Comparative Study In Some Biochemical Parameters In Stored Whole Blood In Standard Blood Bank And Tradi~0docx
=== Comparative study in some biochemical parameters in stored whole blood in standard blood bank and tradi~0 ===
COMPARATIVE STUDY ON SOME BIOCHEMICAL PARAMETERS IN STORED WHOLE BLOOD IN STANDARD BLOOD BANK AND TRADITIONAL REFRIGERATOR
OBISIKE, UCHECHUKWU ACHOR
NOVEMBER, 2015
COMPARATIVE STUDY ON SOME BIOCHEMICAL PARAMETERS IN STORED WHOLE BLOOD IN STANDARD BLOOD BANK AND TRADITIONAL REFRIGERATOR
OBISIKE, UCHECHUKWU ACHOR
PG.2014/01079
BMLS, (RSUST), AMLSCN
A DESSERTATION SUBMITTED TO THE POST GRADUATE SCHOOL, RIVERS STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY PORT HARCOURT, RIVERS STATE, NIGERIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (M.Sc.) IN MEDICAL LABORATORY SCIENCE (CHEMICAL PATHOLOGY OPTION)
SUPERVISOR
PROFESSOR NSIRIM NDUKA
CO-SUPERVISOR
DR BROWN HOLY
DECEMBER, 2015
ABSTRACT
Preservation and long term storage of whole blood or any of its components is needed to ensure a readily available, safe blood supply for transfusion medicine. To ensure therapeutic relevance of the product, strict adherence to favorable storage conditions of the blood bank is crucial. Biochemical integrity in stored blood is a function of prevailing storage temperature. Storage outside the stipulated temperatures could on transfusion lead to severe clinical consequences. This study aims at evaluating the in vitro effect of storage on ten (10) selected biochemical parameters (Na+, K+, Cl-, HCO3-, Total protein, Albumin, Ca2+, pH, Glucose and Hemoglobin concentration) in citrate phosphate dextrose adenine (CPDA-1) whole blood stored in a Standard Blood Bank (SBB) and in a Traditional Refrigerator (TR). A total of 37 apparently healthy volunteer donor subjects were used for the study. 20 donors donated 450mL of whole blood each into CPDA-1 blood bags and were stored in a standard blood bank, while 17 units were collected from 17 donors into the same anticoagulant/preservatives but instead stored in a traditional refrigerator. Both refrigerators were allowed the same relatively stable power supply for 35 days. 5mL of blood was taken at intervals of 7 days (1, 7, 14, 21, 28, and 35) from each of the bags for both SBB and TR methods of refrigeration and analyzed for ten parameters. Plasma Na+, K+, Cl-, HCO3-, and pH were analyzed using an automated clinical biochemistry analyzer while spectrophotometric methods as modified by Randox Diagnostics were used to analyze total protein, albumin, Ca2+ and glucose concentrations. Hemoglobin levels were measured photometrically using the Haemiglobincyanide method. It was observed that for both SBB and TR refrigeration, K+ levels rose drastically from the 1st to the 35th day with mean values of 2.89 ± 0.11mmol/L to 5.12 ± 1.10mmol/L and 2.52 ± 1.72mmol/L to 9.48 ± 0.94mmol/L for SBB and TR refrigeration respectively. A significant increase at p<0.001 was observed for K+ levels when values for SBB were compared with those of TR for days 7, 14, 21, 28 and 35. This statistically shows that the rise in K+ level was more in TR refrigeration. It has been observed that following blood transfusion of stored blood, complications such as hyperkalemia results, this can be exacerbated by higher levels of K+ in the transfused blood. Plasma Na+ levels generally decreased for both groups. Comparison between Na+ values for SBB with those of TR refrigeration showed that Na+ values for SBB were significantly higher than those of TR refrigeration at (p<0.001) for Days 7, 14 and 21. Plasma Cl-, HCO3-, albumin, pH and glucose levels decreased as the days of storage increased for both SBB and TR refrigeration, generally recording significant decreases when compared at p<0.001, 0.01 and 0.05. Significant decreases were recorded for Na+, Cl, HCO3- and total protein (Day 7); Na+, Cl- and total protein (Day 14); Cl- and total protein (Day 21); total protein (Day 28); albumin and total protein (Day 35). The results show that there were significant changes in the levels of almost all ten parameters at different weeks of storage and the changes were more in the units stored in the traditional refrigerator and this could pose a very high health risk to the recipient on transfusion.
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DECLARATION
I, Obisike, Uchechukwu Achor (PG.2014/01079), do hereby declare that this dissertation has not been partly or wholly submitted to this or any other university for the award of any kind of degree. It is entirely my own original work and all assistance are dully acknowledged.
…………………………………. ………..…………………
Obisike, Uchechukwu Achor Date (PG.2014/01079)
CERTIFICATION
We the undersigned hereby certify that this dissertation presented by Obisike, Uchechukwu Achor (PG.2014/01079) partially fulfills the requirements for the award of Master of Science (M.Sc) in Chemical Pathology option at the Rivers State University of Science and Technology, Nkpolu Port Harcourt, Rivers State, Nigeria.
Name Signature Date
Professor Nsirim Nduka ……………………… …………………………
(Project Supervisor)
Dr. Brown Holy ……………………… …………………………
(Co-Supervisor)
Dr. (Mrs) Wokem G.N. ……………………… …………………………
(Ag. Head of Department)
Professor Omubo-Pepple V.B. ……………………….. …………………………
(Dean, Faculty of Science)
………………………. ………………………..
(External Examiner)
ACKNOWLEDGEMENT
It pleases me first and foremost to thank God Almighty for the life, privilege and energy He has given to me to accomplish this task. I wish to also acknowledge with profound gratitude the immeasurable concern and contributions of my research supervisory team chaired by Professor Nsirim Nduka and assisted by Dr. Brown Holy. I would like to sincerely thank you both for your valuable time, support, encouragement and guidance. I would like to also say a big thanks to our current Head of Department Dr. (Mrs.) Wokem G.N. and immediate past H.O.D., Professor C.K. Wachukwu for their unquantifiable contributions to the success of this programme. I will not fail to appreciate the Head of HOD Blood Bank, BMSH as well as all the lecturers that taught me and other staff of this department as well as my colleagues in the programme.
I would also like to appreciate the efforts of my parents Chief (Engr) O.S Nduma and Mrs Charity Chijiago and my elder brother Barr. Obisike Chima. I want to also appreciate the person of Professor Steven Odi-Owei who has been very instrumental to my academic journey as well as my father in-law Dr. Itolima Ologhadien who has always advised me not to give up on my dreams and my spiritual fathers Professor Kingdom Orji and Pastor Tunde Okeleye as well as all members of DLCF RSUST who have never ceased in their prayers for me.
Finally, I will continue to remain grateful and thankful to a friend and mother, my dear wife, Mrs Ologhi Uchechukwu and my lovely daughter Ruhuoma Deborah Uchechukwu who have been of immense value to my life and achievements. I would like to thank them for their motivation, patience, support, understanding, encouragement and endurance in making me achieve this great feat.
DEDICATION
This work is dedicated to God and all mankind with medical and scientific minds.
