Hb Koln [β98(FG5) [GTG → ATG,Val → Met]: The first report from India

Background

The group of unstable hemoglobins are associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule. They often lead to formation of the characteristic inclusion bodies or Heinz bodies.

Aim

To identity the cause of mild anemia, reticulocytosis, and hepatosplenomegly in a case of non-spherocytic hemolytic anemia.

Materials and methods

A 34-year-old female patient originating from Maharashtra, western India presented with mild anemia and jaundice which had persisted since childhood. Investigations included a complete blood count, screening for red cell membrane protein defects, Hb analysis by high-performance liquid chromatography (HPLC) and cellulose acetate electrophoresis (pH 8.9), heat instability test and DNA sequencing.

Results

Hemoglobin analysis by HPLC showed an abnormal peak in the Hb C window (9.8%) with a retention time of 4.90 minutes. Cellulose acetate electrophoresis (pH 8.9) showed a slow moving band (6.15%) between Hb A₂ and Hb S. The heat instability test was positive. DNA analysis of α globin genes showed absence of both deletional and non- deletional α thalassemia. DNA sequencing of the β globin gene revealed heterozygosity for a mutation at codon 98 [GTG → ATG, Val → Met], which gives rise to Hb-Koln.

Conclusion

Hb Koln is the commonest unstable Hb variant reported from many populations in the world. However, this is the first report of this unstable Hb variant from India.     

Molecular and clinical analysis of haemoglobin Lepore in Campania,a region of Southern Italy

Objective: To date in Italy, there is paucity on data about the prevalence, clinical and haematological features of patients carrying the haemoglobin (Hb) Lepore variant in homozygous or in association with other haemoglobinopathies.

Methods: Here we report the results of a retrospective analysis on 33 patients from Campania, a region of Southern Italy, historically followed at ‘UOSD Malattie Rare del Globulo Rosso’ of Cardarelli hospital, Naples, Italy.

Results: We described 33 patients carrying the Hb Lepore variant: 21 compound heterozygotes with a common thalassaemia allele, six patients with homozygous state for Hb Lepore, five patients with Hb Lepore/Hb S and one patient with Hb Lepore/Hb Neapolis were identified. All individuals carried haplotype I or V.

Discussion: These thalassaemic patients showed different phenotypes ranging from severe disease with early blood transfusion dependency to moderate form of thalassaemia intermedia. In most cases, thalassaemia mutation type determined the severity of the disease.

Conclusion: A great variability of clinical phenotype among the same genotypes was also observed suggesting the presence of unknown genetic modifiers acting in combination with Hb Lepore.     

Low oxygen saturation and severe anemia in compound heterozygous Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]

Objective: To investigate the cause(s) of a Thai male proband presenting low oxygen saturation by pulse oximetry (SpO₂) and severe anemia.

Methods: As Hb variant was suspected, Hb typing was determined by high-performance liquid chromatography and capillary electrophoresis, and subsequently Hb variant was identified by DNA sequencing. Complete blood counts were performed using automated blood cell counter and oxygen saturation was measured by pulse oximetry.

Results: Proband was compound heterozygous for Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]. Of the proband’s two sons, one was compound heterozygous for Hb Louisville and Hb E and the other for Hb La Desirade and Hb E. The former son had similar clinical features and laboratory findings with those of the proband while the latter showed had no abnormal clinical manifestations.

Conclusion: This the first report of compound heterozygosity of Hb Louisville and Hb La Desirade in an individual of Southeast Asian ethnicity. Hb variant identification is crucial for genetic counseling and appropriate treatment in regions where hemoglobinopathies are common.     

Acute physiological responses to physiotherapy applications pre and post autologous stem cell transplantation: an experimental study

Objective

We aimed to investigate the acute physiological responses (APR) to physiotherapy applications in patients undergoing autologous stem cell transplantation (ASCT), the difference between pre- and post-ASCT according to APR.

Methods

Twenty-six patients who were hospitalized for ASCT attended regular physiotherapy program. APR was recorded in the beginning and at the end of each exercise session. The differences in APR were calculated for each session. The mean values of the differences in APR were computed in pre-conditioning, pre-, and post-ASCT. Daily complete blood counts were also recorded during ASCT.

