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SYNOPSIS Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. IMMATURE RETICULOCYTE FRACTION AS A USEFUL PARAMETER FOR BLOOD TRANSFUSION ASSESSMENT IN ANAEMIA Name of the candidate : Dr. Susan Maria Mendonca Guide : Dr. Jayaprakash C.S. Course and Subject : M.D (Pathology) Department of Pathology, Father Muller Medical College, Kankanady, Mangalore – 575002. JULY- 2013 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA PROFORMA FOR REGISTRATION OF THE SUBJECT FOR DISSERTATION 1. Name of the candidate and address (in block letters) DR.SUSAN MARIA MENDONCA P.G. RESIDENT. FR. MULLER MEDICAL COLLEGE, KANKANADY, MANGALORE – 575002. 2. Name of the institution FR. MULLER MEDICAL COLLEGE, KANKANADY, MANGALORE – 575002 3. Course of study and subject M.D. (PATHOLOGY) 4. Date of admission to the course 01-06-2013 5. Title of the topic: “ IMMATURE RETICULOCYTE FRACTION AS A USEFUL PARAMETER FOR BLOOD TRANSFUSION ASSESSMENT IN ANAEMIA ‘’ 6. BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED FOR THE STUDY Anaemia is a global public health problem affecting both developing and developed countries with major consequences on human health as well as social and economic development.1 The diagnosis and treatment of anaemia is based on several parameters. One of the sensitive parameter in this condition is the Peripheral blood reticulocyte count. Automated reticulocyte count is much superior to that of the manual Reticulocyte count because many more cells are counted and subjective element inherent in recognizing late reticulocytes is eliminated.2 One of the parameters assessed by the automated haematology analyser is the Immature Reticulocyte Fraction (IRF) . The IRF can be helpful in evaluating bone marrow , erythropoietic response to anaemia and evaluating response to therapy.3 Till date, Haemoglobin has been used as the parameter to assess the need for blood transfusion. Since Immature Reticulocyte Fraction is indicative of erythropoiesis, it could be used to predict the need for blood transfusion. Therefore, the aim of this study is to assess the utility of Immature Reticulocyte Fraction as a useful parameter of anaemia type, which may inform the decision to treat with red cell transfusion.4 6.2 REVIEW OF LITERATURE The term Anemia, as it generally is used in clinical medicine , refers to a reduction below normal in the concentration of hemoglobin or red blood cells in the blood. Before the advent of modern electronic equipment, the concentration was best measured as the volume of packed red cells(VPRC) rather than by enumeration of the red blood cells, because the VPRC offered greater precision and accuracy and also greater ease of standardization and maintenance of equipment. Now, however, the blood hemoglobin concentration is often preferred in part, because technological advancement has improved its accuracy and reproducibility so that it equals or exceeds that of VPRC, and in part because it is the value most indicative of the pathologic consequences of anemia.5 Since the number and characteristics of reticulocytes in the peripheral blood reflect the activity of bone marrow, reticulocyte counting has become a fundamental part of the evaluation of patients with hematopoietic disease.2 Circulating reticulocytes are decreased in patients with impairment of bone marrow function and increased under circumstances of blood loss with normal bone marrow activity. Reticulocyte enumeration has been performed for several decades by light microscopy, with the use of a supravital dye (new methylene blue) which binds to the RNA in the reticulocyte. However, the accuracy and precision of this assay are greatly compromised by its subjective nature, and by limited number of cells that can be counted by a technologist in a reasonable length of time. In contrast, automated techniques of reticulocyte enumeration are more precise , accurate , objective, and cost effective , since 30,000 or more cells can be accurately evaluated in a very short period of time.6 Automated reticulocyte counting based on flow cytometry has provided much objective and exactly measure of percentage and absolute number of reticulocytes than microscopic method.7 Rowan et al, in a review on reticulocyte count, suggest that absolute reticulocyte count and the reticulocyte percentage indicate different aspects of erythropoietic control: absolute count reflects the rate of red cell production, balanced by maturation, while the percentage indicates the balance of production of red cells in relation to their rate of destruction.8 Besides these two traditional parameters, automated reticulocyte counters can detect differences in the amounts of cellular RNA present in red blood cells which reflects their maturational stages. There are three related parameters which describes the reticulocyte maturation: RMI (reticulocyte maturity index), HFR (high fluorescence reticulocytes), IRF (immature reticulocyte fraction).8 Lin and collaborators evaluated automated reticulocyte evaluation in a large group of patients. Very low reticulocyte counts (< 0.03x 10 12 / L) and very low immature fractions (< 0.1) were characteristic of patients with aplastic or megaloblastic anemias, while patients with marrow infiltrative diseases had nearly normal reticulocyte counts , but high immature fractions( >0.3).6 (Reference range IRF 0.2-0.4).9 Studies by Geldard AR et al showed that IRF was found to show a more significant correlation with haemoglobin level than did the absolute reticulocyte count.4 (IRF (0.210.44).10 6.3 Objective of the study: a) To correlate hemoglobin levels and reticulocyte parameters (Immature Reticulocyte Fraction (IRF), Absolute reticulocyte count and reticulocyte percentage) as obtained from automated hematology analyzers . b) To group anemias into proliferative and hypoproliferative types based on IRF obtained from automated hematology analyzers. c) To assess the need for blood transfusion based on IRF values obtained. 