TABLE OF CONTENTS
TABLE OF CONTENTS
Contents Page
Title Page ii
Abstract iii
Declaration iv
Certification v
Acknowledgement vi
Dedication vii
Table of Contents viii
List of Tables xi
List of Figures xii
Abbreviations xiii
Chapter 1
Introduction 1
Background of Study 1
1.1 Aim and Objectives 7
1.2 Hypothesis 7
Chapter 2
Literature Review 9
Blood 9
2.01 Functions of Blood 10
2.02 Components of Blood 12
2.03 Blood Cell Production 14
2.1 Transfusion Medicine 17
2.1.1 Origin of Transfusion Medicine 17
2.2.2 Blood Donation 19
2.1.3 Types of Transfusion 21
2.1.4 Recruitment of Donors 28
2.1.5 Collection of Whole Blood 29
2.1.6 Whole Blood Donor Screening 30
2.1.7 Transfusion Related Reactions 32
2.1.8 Acute Transfusion Reaction 33
2.1.9 Delayed Transfusion Reaction 37
2.1.10 Whole Blood Collection, Storage and Processing 37
2.1.11 Collection of Blood Components for Apheresis 38
2.2 Effects of Storage on Whole Blood 39
2.2.1 Changes in Biochemical Parameters 41
2.2.2 Complications Resulting from Leukocytes 60
2.3 Impact of Blood Storage on Clinical Outcome 62
2.3.1 Impact of Transfusion on Red Blood Cells 62
2.3.2 Consequences of Transfusing New Blood 65
2.3.3 Solutions to Conservative Approach to Blood Transfusion 66
2.4 Blood Components Storage Temperature Range 67
2.4.1 The Blood Bank Refrigerator 71
Chapter 3
Materials and Methods 73
3.1 Description of Study Area 73
3.2 Study Subjects and Enrolment Criteria 73
3.2.1 Inclusion Criteria 74
3.2.2 Exclusion Criteria 74
3.3 Ethical Consideration 74
3.4 Study Design 74
3.4.1 Sampling Method 74
3.4.2 Collection of Blood Samples (Bleeding of Donors) 76
3.4.3 Blood Sample Collection for Analysis 77
3.4.4 Sample Analysis 79
Chapter 4
Results 85
Chapter 5
Discussion 120
Chapter 6
6.1 Conclusion 128
6.2 Recommendations 129
References 130
Appendix 144
LIST OF TABLES
TABLE TITLE PAGE
4.1 Demographic Characteristics of Enrolled Subjects 86
4.2 Mean Values and Significance Levels of SBB Refrigeration for all Parameters 89
4.3 Mean, SD and Significance Levels of TR Refrigeration for all Parameters 92
4.4 Mean, p values and Levels of Significance for Day 1 for all Parameters
Comparing SBB and TR Refrigeration 94
4.5 Mean, p values and Levels of Significance for Day 7 for all Parameters
Comparing SBB and TR Refrigeration 96
4.6 Mean, p values and Levels of Significance for Day 14 for all
Parameters Comparing SBB and TR Refrigeration 98
4.7 Mean, p values and Levels of Significance for Day 21 for all
Parameters Comparing SBB and TR Refrigeration 100
4.8 Mean, p values and Levels of Significance for Day 28 for all
Parameters Comparing SBB and TR Refrigeration 102
4.9 Mean, p values and Levels of Significance for Day 35 for all
Parameters Comparing SBB and TR Refrigeration 104
4.10 Details of outcome of Questionnaire 106
LIST OF FIGURES
FIGURE TITLE PAGE
4.1 Line Graph Showing Temperature Fluctuations from
Day 1 to Day 35 for both SBB and TR 108
4.2 Mean Na+ Values for both SBB and TR Refrigeration 109
4.3 Mean K+ Value for both SBB and TR Refrigeration 110
4.4 Mean Cl- Values for both SBB and TR Refrigeration 111
4.5 Mean HCO3- Values for both SBB and TR Refrigeration 112
4.6 Mean Total Protein Values for both SBB and TR
Refrigeration 113
4.7 Mean Albumin Values for both SBB and TR Refrigeration 115
4.8 Mean pH Values for both SBB and TR Refrigeration 116
4.9 Mean Ca2+ Values for both SBB and TR Refrigeration 117
4.10 Mean Glucose Values for both SBB and TR Refrigeration 118
4.11 Mean Hb Level for both SBB and TR Refrigeration 119
ABBREVIATIONS
AABB American Association of Blood Bank
ACD Acid-Citrate-Dextrose
ADSOL Adenine, Dextrose, Sodium Chloride, and Mannitol
AIDS Autoimmune Deficiency Syndrome
ANOVA Analysis of Variance
AS Additive Solution
ATP Adenosine Triphosphate
BCG Bromocresol Green
BCP Bromocresol Purple
BMSH Braithwaite Memorial Specialist Hospital
BTS Blood Transfusion Service
CD Cluster of Differentiation
CDP Cryoprecipitate-Depleted Plasma
CPDA-1 Citrate Phosphate Dextrose Adenine Formula 1
CRP C Reactive Protein
DEHP Di-(2-ethylhexyl) phthalate
DPG Diphosphoglycerate
eNOS Endothelial Nitric Oxide Synthase
FDA The Food and Drug Administration
FFP Fresh Frozen Plasma
FNHTRs Febrile Nonhemolytic Transfusion Reactions
G-CSF Granulocyte-Colony Stimulating Factor
Hb` Hemoglobin
HCO3- Bicarbonate ion
HiCN Haemiglobicyanide
HIV Human Immunodeficiency Virus
HLA Human Leukocyte Antigen
HTLV Human T- Lymphotropic Virus
ICI Imperial Chemical Industries
IgA Immunoglobulin A
iNOS Inducible Nitric Oxide Synthase
INR International Normalized Ratio
K+ Potassium ion
MHC Major Histocompatibility Complex
MLSCN Medical Laboratory Science Council of Nigeria
Na+ Sodium ion
nNOS Neuronal Nitric Oxide Synthase
NO Nitric Oxide
OCPC Calcium O-Cresolphthalein Complexone
PAS Platelet Additive Solution
PRBC Packed Red Blood Cells
PRP Platelet-Rich Plasma
RBC Red Blood Cell
RCT Randomized Controlled Trial
SAGM Saline, Mannitol, Glucose, and Adenine
SBB Standard Blood Bank
SNO S-nitrosothiol
SNO-Hb S-nitrosohaemoglobin
SNOHb S-nitrosylated Hemoglobin
SPE Serum Protein Electrophoresis
TA-GVHD Transfusion-Associated Graft-Versus-Host Disease
TR Traditional Refrigerator
TRALI Transfusion-Related Acute Lung Injury
TTP Thrombotic Thrombocytopenic Purpura
VWF von Willebrand Factor
WB Whole Blood
WHO World Health Organization
CHAPTER 1
INTRODUCTION
1.0 Background of study
Preservation and long term storage of whole blood is needed to ensure a readily available, safe blood supply for transfusion medicine. In recent years, there has been an evolving argument regarding a functional issue in transfusion medicine: the question is about the effect of storing blood products on outcome in transfusion recipients and, do biochemical parameters increase or decrease when transfused blood has been stored for long periods, and what are the effects of temperature changes on the biochemical parameters of stored whole blood? The controversy is about whether the age and temperature of storage of a given unit of whole blood or its product is a factor in transfusion efficiency. Specifically, about whether “older” blood directly or indirectly increases risk of complication, and what are the changes in whole blood biochemistry at certain temperature fluctuations. Studies have not been consistent on answering these questions. With some showing that blood stored for a longer time is indeed less effective, but with others stating otherwise.