Results

Hemoglobin and platelet counts were significantly lower pre- and post-ASCT. Neutrophil counts were significantly lower post-ASCT. The difference in systolic blood pressure (SBP) in the beginning and at the end of the exercise sessions was significantly higher post-ASCT in comparison to pre-ASCT.

Conclusion

There was no significant change in APR except the SBP which suggests that similar level of exercise intensity could be tolerated in pre- and post-ASCT periods as well as preconditioning.     

Book reviews

The School Years. Current Issues in the Socialization of Young People John C. Coleman (Ed.) London, Routledge, 1992 (2nd edn) 208 pp.; £10.99 (paperback)

AIDS and the Hospice Community Madalon O'Rawe Amenta & Clair B. Tehan Binghampton, NY, Harrington Park Press, 1991 186 pp. + index

Social Care & HIV/AIDS The Hull York Research Team London, HMSO, 1993 214 pp., £13.70 (paperback)

Private Risks and Public Dangers Sue Scott, Gareth Williams, Stephen Platt & Hilary Thomas (Eds) Aldershot, Avebury, 1993 205 pp., £34.95 (hb)

AIDS: a problem for sociological research Michael Pollak, Geneviève Paicheler & Janine Pierret London, Sage Publications, 1992 124 pp., £10.95 (paperback)

AIDS: The Winter War Arthur D. Kahn Philadelphia, Temple University Press, 1993 236 pp., U£27.95 (hardback)

AIDS: Facing the Second Decade Peter Aggleton, Peter Davies & Graham Hart (Eds) Taylor & Francis, 1993 215 pp., £12.95 (Pb)

Women and AIDS: Psychological Perspectives Corrine Squire (Ed.) London, Sage Publications, 1993 196 pp., £12.95 (paperback)     

Angiogenesis and acute myeloid leukemia

Background

Angiogenesis is a word of Greek origin, ‘angeio’ refers to blood vessel, and genesis refers to creation, meaning the generation of new blood vessels. This process is essential for vertebrate development and plays a key role in human diseases. Angiogenesis is generally understood to be essential for the growth and metastasis of solid tumors and is also important in acute myeloid leukemia (AML).

Methods

This review summarizes the essential features of physiological and tumoral angiogenesis and the methods used for their assessment.

Results

Technologies for evaluating angiogenesis in AML are discussed and the prognostic significance of angiogenic factors is considered in the context of optimizing treatment.

Conclusion

As acute myelogenous leukemia and endothelial cells depend on each other for survival and proliferation, therapy directed against several pro-angiogenic factors might help to enhance the AML outcome.     

Book reviews and books received

Practices of Freedom: Selected writings on HTV/AIDS Simon Watney London, Rivers Oram Press, 1994 328 pp., £11.95 (paperback): £30 (hardback)

AIDS: The Challenge—Understanding Education and Care N. Small Aldershot, Avebury, 1993 172 pp.

Sex, Gay Men and AIDS P. M. Davies, F. C. I. Hickson, P. Weatherburn & A. J. Hunt London, Falmer Press, 1993 199 pp., £12.95

AIDS: Working with young people Peter Aggleton, Kim Rivers & Ian Warwick Horsham, West Sussex, Avert, 1993 162 pp., £ 18.95 (paperback)

ABC of AIDS. Third Edition Michael W. Adler (Ed.)London, BMJ Publishing Group, 1993 86 pp., £ 12.95 (paperback)

Guidelines for Management of Children with HTV Infection Diana Gibb & Sam Walters Horsham, West Sussex, AVERT, 1993 29 pp., £ 4 (paperback)

A Guide to HIV Infection and Child-bearing Danielle Mercey, Susan Bewley & Peter Brocklehurst Horsham, West Sussex, AVERT, 1993 25 pp., £ 3 (paperback)

Essential AIDS Information Resources AHRTAG/WHO, 1994 £ 5.00/US $10.00(Free to readers in developing countries)     

Influence of age on the correlations of hematological and biochemical variables with the stability of erythrocyte membrane in relation to sodium dodecyl sulfate

Objectives

To evaluate the influence of age on the relationships between biochemical and hematological variables and stability of erythrocyte membrane in relation to the sodium dodecyl sulfate (SDS) in population of 105 female volunteers between 20 and 90 years.