7.1: Source of data: The EDTA treated venous blood specimens received in hematology laboratory in Father Muller Medical College will be used to measure Hemoglobin levels and automated reticulocyte counts. 7.2 Sample size: A minimum of 150 patients suspected for anaemia will be included in the study. Design of the study: Correlative study. Sampling technique is purposive based on inclusion and exclusion criteria. Duration of the study: The study period will be from November 2013 to January 2015. 7.3 METHOD OF COLLECTION OF DATA Study will include a minimum of 150 patients attending the Out Patients Department and In-Patient’s of Father Muller Medical College Hospital, from November 2013 to January 2015.The data necessary will be collected in the following mannerClinical details comprising of the following will be noted: • age • gender • intake/exposure to chemicals or drugs • bone pain • fever • night sweats • malaise • weight loss A detailed physical examination will be done to look for the following; • hepatomegaly • splenomegaly • lymphadenopathy • sternal tenderness Basic investigations will be performed for each patient including - Peripheral smear Red blood cell count White blood cell count Platelet count Red blood cell indices Red cell distribution width Hemoglobin - Hematocrit - Reticulocyte count ( manual and automated) - Serum biochemical studies will be done whenever required. - Marrow examination whenever needed. Inclusion Criteria • Patients belonging to the age group of 18 to 75 years presenting with anemic features will be included in the study group • Patients showing parameters as Hb< 10g/dL Exclusion Criteria Patients who have been treated for anemia will be excluded from the study. Data Analysis Collected data will be analysed by mean, standard deviation, Karl Pierrson correlation coefficient, T test and ROC analysis. 7.4 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? Yes. The study requires collection of blood samples and bone marrow samples for examination. Voluntary informed consent will be obtained from the subjects of the study. Consent form written in english will be translated into the language the patient understands best. 7.5 Has ethical clearance been obtained from your institution in case of 7.4? Yes. INFORMED CONSENT FORM Name of the volunteer: Date: Sex: Age: Address: This is to declare that I __________ have understood every aspect of the study “Immature reticulocyte fraction as a useful parameter for assessment of blood transfusion in anaemia” being conducted by Dr.Susan Maria Mendonca, post graduate student of the Department of pathology, FMMC, Mangalore. I voluntarily give my consent for the tests to be performed using my blood sample. The details of the study have been explained to me in an understandable manner and to my satisfaction. I have read this completely and I am sure that the data generated will be kept in at most confidence. I completely agree with this and shall follow all medical directions given to me. By my own decision and self interest, I give my consent to take part in this study. I keep my option to leave the study at any time I desire to do so. I am allowed to freely ask questions regarding the research study. I do agree to take part in the study voluntarily. Signature of the volunteer: Signature of the witness: Signature of the principal investigator. 8. LIST OF REFERENCES: 1. World Health Organisation. Worldwide prevalence of anemia 1993-2005:WHO global database on anemia 2005. 2. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Haematology.10th Edition. Philadelphia: Elsevier;2006. Chapter 3, Basic haematological techniques;p52. 3. McKenzie SB, Williams JL. Clinical Laboratory Hematology.2nd Edition: New Jersey:Pearson Education,Inc; 2010. Chapter 8, Introduction to Anemia; p156. 4. Geldard AR, Tobin DJ, Cuthbert A. Immature reticulocyte fraction as a useful parameter for blood transfusion assessment in anaemia.Br J Biomedical Sci 2009;66(2)abstract. 5. Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN. Wintrobe’s Clinical Hematology.9th Edition.London: Lea & Febiger;1993.Chapter 23, The approach to the patient with anemia;p716. 6. Riley SR, Ben-Ezra JM, Tidwell A. Reticulocyte enumeration: past & present. Lab Med 2001;32:599-607. 7. Wysocka J, Turowski D. New reticulocyte indices and their utility in hematological diagnosis.Pol Merkur Lekarski 2000 Jul;8(49):abstract 8. Cortellazi LC, Teixeira SM, Borba R, Gervasio S, Cintra CS, Grotto HZW. Reticulocyte parameters in hemoglobinopathies and iron deficiency anemia. Rev. Bras. Hematol. Hemoter. 2003, June;25(2) : 97-102 . 9. Kessler C, Machin SJ, Pollard Y et al. Reticulocyte performance on the Coulter GEN.S System. Lab Hematol. 1997; 3:41-47. 10. Briggs C, Grant D, Machin SJ. Comparison of the Automated Reticulocyte Counts and Immature Reticulocyte Fraction Measurements Obtained Wentra 120 Retic Blood Analyzer and the Sysmex XE-2100 Automated Hematology Analyzer. Laboratory Hematology 2001;7:75-80. 9. SIGNATURE OF THE CANDIDATE - 10. REMARKS FROM GUIDE- IRF is a simple, readily available parameter. Can be used to evaluate anemia-useful study 11. 11.1NAME & DESIGNATION DR. JAYAPRAKASH C.S., M.D OF GUIDE PROFESSOR DEPARTMENT OF PATHOLOGY FR. MULLER MEDICALCOLLEGE MANGALORE – 575002 11.2 SIGNATURE 11.3 HEAD OF DEPARTMENT DR. HILDA FERNANDES MD PROFESSOR AND HOD OF PATHOLOGY DEPARTMENT OF PATHOLOGY FR. MULLER MEDICAL COLLEGE KANKANADY, MANGALORE – 575002 11.4 SIGNATURE 12 12.1 REMARKS OF THE CHAIRMAN AND DEAN 12.2 SIGNATURE