Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM

Usually packed red blood cells (pRBCs) require specific conditions in storage procedures to ensure the maximum shelf life of up to 42 days in 2–6°C. However, molecular and biochemical consequences can affect the stored blood cells; these changes are collectively labeled as storage lesions. In this study, the effect of prolonged storage was assessed through investigating morphological changes and evaluating oxidative stress. Samples from leukodepleted pRBC in SAGM stored at 4°C for 42 days were withdrawn aseptically on day 0, day 14, day 28, and day 42. Morphological changes were observed using scanning electron microscopy and correlated with osmotic fragility and hematocrit. Oxidative injury was studied through assessing MDA level as a marker for lipid peroxidation. Osmotic fragility test showed that extended storage time caused increase in the osmotic fragility. The hematocrit increased by 6.6% from day 0 to day 42. The last 2 weeks show alteration in the morphology with the appearance of echinocytes and spherocytes. Storage lesions and morphological alterations appeared to affect RBCs during the storage period. Further studies should be performed to develop strategies that will aid in the improvement of stored pRBC quality and efficacy. http://www.hindawi.com/journals/bmri/2016/4529434/

 

Advertisements

Incidence of blood groups among blood donors in Maldives

Prevalence of blood groups among different ethnic groups have variation in different part of the world. There is no published data about the prevalence of blood groups in Maldives. The data shown here is the incidence trend among blood donors in blood bank at Maldivian Blood Services. As expected O Rh+ blood group is most common blood group among Maldivians.

Blood group

Cancer Awareness Program in Hulhmeedhoo

Cancer is alarmingly increasing in Hulhumeedho for the last decade the data shows.

Cancer awareness workshop held in Adducity Hulhumeedhoo on 22-23rd June 2012. The target audience was women of age group 21-45. The areas covered on this workshop were introduction of cancer, and common predisposing factors for cancer. Major areas covered among most common female cancers were cervical cancer, uterine and breast cancer.

Haveeru News Report

The Crisis of Iron in Transfusion Medicine: Improved Iron Chelation Therapy and its Implications For Clinical Practice in the Maldives

Name: Ibrahim Mustafa

Date: Monday, July 18, 2011 at 9:00 AM

Place: Room 203 of the Graduate Student Centre

Abstract
The Maldives has one of the highest incidences of ß thalassemia in the world. Treatment of ß thalassemia is characterized by two distinct phases: treatment of nature’s disease (anemia) and the secondary treatment of transfusional iron overload, a side effect of our cure. Interestingly, excess iron may also exert a negative effect on immune competence thus explaining the recurrent bacterial infections in these patients. Current iron chelation therapy using DesferalÒ (DFO) is challenging due to its short vascular half-life, frequency of injections, toxicity and expense. To address this problem, we have tested novel, low toxicity, high molecular weight (HMW) iron chelators. The utility of these chelators was tested in vitro using iron loaded (0-500 μM ferric (Fe3+) ammonium citrate, FAC; 0- 48 hours) HepG2 and dendritic cells (DC). Iron chelation studies utilized either single or combinational treatment with deferiprone (L1) or DFO (both low molecular weight chelators) and S-DFO (a HMW derivative of DFO) for 0-48 hrs. The efficacy of treatment was assessed by cellular ferritin, Perl’s iron stain, transmission electron microscopy (TEM), antigen presentation assays and cell viability assays. Iron treatment alone resulted in a significant increase in intracellular ferritin, histochemical iron staining and also resulted in a ~65.2% reduction in PBMC proliferation in response to the tetanus toxoid following 14 days of incubation.

Treatment with either L1 or S-DFO alone demonstrated modestly decreased ferritin levels and iron staining. Importantly, combination therapy (L1+S-DFO), resulted in an additive effect resulting in a 79% decrease in FAC-driven ferritin levels after 48 hours and TEM studies of FAC treated, but not control, cells.  FAC treated cells also demonstrated organellular and structural changes with electron dense iron deposits. As hypothesized, iron chelators (e.g., 200 μM DFO or L1) restored the PBMC proliferation in a concentration dependent manner and reversed the ultra structural changes in organelles. The development of HMW chelators may provide better therapeutic value (less toxic and less frequent administration) in developing nations. Consequent to this, iron mediated pathology in patients would be diminished resulting in less cost to already strained public health budgets such as in Maldives.

http://www.pathology.ubc.ca/pathology/News.htm

Next Generation of Transfusion Scientists

Saga of thalassemia-Maldivian perspectives

Ibrahim Mustafa
National Thalassemia Centre, Male’ Maldives

Maldives is a country with a distinctive geography that portrays a model of a small island nation. The total landmass of the 1190 small islands of the country is less than one percent of its national territory. Population of the 200 inhabited islands varies considerably. The country’s 2000 population census shows a total of 270,101 people living in the country.