Blood is stored for the purpose of transfusion. Blood transfusion is generally the process of receiving whole blood or one of its components into the circulation for the purpose of replacing lost components. Early transfusion used whole blood, but modern medical practice commonly uses only components of blood such as Packed Red Blood Cells (PRBC), fresh frozen plasma (FFP), and so on. In Nigeria, most of the blood banks still practice whole blood transfusion (Adias et al., 2012).
The administration of whole blood or units containing cellular elements may pose many risks and potential unfavorable effects. This is basically due to the gradual decomposition of blood components and as a result of the accumulation of product of the cellular metabolism, that is, anaerobic glycolysis, particularly when the components are not stored at the temperature range as required by regulatory bodies, the biochemical composition of the stored whole blood are bound to undergo bizarre changes. The changes are proportional to the storage time, temperature and other factors (Radovan et al., 2011). The effect of time and temperature shall be considered in this study. Large volume of blood transfusion may attribute to changes in the patients plasma biochemical parameters and may therefore be related not only to the volume of blood products but also to storage duration and temperature. Brecher in 2005 reported that during storage, there is an increase in K+ and lactate levels and a simultaneous decrease in pH, glucose and Na+ levels. The storage has no impact on Ca2+ levels in the RBC concentrates (Radovan, et al., 2001).
Other changes include a reduction in RBC deformability, altered RBC adhesiveness and aggregability, and a reduction in 2, 3 – diphosphoglycerate (2, 3 – DPG) and adenosine triphosphate (ATP), bioactive compounds with proinflamatory effects also accumulate in the storage medium. These changes reduce post transfusion viability of RBCs. The clinical effect beyond transfusion are uncertain, but a growing body of evidence suggest that the storage lesion may reduce tissue oxygen availability, have proinflammatory and immunomodutatory effects and influence morbidity and mortality (Ho et al., 2003).
Blood storage is currently a logistical necessity for the maintenance of a readily and adequate blood supply. Whole blood is usually collected in an acidic solution and then stored in a plastic bag of anticoagulant/preservative citrate phosphate dextrose adenine (CPDA-1) in a Standard Blood Bank at 2-6oC. This solution contains citrate as the anticoagulant, phosphate helps in maintaining the pH, dextrose as energy source and adenine for the synthesis of ATP. It is unfortunate that after sampling one hundred and fifty (150) laboratories located in the city of Port Harcourt using a well-structured Questionnaire, an astonishing 65% of the laboratories use the traditional refrigerator as blood bank. This outcome necessitated the carrying out of this study. During storage, Red Blood Cells are metabolically active without any waste disposal system (that is, no renal system or liver), so the stored cells are essentially marinated in a pool of ever-increasing waste products (example, lactate). The obvious fact is that storage is a distinctly unnatural state in which blood cells are exposed somewhat a non-physiological conditions for various periods of time. The overall issue splits into two major questions: (Hess, 2012), to what extent can stored blood cells maintain their functional integrity and therefore constitute an efficacious product? (Hobert and Fergusson, 2004), and are there untoward effects of storage based changes that may result in medical consequences due to the accumulation toxic substances in the blood?
Although, the above questions may sound conceptually simple and straight forward, generating meaningful solutions is a considerable challenge which the field of transfusion medicine has been battling with. These difficulties may stem from several sources, including the current lack of optimal assays and thus the inability to observe certain literatures, substantial donor to donor disparities in blood storage, and the repertoire of disorders for which transfusion is administered. The condition is further exacerbated when the required temperatures are not used due to lack of the adequate facilities and other factors.
The Food and Drug Administration (FDA) criteria by which blood storage solutions are approved, which are the same generally used criteria in biochemical research, do not estimate the function of the blood products that are being administered. Whole blood storage include haemolysis of less than 1% and average 24 hours post transfusion recoveries of 75% or a greater percentage, which remain the only useful metrics that are accurately assessed (Hess, 2012). These metrics are by no means implausible, indeed an Red Blood Cell that does not survive the period and conditions of storage or cannot be useful upon transfusion will in effect not be able to transport oxygen, collect CO2, or participate in other Red Blood Cell functions. Several attempts are certainly made to evaluate the levels of parameters that may correlate to function. For instance, Red Blood Cell storage studies usually measure rheological properties of Red Blood Cells, some Red Blood Cell surface molecules, and Red Blood Cell metabolism (including 2, 3 – Diphosphoglycerate levels that regulate oxygen affinity curves).
There is a strong argument to be made that stored whole blood has decreased function after transfusion. Some have even expressed the view that RBCs have no function immediately after transfusion and do not improve oxygenation (Hebert and Fergusson, 2004; Shah et al., 2009; Tsai et al., 2004). This extreme view seems to be contradicted by the usual observation that patients who are severely anemic and symptomatically hypoxic improve in clinical presentation after transfusion. Some patients also undergo massive transfusion protocols in which essentially every circulating Red Blood Cell was recently transfused. The observation that these patients live shows that stored Red Blood Cells do have at least some post transfusion function. Chronically, anemic patients and those requiring hematological support during reconstitution after bone marrow transplantation hardly survives without transfusion. However, such observations do not in any way indicate that stored blood cells function optimally. It is also shows that even if the transfused Red Blood Cells were optimally functioning, this however does not necessitate that transfusion would have a positive effect upon medical outcome in several settings in which the purpose for transfusion itself is questioned (Hill et al., 2002).
Retrospective cohort studies have found a correlation between RBC storage duration and morbidity and mortality rates after transfusion (Purdy et al., 1997; Zallen et al., 1999; Leal-Noval et al., 2003), suggesting progressive storage lesions may be responsible for adverse outcomes. Despite these observational data no large controlled clinical trials have been conducted to evaluate the relationship between the age of stored Red Blood Cells and clinical outcomes. Red Blood Cell transfused patients had worse outcomes than non-transfused patients matched for clinical variables in several studies (Vincent et al., 2002). Moreover, in randomized clinical trials, a more liberal RBC transfusion strategy failed to benefit paediatric or adult patients with anaemia and critical illness (Hebert et al., 1999; Lacroix et al., 2007), raising the border concern that RBC storage is problematic (Tinmouth, 2006; Hogman and Meryman, 2006) and could be improved. This is particularly relevant because, to date, the development of and outcomes with blood substitutes have been disappointing (Kerner et al., 2003). Although storage induced changes in certain RBC molecular constituents have been studied, less is known about changes in RBC function with storage (Tinmouth, 2006; Valesi and Hirsch, 2009).
As aforementioned, one of the RBC’s principal functions is the O2 delivery, a product of changes in O2 content and blood flow. Increases in O2 affinity in stored RBCs, reflecting progressive decreases in 2,3-DPG over the weeks of storage, are well documented (Valesi and Horsch, 2009), and O2 delivery by stored RBCs is deficient even early after processing and before significant decline in 2,3-DPG (Tsai et al., 2004). However, less is known of how storage influences the role of the RBC in the O2–dependent regulation of blood flow (“hypoxic vasodilatation”) in part because this RBC function was only recently appreciated (McMahon et al., 2002). The O2 sensor role of haemoglobin (Hb) subserves this RBC activity by dispensing vasodilator. S-nitrosothiol (SNO) equivalents in proportion to the degree of hypoxia in the tissues it perfuses (McMahon et al., 2002). In concert with the oxygenation-induced allosteric transition, S-nitrosohaemoglobin (SNO-Hb) forms in human blood when a nitric oxide (NO) equivalent binds to the conserved 93 cysthiol residue of Hb. Conversely, RBCs perfusing tissues release limited fluxes of vasodilator SNO equivalents in proportion to Hb O2 desaturation, matching regional blood flow with metabolic demand.