Methods

The stability of RBC membrane was determined by non-linear regression of the dependency of the absorbance of hemoglobin released as a function of SDS concentration, represented by the half-transition point of the curve (D₅₀) and the variation in the concentration of the detergent to promote lysis (dD).

Results

There was an age-dependent increase in the membrane stability in relation to SDS. Analyses by multiple linear regression showed that this stability increase is significantly related to the hematological variable red cell distribution width (RDW) and the biochemical variables blood albumin and cholesterol.

Discussion

The positive association between erythrocyte stability and RDW may reflect one possible mechanism involved in the clinical meaning of this hematological index.     

Interaction between Hb E and Hb Yala (HBB:c.129delT); a novel frameshift beta globin gene mutation,resulting in Hemoglobin E/β0 thalassemia

Objectives: There are more than 200 known mutations found in patients with β-thalassemia, a possibility to identify an unknown or novel mutation becomes less possible. Here, we report a novel mutation in a patient from Thailand who presented with chronic hemolytic anemia.

Methods: A comprehensive hematology and DNA analysis was applied in the index patient and her mother.

Results: Hematological and hemoglobin analyses were consistent with the clinical diagnosis of Hb E/β⁰-thalassemia. However, we could find only Hb E heterozygous mutation using our common polymerase chain reaction-based mutation detection of the β-globin genes. Furthermore, the molecular analysis demonstrated a novel T-deletion at codon 42 of the second exon of the β-globin gene which we named ‘Hb Yala’ according to the origin of this index family.

Discussion: This mutation was assumed to generate a truncated β-globin chain terminating at codon 60 with possible unstable variant leading to a ‘null’ or β⁰-thalassemia. However, the clinical phenotype was surprisingly mild and no other ameliorating genetic factors, including co-inheritance of α-thalassemia and high propensity of Hb F by Xmn I polymorphism, were found.

Conclusion: This report has provided evidence that genotype–phenotype correlation in thalassemia syndromes is highly complex and a correct clinical severity classification of thalassemia should be mainly based on clinical evaluation.     

Book Review

Acquainted with the Night Allegra Taylor, 1989 London, Fontana £3.99 (paperback)

The AIDS Handbook Carl Miller 1990 Penguin Books 159pp. £4.99 (paperback)

Caring for Someone with AIDS Edited by David Yelding, 1990 Published for the Research Institute for Consumers Affairs by the Consumers Association and Hodder & Stoughton £6.95 (paperback)

AIDS. A Pocket Book of Diagnosis and Management Edited by A. Mindel, 1990 London, Arnold 244 pp. £12.95

AIDS and its Metaphors Susan Sontag, 1989 Harmondsworth, Penguin 98 pp. £3.99 (paperback)     

Selective apoptosis of multiple myeloma cells in primary samples induced by arsenic trioxide

Objectives

Currently, multiple myeloma (MM) is an incurable disease. Despite the fact that arsenic trioxide (ATO) shows promising results in vitro, data from treatment of patients with MM are disappointing. Due to these discrepancies, we compared the efficacy and selectivity of ATO at two different concentrations in samples from MM patients.

Methods

The extent of apoptosis induced by 2 and 5 µM ATO was evaluated by flow cytometry using annexin V. 34 diagnostic bone marrow samples obtained from MM patients were analysed.

Results

5 µM ATO efficiently induced apoptosis in primary samples. Besides efficacy, also selectivity of action on MM cells in comparison to remaining haematopoietic cells was demonstrated for 5 µM ATO but not for 2 µM ATO.

Discussion

Our study on primary samples confirmed that ATO has a potential role in therapeutic management of MM. Further controlled studies on MM patients are needed.     

Nucleostemin knocking-down causes cell cycle arrest and apoptosis in human T-cell acute lymphoblastic leukemia MOLT-4 cells via p53 and p21Waf1/Cip1 up-regulation

Objectives

Nucleostemin (NS), a recently discovered nucleolar protein, is essential for maintaining self-renewal and proliferation of embryonic and adult stem cells as well as cancerous cells. The aim of this study was to determine biological function of NS in MOLT-4 cells as a human T-cell acute lymphocytic leukemia (T-ALL) model.