Origins of the Maldivians are not very certain. However, it is believed that the history goes as far back as BC 1000. Early settlers were travelers on the Silk Route, and from the Indus Valley Civilization. The Maldivians are innately warm, friendly and generous by nature, and anyone can easily establish a casual conversation with the Maldivians.

It is believed that the genetically inherited heterogeneous disease “Thalassaemia” have been imported to Maldives through traders’ route, how ever there is no scientific evidence to prove this, or the gene is due to a mutation in the midst of Maldivian population. The earliest case of Thalassaemia diagnosed goes back to early 1970s. During 1970 – 1980 few cases registered in Government Hospital where there was no proper regimen for transfusion and proper management of this disease. During 1980 -1990 there was dramatic increase of Thalassaemia patients in the country, due to this special clinics for these patients were been made in the paediatric ward of the hospital. The impact of Thalassaemia and its social stigma has been recognized by the government and many experts from various countries have been brought in collaboration with WHO during 1980s. Ever since 1992 one of the leading NGO in the country ,Society for Health Education (SHE) working towards well being of Maldivian’s health has started one of its essential element, Thalassaemia Prevention Program, comprising ,health education , population screening and genetic counseling .During 1993 National Thalassaemia Program has been formulated and in December 1994 National Thalassaemia Centre has been inaugurated with 17 beded ward , blood banking facilities and a diagnostic laboratory services.

The enormous contribution of many people in both Government and NGOs across the country, thalassaemia awareness has increased dramatically and now the word thalassaemia is a house hold story amid all the alliance of communities. The combine effort of these institutions has screened thalassaemia and other hemoglobinopathies over 30 % of the entire population in the archipelago using modern diagnostic methodologies, including hemoglobin electrophoresis, automatic cell counting and quantification of hemoglobin. Some collaborative studies has been done with International Thalassaemia Research Institutions abroad, such as molecular defect of Beta Thalassaemia in the Maldives has been studies and their prevalence has been identified (74% being IVS 1 – 5 G – C ) ( Journal of Hemoglobin 1998, Fucheron et al ). This knowledge has contributed and paves the way to guiding principle for the prevention of thalassaemia for the sustainable development of health sector in the country.

Around 18% of the Maldivian population is found to be carriers for hereditary blood disorder, beta thalassaemia. This prevalence level leads to a birth of a thalassaemic child (thalassaemia major) for every 120 births. At the end of the year 2002, nearly 504 thalassaemia majors were registered at the National Thalassaemia Center. It has been estimated that for the eight-year period 1990-1998 more than 50% of the Thalassaemic children born in Maldives would have died in infancy or before their third birthday.

Due to the impact of emerging issues of thalssaemia and its impact to the community directly and indirectly, a six year National Thalassaemia Program has been formulated, many areas have been highlighted and it’s on the way for implementations. This includes, population screening, thalassaemia education, prenatal diagnosis (PND) and research. In near future PND can be done in Maldives with the establishment of molecular biology laboratory in the country. The couples at risk can be counseled and if they desire medical termination of pregnancy (MTP) can be recommended.

Screening for thalassaemia, as well as treatment of thalassaemics, is costly but at the moment it’s free in the Maldives. Thalassaemic children require continuous and regular care and treatment to stay alive. They require monthly transfusions and treatment with the drug Desferrioxamine, injected five times a week. The annual cost of treatment exceeds US$6,000. At present only, Bone Marrow Transplant (BMT) ensures permanent cure for Thalassaemics. But the cost of this treatment ranks between US$30,000 and US$50,000. Due to the low income of average people, this costly treatment BMT cannot be afforded by many families.

The perspective of Thalassaemia seems to be a successful story in Maldives, as it is integrating government and non governmental efforts to control thalassaemia in Maldives. A look at the expectations therefore indicates the need for a more comprehensive incorporated program to strengthen the existing agenda for the prevention of thalassaemia in Maldives.

Note: This article was written on 2002 and published on Thalassemia Awareness Week supplement at Baqai Medical University.

References:

1. Ministry of Planning National Development: Population and Housing Census of Maldives 2000, Population and Housing Tables (1st Edition) Novelty Press. Male’
2. Society for Health Education (2002),Thalassaemia Status in Maldives: a report from retrospective studies
3. National Thalassaemia Centre(2002) Laboratory data ,Male’ Maldives
4. UN Building, WHO Reference Library, Reports on Thalassaemia in Maldives
5. Health Master Plan, Sustainable Development of Health 1996
6. National Thalassaemia Program 2001 – 2006, Ministry of Health, Maldives.
7. Daniel W. Byrne(1988) Publishing Your Medical Research Paper(1stEdition)Williams

Canadian Society For Transfusion Medicine Annual Meeting 2011,Toronto, Canada

Effect of Iron on Antigen Presenting Cells: Implications For Transfusion Dependent Hemoglobinopathies.