Previous studies have looked at particular aspects of storage alone and have not collected, processed, and stored blood consistent with standards of the American Association of Blood Bank (AABB). Studies have also been conducted to simultaneously quantify multiple biochemical components of the blood storage lesion and related storage-induced, changes in RBC physiologic functions critical to O2 delivery, particularly deformability and RBC-dependent vasoactivity. It was reasoned that an “impairment in RBC-dependent hypoxic vasodilatation might underlie the functional RBC storage lesion. In particular, the hypothesis was tested that the processing and storage of RBCs for transfusion may disturb sNO-Hb stability (e.g. by oxidation, degradation, or release into the storage medium) and thus compromise RBC-dependent vasoregulation (McMahon et al., 2002).
Clinical implications, is in part related to bioreactive substances released by leucocytes in the storage medium, such as histamine, lipids, and cytokines, which may exert direct effect on metabolic and physical changes associated with the senescence, such as membrane reticulation, decrease in cell size, increase of cell density, alteration of cytoskeleton enzymatic desilytion, and phosphatidylsetine exposure, RBCs lose potassium. 2, 3-DPG, ATP stores, lipids and membrane while becoming more rigid and demonstrating reduced oxygen off-loading (Hess, 2006). However, stored units become more acidotic and the suspending fluid has highest concentrations of free haemoglobin and biologically active lipids and contains greater quantities of negatively charged micro vesicles with pro-inflammatory and pro-coagulant activity (Hess, 2006). Platelets circulate longer when stored at room temperature and are more activated and able to form a lot more effectively when stored at 40C. White cells lose their phagocyte property within 4-6 hours of collection and become non-functional after 24 hours of storage (Thon et al., 2008). It is important to remember they do not lose their antigenic property and are capable of sensitizing the recipient to produce non-haemolytic febrile transfusion reactions. Few lymphocytes may remain viable even after 3 weeks of storage. This is limited evidence to suggest that some biochemical analytes (such as cholesterol) may be stable in whole blood for several days at ambient temperature. The only important electrolytic change in stored blood is that of potassium. During storage there is a slow but constant leakage of K + from cells into surrounding plasma. In severe kidney disease even small amount of K+ fluctuations can be dangerous and relatively fresh or washed red cells are indicated. Due to a higher K+ content of stored blood <5 days old is recommended by Ono et al (Ono et al., 1981) for neonatal exchange and top-up transfusion.
Justification of Study
Whole blood transfusion is still a common practice in this part of the world. Evaluation of changes in the levels of these selected biochemical parameters in CPDA-1 stored whole blood, comparing values from units stored in a Standard Blood Bank (SBB) and Traditional Refrigerator (TR) had not been reported.
A well-structured and adequately distributed questionnaire conducted by the same study has shown that out of eighty four (84) sampled medical laboratories in Port Harcourt city that operate blood banks, an unimaginable 61.9% (52 laboratories) used traditional refrigerator in blood banking. This prompted the need for this study.
1.1 Aim and Objectives
Aim
This study aims at evaluating the in vitro effect of storage on some selected biochemical parameters in citrate phosphate dextrose adenine (CPDA-1) whole blood stored in a standard blood bank and to compare values of same with those stored in a traditional refrigerator.
Objectives
The objectives of the study are:
1. To determine the baseline values for selected biochemical parameters in whole blood stored in both traditional refrigerator and standard blood bank prior to storage
2. To determine the changes in the levels of ten (10) selected biochemical parameters (pH, Total protein, Na+, K+, Cl-, HCO3-, Glucose, Total Calcium, Albumin and Hb) in CPDA-1 stored whole blood for both Traditional Refrigerator and Standard Blood Blank.
3. To compare the changes (if any) in the ten listed biochemical parameters for the whole blood stored in a Traditional Refrigerator and those stored in a Standard Blood Bank.
1.2 Hypothesis
Part 1:
Ho; there are no significant differences between the mean values of ten biochemical parameters in SBB and TR CPDA-1 stored whole blood.
H1; there are significant differences in the mean values of ten biochemical parameters in SBB and TR CPDA-1 store whole blood.
Part 2:
Ho; there are no significant differences in the mean values of ten biochemical parameters from day 1 to 35 for both SBB and TR (separately) in CPDA-1 stored whole blood.
H1; there are significant differences in the mean values of ten biochemical parameters from day 1 to 35 for both SBB and TR (separately) in CPDA-1 stored whole blood.
CHAPTER 2
LITERATURE REVIEW
2.0 Blood
Blood is a constantly circulating fluid that provides the body with nutrition, oxygen, and waste removal. Blood is mostly liquid, with numerous cells and proteins suspended in it, making blood more viscous than pure water. The liquid part of blood called plasma makes up about half of the content of blood. One prime function of plasma is to help in blood to clotting, transport substances through the blood, and perform other functions due to the presence of proteins. Glucose and nutrients are also dissolved in blood plasma. In animals, blood delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells. Blood in vertebrates is bright red when its hemoglobin is oxygenated and dark red when it is deoxygenated. Blood accounts for 7% of the human body weight, with an average density of approximately 1060 kg/m3, this is very close to pure water's density of 1000 kg/m3. The average adult has a blood volume of roughly 5 litres, which is composed of plasma and several kinds of cells. These blood cells (also called corpuscles or "formed elements") consist of erythrocytes (red blood cells, RBCs), leukocytes (white blood cells), and thrombocytes (platelets). By volume, the RBCs constitute about 45% of whole blood, the plasma about 54.3%, while white cells about 0.7%. It is made of blood cells suspended in blood plasma. Plasma which constitutes 55% of blood fluid, is mostly water (92% by volume), and contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation), and blood cells themselves. Albumin being the main protein in plasma functions to regulate the colloidal osmotic pressure of blood. The blood cells are mainly red blood cells or erythrocytes, white blood cells or leukocytes and platelets. The most abundant cells in vertebrate blood RBCs. These contain hemoglobin, an iron-containing protein, which facilitates oxygen transport by reversibly binding to this respiratory gas and greatly increasing its solubility in blood. In contrast, carbon dioxide is almost entirely transported extracellularly dissolved in plasma as bicarbonate ion.
Blood is circulated around the body through blood vessels by the pumping action of the heart. In animals with lungs, arterial blood carries oxygen from inhaled air to the tissues of the body, and venous blood carries carbon dioxide, a waste product of metabolism produced by cells, from the tissues to the lungs to be exhaled. In terms of anatomy and histology, blood is considered a specialized form of connective tissue, given its origin in the bones and the presence of potential molecular fibers in the form of fibrinogen.