Methods

Efficacy of a specific small interference RNA on NS depletion was studied by quantitative polymerase chain reaction and western blotting. The growth rate and viability were analyzed by trypan blue exclusion test. Fluorescent microscopy was used for detecting apoptosis. Cell cycle and apoptosis were mechanistically studied by flow cytometry and western blotting.

Results

Knockdown of NS inhibited proliferation, arrested the cell cycle, and induced apoptosis through p53 and p21^Waf1/Cip1 pathways in MOLT-4 cells.

Discussion

These findings demonstrate critical roles of NS in MOLT-4 cells and may implicate on its therapeutic potential in this human T-ALL model.     

Bleeding manifestations apparently unrelated to coagulation or other organic disorders: A tentative classification and diagnostic clues

Objective

To study the features of bleeding conditions apparently not associated with vascular, platelet, or clotting dysfunctions.

Method

Conditions that may meet these criteria are: Münchausen syndrome factitious or fictitious, suicidal or homicidal bleeding, bleeding due to self-punishment, stigmatization, the battered child syndrome, and psychogenic bleeding.

Results

The importance of these variegate conditions is not trivial in clinical practice. Differential diagnosis may be difficult and involve other specialists besides hematologists. Occasionally, invasive procedures are involved.

Discussion

The occurrence of bleeding in patients, without a clotting defect or a systemic disorder and a negative family history for bleeding represents a diagnostic challenge. A careful examination of the physical and psychological status of the patient and an appropriate evaluation of the environment in which bleeding occurs, is always needed.     

A killer revealed: 10-year experience with beta-thalassemia intermedia

Objectives

Patients with beta-thalassemia intermedia tend to present later in life with milder anemia than beta-thalassemia major patients. The incidence of mortality and its causes in this patient population remains unknown. We aim to reveal the incidence and most common causes of death in this population.

Methods and results

We reviewed the charts of all of the beta-thalassemia intermedia patients who had been followed at the Chronic Care Center in Hazmieh, Lebanon during a 10-year period. A total of 18 patients out of 127 had died during the follow-up period giving a cumulative 10-year mortality incidence of 14%. The most common causes of cardiac deaths were due to renal and cardiac causes.

Discussion

Most causes of death have been linked to the high levels of iron coupled with anemia present in this patient population. Many of deaths could be prevented by adequate treatment.

Conclusion

Larger studies with more comprehensive data capture on risk factors of mortality in this patient population are called for.     

Heterogeneity in clinical course of EBV-associated lymphoproliferative disorder after allogeneic stem cell transplantation

Objective and Importance

Post-transplant lymphoproliferative disorder (PTLD) is a severe complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) associated with Epstein–Barr virus (EBV).

Clinical presentations

Among 263 individuals treated with allo-HSCT for severe aplastic anemia, pure white cell aplasia, T-prolymphocytic leukemia, and relapsed Hodgkin lymphoma, we diagnosed EBV-PTLD in 5 patients. Median age was 29 years (range 19–70 years) and four of five patients were EBV-seropositive prior to HSCT. All five had unrelated EBV-positive donors. In all cases, PTLD occurred within the first year post-transplant (median 4 months).

Intervention

There were two rapidly fatal courses with extensive organ involvement. Both patients showed lymphopenia and thrombocytopenia. In contrast, the three surviving patients had higher lymphocytes and normal platelet counts, while PTLD was restricted to one site and resolved after 2–4 cycles of rituximab.

Conclusion

In this case series courses of PTLD showed substantial diversity.     

Incidence of internal tandem duplications and D835 mutations of FLT3 gene in chronic myeloid leukemia patients from Southern India

Objective

To screen two important FLT3 mutations (internal tandem duplication (ITD) and D835 point mutations) in chronic myeloid leukemia (CML) patients from Southern India and report their incidence.

Methods

Screened 350 CML patients and 350 controls for the two FLT3/mutations through polymerase chain reaction and restriction fragment length polymorphism methods.

Results

ITDs were detected in 12 of the 350 CML patients (3.4%) and D835 mutations in only four cases (1.14%), relatively low in frequency as compared to those reported earlier from non-Indian populations. None of the cases showed simultaneous occurence of both ITD and D835 mutations.

