Changes in RBC and Platelet indices in CPDA stored BLOOD

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Sharon Daniels
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1 Original article: Changes in RBC and Platelet indices in CPDA stored BLOOD Dr. Sonia Chhabra*, Dr. Saurav Chaudhary #, Dr. P.K. Sehgal**, Dr. Sunita Singh ##, Dr. Monika Gupta***, Dr. Rajeev Sen ### Professor*, Junior Resident#, Professor and Head**, Senior Professor ##, Assistant Professor***, Senior Professor and Head ### Department of Pathology* and Department of Transfusion Medicine** Pt. B.D. Sharma PGIMS, Rohtak , Haryana (India) Correspondence: Dr. Saurav Chaudhary Abstract Whole blood is still commonly transfused in developing countries where blood components are not easily available. This study aims to study the efficacy of stored whole blood for a period of 28 days and to delineate the changes that occur in RBC indices and platelet count in CPDA stored whole blood. Samples were collected and tested for various hematological parameters (haemoglobin, RBC count, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution- coefficient of variance, platelet count) at days 1 and 28 respectively on Mindray BC 5800 (5 part analyser). Statistically significant changes were observed in mean corpuscular volume and platelet count (p <0.05) while statistically non significant changes were observed in other parameters (p >0.05). Key words: Whole blood, RBC indices, platelet count Introduction The ultimate aim of transfusing blood is the maintenance or restoration of a medium for transport of oxygen to body tissues. 1 Whole blood still remains a commonly employed blood product at some places due to the lack of facility for component separation. 2 Blood is stored to achieve a good post transfusion survival. 3 Blood donations are of three main types: Whole blood donations, autologous donation, donor pheresis. One unit of whole blood is donor blood collected in a suitable anticoagulantpreservative solution (citrate phosphate dextrose adenine; CPDA) and its total volume is about 400 ml (350 ml of blood + 49 ml of anticoagulant). This blood is stored in an approved blood bank refrigerator at C. Shelf life of such blood is 35 days. 2,4 Whole blood can be separated into cellular and plasma components. 4,5 Red blood cell storage changes: A variety of changes have been identified in red blood cells during red blood cell (RBC) preservation. They are collectively termed as storage lesion and include extensive biochemical and biomechanical changes. Over time the glucose in stored blood is consumed, levels of 2,3-diphosphoglycerate (DPG) and ATP decrease, leading to reduced structural integrity of cells. Thus red cells become less deformable and more fragile as they age. 6,7 This fragility leads to the release of cell free haemoglobin and formation of microparticles, sub-micron haemoglobin containing vesicles and additional haemolysis. 8,9 69