2.0.1. Functions of Blood
Blood performs many important functions within the body including:
2.0.1.0 Transportation
Blood is the primary means of transport in the body that is responsible for transporting important nutrients and materials to and from the cells and molecules that make up the human body. It is the duty of blood to first take the oxygen processed by the lungs to all the cells of the body and then to collect the carbon dioxide from the cells and deliver it to the lungs. It is also tasked with the job of collecting metabolic waste from up and down the body and take it to the kidneys for excretion. Blood also has to perform the task of delivering the nutrients and glucose generated by the organs of the digestive system to the other parts of the body including the liver. In addition to these tasks, blood also has to carry out the transportation of hormones produced by the glands of the endocrine system.
2.0.1.1 Protection
Blood performs the important task of protecting the body from the threat of infections and disease causing bacteria. The white blood cells found in blood are responsible for safeguarding the different organs of the body by producing antibodies and proteins which are capable of fighting off and killing the germs and viruses that can causes serious damage to the body cells. The platelets present in blood handle the task of limiting blood loss in the wake of an injury by helping the blood to clot quickly.
2.0.1.2 Regulation
Blood is also a regulator of many factors in the body. It oversees the temperature of the body and maintains it to a level that is tolerated by the body with ease. Blood is also responsible for controlling the concentration of Hydrogen ions in the body, which are also known as pH balance. The administration of the levels of water and salt required by each cell of the body also falls under the regulation duties of blood. Another regulatory task performed by blood is to control the blood pressure and restrict it under a normal range. Other functions include:
Supply of oxygen to tissues (bound to hemoglobin, which is carried in red cells) Supply of nutrients such as glucose, amino acids, and fatty acids (dissolved in the blood or bound to plasma proteins (for example, blood lipids)) Removal of waste such as carbon dioxide, urea, and lactic acid Immunological functions, including circulation of white blood cells, and detection of foreign material by antibodies Coagulation, the response to a broken blood vessel, the conversion of blood from a liquid to a semi-solid gel to stop bleeding. Messenger functions, including the transport of hormones and the signaling of tissue damage Regulation of body pH Regulation of core body temperature Hydraulic functions.
2.0.2 Components of Blood
As stated earlier, about 7% of human body weight is from blood. In adults, this amounts to 4.5-6 quarts of blood. This essential fluid carries out the critical functions of transporting oxygen and nutrients to our cells and getting rid of carbon dioxide, ammonia, and other waste products. In addition, it plays a vital role in our immune system and in maintaining a relatively constant body temperature. Blood is a highly specialized tissue composed of more than 4,000 different kinds of components. Four of the most important ones are red cells, white cells, platelets, and plasma. All humans produce these blood components–there are no populational or regional differences.
2.0.2.0 Red Blood Cells
Red cells, or erythrocytes, are relatively large (about 0.0003 inches in diameter) microscopic cells without nuclei. Red cells normally make up 40-50% of the total blood volume. They transport oxygen from the lungs to all of the living tissues of the body and carry away carbon dioxide. The red cells are produced continuously in the bone marrow from stem cells at a rate of about 2-3 million cells per second. Hemoglobin is the gas transporting protein molecule that makes up 95% of a red cell. Each red cell has about 270,000,000 iron-rich haemoglobin molecules. People who are anemic generally have a deficiency in red cells, and subsequently feel fatigued due to a shortage of oxygen. The red colour of blood is primarily due to oxygenated red cells. Human foetal haemoglobin molecules differ from those produced by adults in the number of amino acid chains. Foetal hemoglobin has three chains, while adults produce only two. As a consequence, foetal hemoglobin molecules attract and transport relatively more oxygen to the cells of the body.
2.0.2.1 White Blood Cells
White cells, or leukocytes, exist in variable numbers and types but make up a very small part of blood's volume, normally only about 1% in healthy people. Leukocytes are not limited to blood. They occur elsewhere in the body as well, most notably in the spleen, liver, and lymph glands. Most are produced in the bone marrow from the same kind of stem cells that produce red blood cells. Others are produced in the thymus gland, which is at the base of the neck. Some white cells (called lymphocytes) are the first responders for the immune system. They seek out, identify, and bind to alien protein on bacteria, viruses, and fungi so that they can be removed. Other white cells (called granulocytes and macrophages) then arrive to surround and destroy the alien cells. They also have the function of getting rid of dead or dying blood cells as well as foreign matter such as dust and asbestos. Red cells remain viable for only about 4 months before they are removed from the blood and their components recycled in the spleen. Individual white cells usually only last 18-36 hours before they also are removed, though some types live as much as a year. The description of white cells presented here is a simplification. There are actually many specialized sub-types of them that participate in different ways in our immune responses.
2.0.2.2 Platelets.
Platelets, or thrombocytes, are cell fragments without nuclei that work with blood clotting chemicals at the site of wounds. They do this by adhering to the walls of blood vessels, thereby plugging the rupture in the vascular wall. They also can release coagulating chemicals which cause clots to form in the blood that can plug up narrowed blood vessels. Thirteen different blood clotting factors, in addition to platelets, need to interact for clotting to occur. They do so in a cascading manner, one factor triggering another. Hemophiliacs lack the ability to produce either blood factor VIII or IX.
Platelets are not equally effective in clotting blood throughout the entire day. The body's circadian rhythm system causes the peak of platelet activation in the morning. This is one of the main reasons that strokes and heart attacks are more common in the morning.
Recent research has shown that platelets also help fight infections by releasing proteins that kill invading bacteria and some other microorganisms (Klinger and Jelkmann, 2002). In addition, platelets stimulate the immune system. Individual platelets are about 1/3 the size of red cells. They have a lifespan of 9-10 days. Like the red and white blood cells, platelets are produced in bone marrow from stem cells.
2.0.2.3 Plasma.
Plasma is the relatively clear, yellow tinted water (92+%), sugar, fat, protein and salt solution which carries the red cells, white cells, and platelets. Normally, 55% of our blood's volume is made up of plasma. As the heart pumps blood to cells throughout the body, plasma brings nourishment to them and removes the waste products of metabolism. Plasma also contains blood clotting factors, sugars, lipids, vitamins, minerals, hormones, enzymes, antibodies, and other proteins. It is likely that plasma contains some of every protein produced by the body approximately 500 have been identified in human plasma so far.
2.0.3. Blood Cell Production
Hematopoiesis is the formation of blood cellular components. All cellular blood components are derived from hematopoietic stem cells. In a healthy adult person, approximately 1011–1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation. Hematopoiesis generates a variety of distinct blood cell types from a common stem cell. Secreted signaling molecules called cytokines modulate the survival, proliferation and differentiation of all the blood cell lineages, mediated by defined sets of transcription factors. Haematopoiesis is also influenced by external cues such as oxygen concentration. Haematopoiesis is an ongoing process continuing throughout lifetime, although the location of stem cells, and the specific cell types derived from them, changes during embryonic, foetal and early postnatal development. In the adult, haematopoiesis occurs primarily in the bone marrow, in association with a supportive niche. Haematopoietic stem cells are derived from cells bipotential for blood and endothelial cells, or directly from specialised endothelium. Blood lineages have a hierarchical relationship, but there is some flexibility among progenitors for deriving specific fates. Defects in haematopoiesis result in common and serious human diseases including anaemia and leukaemia.
Key concept are:
Haematopoiesis is the process of forming blood cells, which occurs during embryogenesis and throughout life.
Defects in haematopoiesis can result in some of the most common and serious human diseases, including anaemia and leukaemia.
Blood consists of many different kinds of cells with a diverse range of functions, controlling gaseous exchange and clotting and comprising the immune system.