Discussion

These FLT3 mutations seem to be very rare in CML, and it is possible that these could be found only in a subset of patients who are in the progressive stage and/or with varied drug response. Prospective studies are needed to confirm the role of FLT3 mutations in CML pathogenesis, which may help devising therapeutic interventions.     

Optimal length of time of cryopreserved umbilical cord blood infusion after thawing

Objective

Dimethyl sulfoxide (DMSO) can damage hematopoietic progenitor cells (HPCs) at room temperature. To minimize the time of contact between DMSO and the thawed umbilical cord blood (UCB), there is an incentive to infuse the UCB as quickly as possible. However, the infusion of cryopreserved UCB also results in side effects. Currently, it is difficult to determine the optimal length of time for cryopreserved UCB infusion, not only to ensure the maximum effect of engraftment but also to reduce the toxicity of the cord blood infusion.

Methods

Ten units of cord blood were thawed to assess viability, apoptotic events of CD34⁺ cells and CD45⁺ cells, and the number of colony-forming units (CFUs) at four time points: 0, 10, 20, and 30 minutes post-thaw (PT). The infusion time, side effects, and the speed of platelet and neutrophil engraftment of the 10 patients were monitored.

Results

Within 30 minutes, the viability decreased (P < 0.01), the percentage of early apoptotic CD34⁺ cells and CD45⁺ cells was unchanged. At time point PT30, the number of CFUs was decreased compared to PT0 (P < 0.05), but it was unchanged within 20 minutes. All the 10 UCB cells engrafted well in patients.

Conclusion

This study indicates that post-thawed UCB HPCs are preserved for less than 30 minutes at room temperature; thus, the optimal length of time of cryopreserved cord blood infusion should be no more than 20 minutes after thawing.     

BCR-ABL inhibitors: Updates in the management of patients with chronic-phase chronic myeloid leukemia

Objectives

This article reviews recent clinical experiences with first-line and second-line second-generation BCR-ABL inhibitors and discusses considerations for selection of therapy for patients with chronic-phase chronic myeloid leukemia.

Methods

We reviewed recent publications on PubMed and abstracts from major congresses relevant to the topic.

Results

Therapeutic options for front-line treatment have increased with the approval of two second-generation BCR-ABL inhibitors, dasatinib and nilotinib. Both agents are also treatment options for patients with resistance or intolerance to front-line imatinib. More recently, bosutinib, ponatinib, and omacetaxine have also been approved for patients with resistance or intolerance to prior therapy.

Discussion

Expanded treatment options coupled with rapidly changing treatment guidelines have led to numerous questions regarding the selection and monitoring of therapy. Common concerns include how to best select therapy based upon patient-specific comorbidities, monitoring and interpretation of treatment outcomes, and optimization of dosing when side effects occur.     

‘Reflex’ HPLC testing as a screening modality for variant hemoglobins: A pilot study of 1310 cases at a pediatric referral hospital

Objective

High-performance liquid chromatography (HPLC) has been evaluated as useful technique for detection of variant hemoglobins in newborn screening, ethnicity-based screening, and patients with abnormal hemoglobin electrophoresis. This study aimed at evaluating this technique as ‘reflex’ testing in a pediatric referral center.

Methods

This study included 1317 children visiting the pediatric hospital, who underwent HPLC analysis on anticoagulated blood samples. These patients were divided into clinician-requisitioned HPLC (group A), ‘reflex’ testing for anemia (group B), and unrelated etiologies (group C). HPLC graphs were evaluated for various hemoglobins. Statistical analysis was performed for comparison between these groups for frequency of abnormal HPLC assay and various red cell parameters.

Results

The frequency of abnormal HPLC assay was 22.89% in group A (125 of 546 cases), 26.89% in group B (78 of 290 cases), and 31.8% in group C (153 of 781 cases) with statistically significant difference. The most frequent variant in all three groups was thalassemia trait. Thalassemia intermedia and major, both were detected in few patients in groups B and C.

Conclusion

‘Reflex’ testing for hemoglobin variants can be undertaken for pediatric samples to enhance detection of these variants and avoid an additional venepuncture.