2 Platelet storage changes: The changes that occur in platelet under blood bank storage conditions are collectively known as platelet storage lesions (PSL) The platelets are affected by hydrolytic enzymes released by leucocytes during storage which affects platelet membrane and may lead to their destruction. 4 Aims and Objectives : To study storage related changes in RBC indices and platelet count in stored blood with duration of storage up to a maximum of twenty eight days. Materials and Methods : This study was conducted in department of Pathology, Pt. B.D. Sharma PGIMS, Rohtak with the aim to delineate changes occurring in various haematological parameters on prolonged storage of whole blood in minimum of 100 samples. The samples were obtained from healthy voluntary donors donating blood at the model blood bank in our institute. All mandatory tests required prior to collection were conducted. The blood was collected under all aseptic conditions in sterile Citrate Phosphate Dextrose Adenine (CPDA) containing blood collection bags. These blood units were screened for various infectious diseases and stored at 2-6 C in the blood bank refrigerator. Ten ml of blood was transferred in the satellite bag to study various haematological (haemoglobin, RBC count, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distributioncoefficient of variance, platelet count) parameters on day 1 and 28. In order to qualify as a donor in our study the following conditions were met. 4 Age: between 18 and 60 years. Weight: Minimum weight 45 kg. Blood pressure: Systolic BP between 100 to 130 mm of Hg and diastolic between 60 to 90 mm Hg. Pulse: Pulse rate between 50 to 100/min and regular. Temperature: normal. Equipments and materials for blood collection : Blood collection bag: Blood from a donor was collected in a sterile, disposable blood collection bag (double, triple or quadruple bag system with a capacity to hold 350 ml of blood sample. These bags contain a standard amount of anticoagulant preservative solution for specified amount of blood. The anticoagulant used was citrate phosphate dextrose adenine (CPDA) and 49 ml of anticoagulant was used for 350 ml of blood. 2,4 The other necessary equipments included refrigerator (The blood was stored in the blood bank refrigerator at a temperature between 2-6 C). Sphygmomanometer, weighing balance sealing clips artery forceps, vacutainers, ethanol and emergency drugs. Technique for blood collection 2 Blood collection bag was labelled with donor identification number before withdrawal of blood. Ten ml of blood was transferred in the satellite bag for the study. Blood bags after being given a unique blood bag number were stored in the refrigerator at 2-6 C. Out of ten ml of blood transferred to a satellite bag, two ml was taken to study various haematological parameters at days 1 and 28 in five part analyzer (Mindray BC 5800). Each batch of test samples were run with two level controls. The data was subjected to paired t test. p values were obtained and <0.05 were considered significant. The vacutainers were discarded in blue bags and were incinerated in the institute s incinerator. 70

3 Observations The present prospective study was conducted in the Maximum donors eligible for blood donation department of Pathology and Transfusion Medicine, belonged to age group (47%) followed age Pt. B.D. Sharma, PGIMS Rohtak. One hundred group of years (23%). Only 2 of the donors samples of donor blood constituted the material for were female Maximum donors belonged to A+ study. Ten ml of blood from the donated unit of (29%) blood group followed by B+ (27%) group. blood was taken in the satellite bag. Two ml of Only 5% of the donors were Rh negative. stored blood in satellite bag was used to study Table 3 : Changes in various haematological various hematological parameters at days 1 and 28 in parameters on days 1 and 28 of storage in CPDA five part analyzer. stored blood. (day 1 taken as control) The overall observations and result are as follows- Table 1 : Frequency distribution of age of donors Mea Std Mea Std p- Sign Age Number Percentage n Deviat n Deviat val ifi- Distribution (%) Val ion Val ion ue canc ue (Day ue (Day e (Da 1) (Da 28) y y ) 28) Hb Non RBC Non- Cou Sig Total nt (10 12 Table 2 : Distribution of blood group of donors. /l) Blood group Number of Percentage (%) Hct Nondonors (%) Sig A MC Sig B V O (fl) AB MC Non- A- 2 2 H Sig B- 2 2 (pg) AB- 1 1 MC Non- Total HC Sig 71 (g/dl Sig )

4 (g/dl ) RD Non- W Sig CV (%) Plate Sig let Cou nt Statistically non-significant (p>0.05) changes were observed in Hb (13.061g/dl on day 1 and g/dl on day 28) RBC count ( /l on day 1 and /l on day 28), Hct (40.184% on day 1 and % on day 28), MCH (29 on 325 on day 1 and day 28), MCHC ( on day 1 and on day 28), RDW- CV( % on day 1 and % on day 28), while statistically significant (p<0.05) changes were observed in MCV (13.06 fl on day 1 and fl on day 28) and platelet count ( lacs on day 1 and lacs on day 28) in our study. Discussion In our study, a total of hundred voluntary donors satisfying the inclusion criteria were taken. The maximum number of donors (97%) were in the age group of years with 47% being in the age group of years, while only 3% of the donors were above 45 years of age. A similar sample observation was made by Ahmed Y et al in their study where 100% of male donors were in the age group of years while 100% of female donors were in the age group of years. 10 In our study, 98 donors were male and only 2 were female. The gender distribution was different in the study conducted by Karama MI et al (30 males and 30 females). 11 Ahmed Y et al (25 males and 25 females). 10 This difference may be attributed to the selection of sample group, social conditions prevailing in India and the increased incidence of anemia in women in India making them unfit for blood donation. The maximum number of donors in our study belonged to A+ blood group (29%) followed by B + blood group (27%). Next most common group was O+ (26%) followed by AB+ (13%). Rh- blood group constituted only 5% of our study sample. MediEdunet Youtube Channel. Networking in Medical Education Dedicated to Basic concepts in Medical sciences Subscribe & share A project of Medworld asia Internationals 72