All blood cells are derived from a common progenitor, the haematopoietic stem cell.
The sites where haematopoiesis occurs change during embryonic development, but in adult mammals, the bone marrow is the major site of haematopoiesis.
Haematopoietic stem cells in the bone marrow reside in a specialised microenvironment known as the haematopoietic stem cell niche, composed of osteoblasts, mesenchymal cells and sinusoidal vessels.
Growth factors called cytokines control the survival, self‐renewal and differentiation of haematopoietic stem cells and their progeny.
Blood cells have a close developmental relationship with endothelial cells, and haematopoietic stem cells appear to derive from ‘haemogenic endothelium’.
Haematopoiesis is also regulated by external factors. For example, hypoxia results in a compensatory increase in the number of erythrocytes.
Much effort is currently focused on generating and manipulating haematopoietic stem cells in vitro, to develop new regenerative therapies.
In developing embryos, blood formation occurs in aggregates of blood cells in the yolk sac, called blood islands. As development progresses, blood formation occurs in the spleen, liver and lymph nodes. When bone marrow develops, it eventually assumes the task of forming most of the blood cells for the entire organism. However, maturation, activation, and some proliferation of lymphoid cells occurs in secondary lymphoid organs (spleen, thymus, and lymph nodes). In children, haematopoiesis occurs in the marrow of the long bones such as the femur and tibia. In adults, it occurs mainly in the pelvis, cranium, vertebrae, and sternum (Fernández and de Alarcón, 2013). In some cases, the liver, thymus, and spleen may resume their haematopoietic function, if necessary. This is called extramedullary haematopoiesis. It may cause these organs to increase in size substantially. During fetal development, since bones and thus the bone marrow develop later, the liver functions as the main haematopoetic organ. Therefore, the liver is enlarged during development (Georgiades et al., 2002). As a stem cell matures it undergoes changes in gene expression that limit the cell types that it can become and moves it closer to a specific cell type. These changes can often be tracked by monitoring the presence of proteins on the surface of the cell. Each successive change moves the cell closer to the final cell type and further limits its potential to become a different cell type.
2.1 Transfusion Medicine
Blood transfusion is generally the process of receiving blood products into one's circulation intravenously. Transfusions are used for various medical conditions to replace lost components of the blood. Early transfusions used whole blood, but modern medical practice commonly uses only components of the blood, such as red blood cells, white blood cells, plasma, clotting factors, and platelets.
2.1.1 Origin
The use of blood as a product can be traced back to the 17th century, although the greatest advances in its therapeutic was prompted by the worldwide conflicts of the first half of the 20th century. One of the earliest accounts of the circulation of blood was by the Arabic scholar, mathematician and physician Ibnal- Nafis (Khairallah and Haddad, 2000) who, in 1260 AD, described the 'minor circulation' of blood in the body. This was more than 250 years before William Harvey described the continuous circulation of blood around the body in 1616, (Matthew, 2005) which he published in 1628. The advent of the understanding of human anatomy and the circulation of blood gave rise to experimentation in transfusion techniques involving animal-to-animal and animal-to-human procedures. This eventually resulted in human-to-human transfusion. In 1657, Dr (later Sir Christopher) Wren, now better known as the renowned Architect, performed a series of experiments involving the injection of various fluids into the veins of animals, with mixed results. Subsequently, in 1665, at a meeting of the Royal Society of London, of which Wren was a founder member, he demonstrated the transfusion of blood from one animal to another.
In 1864 Dr Roussel in France and Dr James Aveling in London both used India rubber tubes to carry out direct human-to-human transfusions. James Aveling's apparatus consisted of two silver tubes that were used to enter the donor and recipient blood vessels, connected to a length of India rubber tubing, with a stopcock at both ends and a bulb in the middle. When squeezed, the bulb acted as a pump to expedite the flow of blood. In the last decade of the 19th century there was considerable debate about the benefit of using blood rather than saline. George Washington Crile carried out studies in 1898 to compare the efficacy of blood versus saline in maintaining blood pressure in shock. His conclusions kept alive the quest to find better and safer ways of transfusing blood, which did not became apparent until well into the second decade of the new century.
The discovery of a new polymer by a research team at Imperial Chemical Industries (ICI) in 1930 resulted in its rapid adaptation as a new electrical insulating material. It was light, flexible and waterproof, and the company named the substance polythene. The first reported clinical use of polythene in the UK, in 1948, was as a fine, flexible catheter for neonatal use. In 1952, Walter designed a polythene blood collection bag with integral donor line and giving set. Walter's invention was soon recognised and the Fenwal Company was set up to mass produce the bags for use in American and Canadian hospitals. By 1960 they were in widespread use throughout North America. Owing to the adverse economic situation in the UK during this period, and disagreement among directors of the regional transfusion centres, these bags were not introduced throughout the UK until 1975. Baxter (now owning Fenwal) in the USA, Biotest in Germany, Terumo in Japan and Tuta in Australia further developed plastic transfusion sets to enable more blood components and plasma fractions to be aseptically separated and stored for transfusion.
Blood transfusion is obviously a lifesaving procedure, but has risks, including infectious, immunological, and noninfectious complications. There is debate in the medical literature concerning the appropriate use of blood and blood products. Clinical trials investigating their use suggest that transfusing at lower hemoglobin levels is beneficial. (Hébert et al., 1999; Lacroix et al., 2007).
2.1.2 Blood Donation
A blood donation is said to have occurred when a person voluntarily has blood drawn and used for transfusions or for biopharmaceutical medications (fractionation). Donation of blood may be of whole blood, or any of its components directly.
Potential donors are assessed for things that might make their blood unsafe. Screening may include testing the donor for diseases that can possibly be transmitted by blood transfusion, including viral hepatitis and Human Immunodeficiency Virus. Donor’s medical history must also be checked a short physical examination carried out to make sure the donation is not hazardous to his/her health. The frequency of donation varies from days to months based on what he/she donates and the laws governing the country where the donation takes place. For example in the United States of America, donors must wait eight weeks between whole blood donations but only seven days between plateletpheresis donations. (ARCBS, 2009).
The quantity of blood drawn and the methods used vary. The collection of blood can be manual or automated, with the latter only taking specific portions of the blood.
2.1.2.1. Types of Blood Donation.
Blood donations are divided majorly into groups based on the recipient of the donated blood. (Brecher, 2005). An 'allogeneic' donation is carried out when a donor gives blood for storage at a blood bank for transfusion to an anonymous recipient. A 'directed' donation is when a person, often a family member, donates blood for the purpose of transfusing into a specific individual. (Alberts, 2012). Directed donations are relatively rare when there is an established supply. A 'replacement donor' donation is a hybrid of the two and is common in developing countries such as Ghana. (Shmukler, 2004). In this case, a friend/family member of the recipient donates blood to replace the stored whole blood or components used in a transfusion, ensuring a consistent supply. 'Autologous' donation is when a person has blood stored in the bank that will be transfused back to the donor at a later time/date, usually after surgery. Blood that is used for the purpose of making medications can be made from allogeneic donations or from donations exclusively used for manufacturing. (Robert et al., 2006).
