describe the molecular studies on 22 patients with Glanzmann Thrombasthenia at our institute. OBJECTIVES: The main objective was to identify the mutations present in our GT population in order to establish a strategy for genetic counseling and antenatal diagnosis. METHODS: Twenty-two patients with GT were included in the present study. Complete blood count (CBC), platelet aggregation, flow cytometry, Western blot, single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) were performed in all the patients. The patients showing an abnormal migration pattern in SSCP or DGGE were sequenced further on an automated sequencer. RESULTS: Of the 22 patients studied, mutations were detected in 12 individuals. Of these, 11 were novel mutations and one mutation Y115C was reported earlier. Flow cytometric analysis showed the absence of receptors in type I GT, highly reduced levels in type II GT and normal levels in type III GT. The DGGE analysis and SSCP analysis of the patients showed different migration patterns. Sequencing was performed in all patients showing an abnormal migration pattern. Of the 22 cases studied mutations could be detected in 12 cases of GT. We could detect six patients with point mutations, four patients with insertions and five patients with deletion mutations. Exon 4 has been found to be the most common site for mutations in our patients. CONCLUSION: This study has shown a wide array of mutations present in our GT patients which would be extremely useful in genetic counseling and prenatal diagnosis, essential in preventing these disorders in succeeding generations.