5 Table 4: Changes in RBC indices in various studies Stud y/ Year Hb RB C Co unt Hc t M CV M CH MC HC Adia N.S N.S N. N. N. N.S s TC P=. S. S S. p= et al 0.95 p= p= p= p= 0.90 ( ) Kar Sig Sig ama p P MI <0. <0. et al (200 8) Chol Sig e SD p et al <0. ( ) Ahm Sig Sig ed p<0 P < Y et al 5 (200 8) Pres N.S N.S N. Sig N. N.S ent p p S p S p Stud >0. >0. p <0. p >0.0 y >0. 05 >0. 5 ( ) RD W- CV N.S. p= N.S. p >0. 05 N.S. = Non Significant Sig= Significant In our study, no statistically significant fall in haemoglobin concentration was observed during the 28 days storage period (p>0.05). This is in concordance with the observation by Adias TC et al (p = 0.952). 12 Though significant fall in haemoglobin from 10 days onwards of storage (p < 0.05) were observed in the study conducted by Karama MI et al, this decrease can be attributed to hemolysis which occurs during storage. 10,12 Significant fall from 7 th day onward was also observed by Ahmed Y et al (p<0.05). 10 However in our study, subtle statistically non significant changes were observed during storage. Improper mixing of blood and exposure of blood bag to high ambient temperature, leakage of haemoglobin during storage, can be the contributing factors. 10 No statistically significant changes in RBC Count were observed in our study (p >0.05) during the 28 day storage period. Similar observation were made by Adias TC et al (p = 0.376). 12 In our study, RBC count showed minor variations. The statistically non significant rise in mean RBC count on day 28 may be due to delay in sample processing or improper mixing of blood. Statistically insignificant changes were observed in haematocrit (p > 0.05) during the 28 day storage period in our study. Similar observation were made by Adias TC et al (p = 0.312). 12 However, significant change in HCT (p = 0.01) were noted by Karama MI et al 11 and by Ahmed Y et al 10 from 7 day onwards of storage (p <0.01). In our study mean HCT was % on day 1 and then rose to % on day 28. The rise may be due to increase in MCV. A statistically non significant rise in mean HCT 73