Whole blood is sometimes collected using similar methods for therapeutic phlebotomy, methods similar to the ancient practice of bloodletting, which is used for the treatment of conditions such as hereditary hemochromatosis or polycythemia vera. The actual process varies according to the laws of the country, and recommendations to donors vary according to the collecting organization. (Ganong, 2003) (Waugh and Grant, 2007). The WHO gives recommendations for blood donation policies, (Romer and Parsons, 1977) although in most developing countries these are not followed. For instance, the recommended testing requires adequate laboratory facilities, well-trained staff, and specialized reagents, all of which may not readily be available or too expensive to be procured in developing countries. (WHO, 2006). An event where donors come to donate allogeneic blood is sometimes called a 'blood drive' or a 'blood donor session'. These can occur at a blood bank, but they are often set up at a location in the community setting such as a workplace, shopping center, house of worship or school. (American Red Cross, 2006).
2.1.3. Types of Transfusions
2.1.3.1. Red blood cell transfusions.
Packed red blood cells (pRBC) are prepared from previously donated whole blood by removing approximately 250 mL of plasma. One unit of pRBC should increase levels of hemoglobin by 1 g/dL (10 g/L) and haematocrit by 3%. Usually, pRBC units are filtered to reduce white blood cells before storage, which limits febrile nonhemolytic transfusion reactions (FNHTRs), and are considered cytomegalovirus (CMV) safe. (King and Bandarenko, 2008). Red Blood Cell transfusions are used to treat hemorrhage and to improve oxygen delivery to tissues. Transfusion of Red Blood Cells should be based on the patient's clinical condition. (Klein et al., 2007). Indications for Red Blood Cell transfusion may include acute sickle cell crisis or acute blood loss of greater than 1,500 mL or 30% of blood volume (Klein et al., 2007). Patients with symptomatic anaemia should be transfused if the transfusion appears clinically indispensable. (Ferraris et al., 2007; Klein et al., 2007).
In 1999, a randomized, multicenter, controlled clinical trial evaluated a restrictive transfusion trigger (hemoglobin level of 7 to 9 g/dL [70 to 90 g/L]) versus a liberal transfusion trigger (hemoglobin level of 10 to 12 g/dL [100 to 120 g/L]) in patients who were critically ill. (Hébert et al., 1999). Restrictive transfusion practices resulted in a 54% relative decrease in the number of units transfused and a reduction in the 30-day mortality rate. The authors recommended transfusion when hemoglobin is less than 7 g/dL, and maintenance of a hemoglobin level between 7 to 9 g/dL (Hébert et al., 1999). A recently updated Cochrane review supports the use of restrictive transfusion triggers in patients who do not have cardiac disease (Carless et al., 2010). A similar study was carried out in critically ill children (Lacroix et al., 2007). The restrictive transfusion trigger was a hemoglobin level of 7 g per dL, with a target level of 8.5 to 9.5 g per dL (85 to 95 g/L). (Lacroix et al., 2007).
Red cell components are stored at 4 ± 2°C for a maximum of 35-49 days in additive solution or 28-35 days in plasma. The shelf life depends upon the combination of anticoagulant, storage medium, blood pack and whether any further processing steps are performed on the red-cell component (e.g. irradiation of the component). For the vast majority of red-cell units processed, an additive solution containing adenine is added following separation to achieve a hematocrit of 50-70% and maintain red-cell quality during storage. The amount of residual plasma in a red-cell unit in additive solution is dependent on the hematocrit of the donor and how hard red cells have been centrifuged; it is between 5 and 30 mL. Red cells used for intrauterine transfusions and exchange or large-volume transfusion to neonates are normally stored or reconstituted in 100% plasma because of concerns over potential toxic effects of some of the constituents of additive solutions. For patients with immunoglobulin A deficiency or severe allergic or anaphylactic reactions to red cells, it may be necessary to remove > 90% of plasma by washing and re-suspending red cells in saline. Red cells from donors with rare phenotypes may be stored frozen for up to 30 years and are washed prior to transfusion to remove the cryoprotectant used to store them.
2.1.3.2. Plasma Transfusion.
Plasma products available in some developed countries include fresh frozen plasma (FFP) and thawed plasma that may be stored at 1 to 6°C for up to five (5) days. Plasma contains all of the blood coagulation factors. FFP infusion can be used for reversal of anticoagulant effects. Thawed plasma has lower levels of factors V and VIII and is not indicated in patients with consumption coagulopathy (diffuse intravascular coagulation) (King and Bandarenko, 2008). Plasma transfusion is recommended in patients with active bleeding and an International Normalized Ratio (INR) greater than 1.6, or before an invasive procedure or surgery if a patient has been anticoagulated. (Bolt, 1994; Holland and Brooks, 2006). In most cases plasma is often inappropriately transfused for correction of a high INR when there is no bleeding. Supportive care can decrease high-normal to slightly elevated INRs (1.3 to 1.6) without transfusion of plasma (Bolt, 1994; Holland and Brooks, 2006; Liumbruno et al., 2009).
Plasma from whole blood donations or apheresis is used to either prepare plasma components for clinical transfusion or fractionate to produce pure plasma proteins. FFP is produced by rapidly freezing the plasma removed from a whole-blood donation or collected by apheresis. This is usually performed within 8 hours of donation, in order to preserve the activity of coagulation factors V and VIII which are relatively labile. However, FFP can be produced from whole blood that has been stored at 4°C or 22°C for 24 hours. FFP is now only used to replace congenital single coagulation factor deficiencies where purified factor concentrates are not available (factors V and XI). Most FFP is used to treat acquired multiple coagulation factor deficiencies, usually in a clinical setting of massive transfusion, liver disease or disseminated intravascular coagulation (O'Shaughnessy et al., 2004).
Cryoprecipitate is produced by slowly thawing FFP at 4°C. This causes the so-called cryoproteins to precipitate out: factor VIII, fibrinogen, von Willebrand factor (VWF), fibronectin and factor XIII. By centrifuging and removing the supernatant plasma, the cryoprecipitate left is a rich source of these proteins in a small volume of plasma. Because of the widespread availability of purified or recombinant concentrates of factor VIII and VWF, cryoprecipitate is rarely used in the developed world to replace these factors and is mainly used in the treatment of hypo or dysfibrinogenemia. Because of its high fibrinogen content, cryoprecipitate is also used as a starting material for the production of fibrin glue.
The supernatant plasma removed from cryoprecipitate (CDP, cryoprecipitate-depleted plasma) has been used as a replacement fluid for plasma-exchange treatment of patients with thrombotic thrombocytopenic purpura (TTP), as an alternative to FFP. There are theoretical advantages of using CDP as it contains lower levels of high-molecular-weight multimers of VWF, but this benefit has not been proven clinically. In the UK, however, solvent-detergent-treated FFP is now recommended for the treatment of TTP because it is subject to pathogen inactivation during its manufacture and carries a lower risk of transfusion-related acute lung injury (TRALI) because of plasma pooling, which dilutes down the donor antibodies. Frozen-plasma components can be stored for up to 36 months depending on the storage temperature, which is usually below -30°C. Once thawed, FFP should be used immediately but can be stored for up to 24 hours at 4°C.