Ibrahim Mustafa, Duncheng Wang and Mark D. Scott

Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: The thalassemias and Sickle Cell Disease (SCD) are characterized by destabilized hemoglobin that can lead to a potentially life-threatening anemia. This anemia arises due to iron-driven destruction of the RBC. Removal of oxidatively damaged RBC in vivo occurs primarily via erythrophagocytosis by the mononuclear phagocytic system (MPS). This clearance mechanism may result in negative immune consequences such as the observed increased risk of bacterial infections in these patients. Methods: To determine the functional consequences of iron and iron chelators on the MPS, the effects of ferric iron (Fe3+; ferric ammonium citrate, FAC) on dendritic cells (DC) antigen presentation [tetanus toxoid (TT Ag)] and the proliferation of peripheral blood mononuclear cells (PBMC) were examined. The iron chelators tested included Desferal (DFO) and Deferiprone (L1). PBMC were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) to measure cell proliferation. In addition, the effects of iron +/- iron chelators on the expression of CD83, CD80, CD86 and HLA-DR on mature DC were examined. Results: Importantly, iron significantly inhibited antigen presentation and PBMC proliferation. Treatment of DC cells with 200 µM FAC for 24 hours resulted in a ~70% reduction in PBMC proliferation in response to the TT Ag following 14 days culture. However, inclusion of iron chelators (e.g., 200 µM DFO or L1) restored near normal proliferation. Similarly, CD83 an important co-stimulatory molecule expressed on DC cells was also negatively affected by FAC in a dose (0-200 µM) dependent manner. Following 24 hours treatment with 200 µM FAC, a ~30% reduction in the mean fluorescence of CD83 was observed via flow cytometric assay. Treatment with DFO or L1 overcame the effects of iron on CD83 expression. There were no significant effects of iron on CD80 or CD86. Conclusions: As shown, iron has significant immunosuppressive effects on antigen presentation and lymphocyte proliferation. Iron chelators can effectively bind and remove free and complexed iron and reverse iron-mediated immunosuppression. These data suggest that iron chelation may provide a mechanism to diminish the risk of recurrent bacterial infections in patients with unstable hemoglobins or with iron-overload (hemochromatosis or secondary iron overload).

Note: This abstract was published on conference proceedings, of Canadian Society for Transfusion Medicine 2010 held at Vancouver

Immunological Inhibition Arising From Misplaced Iron: Implications for Thalassemia and Sickle Cell Disease

I. Mustafa, D. Wang and M.D. Scott

Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: The thalassemias and Sickle Cell Disease (SCD) arise from mutations to the globin subunits of adult hemoglobin (HbA) resulting in destabilized hemoglobin and, potentially, a life-threatening anemia due in part to iron-driven redox reactions. While transfusions corrects the anemia, secondary iron overload can occur. Thus, both the primary and secondary pathology of thalassemia and SCD arise from “misplaced” iron. Removal of oxidatively damaged RBC in vivo occurs primarily via erythrophagocytosis by the mononuclear phagocytic system (MPS). This clearance mechanism may result in negative immunoregulatory effects such as the observed increased risk bacterial infections in these patients.

Methods: To determine the functional consequences of iron on the MPS, the effects of ferric iron (Fe3+; ferric ammonium citrate, FAC), heme, purified HbA and oxidized RBC on antigen presentation/proliferation by PBMC and cultured dendritic like (DC) cells was examined. Antigens examined included tetanus toxoid (TT Ag), formalin-fixed Streptococcus mutans (SM Ag) and RhD peptide. PBMC proliferation was determined by 3H-thymidine incorporation or via flow cytometry using carboxyfluorescein diacetate, succinimidyl ester (CFSE) stained cells. To determine if iron-driven immunomodulation could be reversed, an iron shuttle chelation system using Desferal (DFO; shuttle chelator) and S-DFO (a high molecular weight DFO-starch conjugate) was examined.

Results: Importantly, all forms of iron, including oxidized RBC, significantly inhibited antigen presentation and PBMC proliferation. For example, 100 µM hemin resulted in a >98% reduction in proliferation in response to the TT or SM Ag. Similarly, phagocytosis of oxidized RBC virtually abolished the ability of antigen presenting cells within the PBMC to present antigen and abolished the response to the TT and SM antigens. DC cells were similarly affected by FAC (200 µM) exposure (7 days) with a ~78% reduction PBMC response to an immunodominant RhD peptide. Iron chelators could partially overcome the effects of the bioreactive iron. Of interest, prolonged treatment with S-DFO (unlike DFO) did not adversely affect purified hemoglobin.

Conclusions: As shown, iron has significant immunodepressive effects on immune function (antigen presentation and lymphocyte proliferation). Iron chelation can effectively bind and remove free and complexed iron /heme preventing both redox-driven damage and immuosuppression. These data suggest that a two component iron shuttle chelation system may effectively slow/prevent iron-driven damage within cells and may also protect immune competency.

Note: This was published Transfusion AABB Annual Meeting, New Orleans, LA USA 24-27 Oct 2009.