6 between day 14 (33.72%) and day 28 (34.20%) was also observed by Adias TC et al 12 which may be due to similar reasons. Statistically significant changes were observed in mean MCV (p < 0.05) in our study. The rise in MCV is attributed to the swelling of RBC during the storage period. However statistically nonsignificant changes were observed by Adias TC et al (p=0.677) 12 his study revealed rise in MCV values during the later phase of storage. No other studies has been done to note the changes in MCV values in CPDA stored whole blood, however Chole SD et al studied changes in MCV in EDTA stored blood over a five day period and found a significant change (p<0.01) in MCV. 13 No statistically significant changes were observed in mean MCH (p > 0.05) in our study. Similar statistically non significant changes were observed by Adias TC et al (p = 0.805) 12 during the 28 day storage period. No statistically significant changes were observed in mean MCHC (p >0.05) during the 28 day storage period in our study. Similar non significant findings were observed by Adias TC et al (p = 0.470) 12 during the 28 day storage period. In our study the mean value of MCHC was g/dl on day one and fell to g/dl on day 28.This may be attributed to slight fall in Hb during storage and gradual rise in haematocrit after third day of storage. In our study, the mean value of RDW-CV was % on day one and rose to % on day 28 however the change was statistically non significant (p>0.05). Similar statistically non significant changes were observed by Adias TC et al (p = 0.316) during the 28 day storage period. 12 The subtle variations in our study may be due to cell lysis/ swelling of some RBC s. Statistically significant fall in platelet count was observed in our study (p < 0.05). The platelets are affected by hydrolytic enzymes released by leucocytes during storage which affect platelet membrane and may lead to their destruction. This is similar to the observation made by Karama MI et al from second day onwards of storage (p < 0.05). 11 In another study Ahmed S concluded that a significant fall in platelet count occurred after two days of storage. 14 However Adias TC et al did not observe a statistically significant change in mean platelet count (p = 0.195). 12 CONCLUSION Donor blood is always in short supply and inadequate to meet clinical requirements. Whole blood is commonly transfused in developing countries due to lack of facility for component separation; hence there is a need to study the efficacy of stored blood. Considering the non significant changes in RBC indices (Hb, RBC count, HCT, MCH, MCHC and RDW-CV) on long storage, whole blood can be used as a medium for increasing the Hb levels and raising HCT in the recipient, however keeping in view the significant changes in MCV on storage and the minor statistically non significant changes in RBC indices in stored blood, component therapy (packed cells) may be better option. Considering the significant changes in platelet count in stored blood, component therapy/platelet-pheresis might be a better option. REFERENCES 1. Bun FH, May MH, Kocholaty WF, Shields CE. Haemoglobin function in stored blood. J Clin Invest. 1969;48: Kawthalkar SM. Whole Blood, Blood Components and Blood Derivatives. In: 74

7 Kawthalkar SM, editor. Essentials of Hematology. 2nd ed. New Delhi: Jaypee; p Oluyombo R, Oluyombo 0, Uchegbwu OO, Adegbamigbe O, Ayodele OE. Quantitative assessment of erythrocytes and leuocytes in CPD-A stored blood. Biomed Res. 2013;24 (4): Saran RK. Transfusion Medicine Technical Journal. 2nd ed. New Delhi: Director General Health Services p Kawthalkar SM. Blood Component. In: Kawthalkar SM, editor. Essentials of Clinical Pathology. 1st ed. New Delhi: Jaypee; p Daniel B, Shapiro K, Lee J, Gladwin MK. Storage Lesion-Role of red cell breakdown. Transfusion. 2011;51(4): Hogman CF, Verdier CH, Borhstrom L. Studies on mechanism of human red cell loss of viability during storage at 4 0 C. Vox Sang. 1987;52: Kor DJ, Buskirk CM, Gajic O. Red Blood Cell Storage Lesion. Bosnian J Med Sc. 2009;9(1): Greenwalt TJ, Sostok CZ, Dumaswala UJ. Studies in red cell preservation. Vox Sang. 1990;(58): Ahmed Y, Dallal B, Bashar M. Effect of blood storage on certain hematological parameters. Tikrit Med Jour. 2009;15(1): Karma MI, Nuamy AL. Hematological changes in stored blood. Jour Edu Sci. 2008;21(4): Adias TC, Moore-Igwe B, Jeremiah ZA. Storage related Hematological and Biochemical changes of CPDA-1 stored blood in a limited Resource Setting. J Blood Disorders. 2012;3(3): Cohle SD, Saleem A, Makkaoui DE. Effects of storage of blood on stability of haematological parameters. Am J Clin Pathol. 1981;76(1): Ahmed S, Orakah J. Cellular changes in stored whole blood and their implications on efficacy on transfusion therapy in Nigeria. Internet J Med. 2008;4(2):

Collect and label sample according to standard protocols. Gently invert tube 8-10 times immediately after draw. DO NOT SHAKE. Do not centrifuge.

Collect and label sample according to standard protocols. Gently invert tube 8-10 times immediately after draw. DO NOT SHAKE. Do not centrifuge. Complete Blood Count CPT Code: CBC with Differential: 85025 CBC without Differential: 85027 Order Code: CBC with Differential: C915 Includes: White blood cell, Red blood cell, Hematocrit, Hemoglobin, MCV,