2.1.3.3. Platelet Transfusion.
Platelet transfusion may be indicated for the prevention of hemorrhage in patients with thrombocytopenia or defects in their platelet function. Contraindications to platelet transfusion include thrombotic thrombocytopenic purpura and heparin induced thrombocytopenia. Transfusion of platelets in these conditions can result in further thrombosis (Schiffer et al., 2001; BCSH, 2003). One unit of apheresis platelets should increase the platelet count in adults by 30 to 60 × 10/μL (30 to 60 × 10 per L) (King and Bandarenko, 2008). In neonates, transfusing 5 to 10 mL per kg of platelets should increase the platelet count by 50 to 100 × 10 /μL (50 to 100 × 10 per L) (Poterjoy and Josephson, 2009). One apheresis platelet collection is equivalent to six pooled random donor platelet concentrates (Slichter, 2007). Spontaneous bleeding through intact endothelium does not occur unless the platelet count is no greater than 5 × 10 per μL (5 × 10 per L) (Liumbruno et al., 2009).
There are two basic methods for producing platelets from whole-blood donations: the 'buffy-coat' method favored in Europe or the platelet-rich plasma (PRP) method favored in North America. Specifications for platelet components are given. In the PRP method, whole blood is separated into PRP and red cells following a 'soft spin'. The PRP is then subjected to a 'hard spin' to remove plasma and concentrate the platelets. In the buffy-coat method, whole blood is subjected to a 'hard spin' and separated into plasma, red cells and a buffy coat that contains most of the platelets but also some leukocytes and red cells. Buffy coats from four to six donations are then pooled with a unit of plasma from one of the donations (or PAS, platelet additive solution), subjected to a 'soft spin' and the PRP removed. The main difference between platelet concentrates collected by apheresis and PRP or buffy-coat platelets is that one or more adult therapeutic doses can be collected by apheresis from a single donor, which is not possible from one whole-blood donation.
For either buffy-coat-derived or apheresis platelets, the majority of plasma (70%) in the platelet concentrate can be replaced with an artificial PAS designed to maintain platelet function during storage. PAS differ in their composition; key elements are the use of acetate or glucose as a substrate for platelet metabolism, phosphate that buffers lactate production, citrate to prevent coagulation and lactate production and the inclusion of potassium and magnesium to improve platelet function during storage. Three different PAS are CE marked in Europe for platelet storage, and some European blood centers routinely produce and store platelets in PAS. Platelets are stored with agitation at 22 ± 2°C for up to 5 days, although in some countries this is extended to 7 days, provided platelets are screened for bacterial contamination. For some patients with severe anaphylactic reactions to platelets because of contaminating plasma proteins, platelets can be re-suspended in 100% additive solution. However, these 'washed' platelets have a reduced shelf life of 24 hours because of the rapid deterioration of platelet quality in the complete absence of plasma, and a proportion of the platelets may be lost during the process.
Platelet transfusion are associated with many complications. Paradoxically, most complication are not caused by the platelets themselves but by the contaminating leucocytes, leucocytes derived cytokines, red cells, plasma proteins and microorganisms.
2.1.3.4. Cryoprecipitate.
Cryoprecipitate can be prepared by thawing fresh frozen plasma and collecting the precipitate. Cryoprecipitate contains high concentrations of factor VIII and fibrinogen. Cryoprecipitate is used in cases of hypofibrinogenemia, which often occurs in the setting of massive hemorrhage. Each unit will raise the fibrinogen level by 5 to 10 mg/dL (0.15 to 0.29 μmol per L), with the goal of maintaining a fibrinogen level of at least 100 mg per dL (2.94 μmol per L) (Callum et al., 2009). The usual dose in adults is 10 units of pooled cryoprecipitate (King and Bandarenko, 2008; Callum et al., 2009). Recommendations for dosing regimens in neonates vary, ranging from 2 mL of cryoprecipitate per kg to 1 unit of cryoprecipitate (15 to 20 mL) per 7 kg.
2.1.3.5. Preparation and Storage of Granulocytes.
Granulocytes may be transfused to patients with a severe deficiency or dysfunction of neutrophils which have developed or are at risk of developing life-threatening infections. There is anecdotal evidence of benefit, but few randomized controlled trials have been performed, and a recent systematic review found that there is inconclusive evidence from randomized controlled trials (RCTs) to support or refute the generalized use of granulocyte transfusion therapy in neutropenic patients (Stanworth et al., 2004). Granulocytes are normally collected by apheresis and contain mainly neutrophils but also significant numbers of lymphocytes, red cells and platelets; hence they need to be crossmatched prior to transfusion. Preadministration of steroids and granulocyte-colony stimulating factor (G-CSF) to donors can considerably increase the yields collected (1-10 × 1010), but this is not permitted in volunteer donors in some countries. Yields in unstimulated donations rarely exceed 0.5 × 1010, which is below the dose generally considered adequate for adults (> 1 × 1010). Because of the logistical and ethical constraints in providing apheresis granulocytes, some countries issue buffy coats as a source of granulocytes. Ten to twelve buffy coats are transfused to provide a dose of 1 × 1010 neutrophils. Granulocytes should be transfused as soon as possible after collection or preparation but can be stored at 22°C for up to 24 hours without agitation and are irradiated prior to transfusion to prevent transfusion-associated graft-versus-host disease (TA-GVHD).
2.1.4. Recruitment of Donors
Although most Americans will require a blood transfusion at some time in their lives, only about one-third of the United States population is eligible to donate blood (Riley et al., 2007) and only a small portion of those actually donate. Blood donors are likely than the general population to be male, age 30 to 50 years (McCullough, 2005) it is generally believed that the most effective way to get someone to donate blood is to ask him or her personally (Piliavin and Callero, 1991). Factors such as the convenience of donation, peer pressure, receipt of blood by a family member and perceived community needs are important factors that are superimposed onto the individual’s basic social commitments (Piliavin and Callero, 1991). Usually blood donors are asked to give to the general community supply. Some donors are asked to give for a specific patient, which is referred to as directed donation. Such donation may be easier to obtain and leave the donor with a stronger sense of satisfaction because of the personal nature of the donation.
The heightened concern about blood safety since the onset of the AIDS epidemic has resulted in expanded requirement for the suitability for blood donation. Thus a larger proportion of the population of potential donors is being excluded and the most common reason is a low hemoglobin.
2.1.5. Collection of Whole Blood
There are majorly two methods of obtaining blood from a donor. The most frequent is to simply take the blood from a vein as whole blood. This blood is typically separated into two parts, usually RBCs and plasma, since most recipients need only a specific component for transfusions. A typical donation is 450mls of whole blood, though 500mls donations are also common. Historically, blood donors in India would donate only 250 or 350 millilitre and donors in the People's Republic of China would donate only 200 millilitres, though larger 300 and 400 millilitre donations have become more common (Wang and Nan, 2010).
The other method is to draw blood from the donor, separate it using a centrifuge or a filter, store the desired part, and return the rest to the donor. This process is called apheresis, and it is often done with a machine specifically designed for this purpose. This process is especially common for plasma and platelets. For direct transfusions a vein can be used but the blood may be taken from an artery instead (AABB, 2010). In this case, the blood is not stored, but is pumped directly from the donor into the recipient. This was an early method for blood transfusion and is rarely used in modern practice. (James, 2007).
The blood is drawn from a large arm vein close to the skin, usually the median cubital vein on the inside of the elbow. The skin over the blood vessel is cleaned with an antiseptic such as iodine or chlorhexidine (Lee et al., 2002) to prevent skin bacteria from contaminating the collected blood (Lee et al., 2002) and also to prevent infections where the needle pierced the donor's skin.
2.1.6. Whole Blood Donor Screening
The approach to the selection of blood donors is designed to ensure the safety of the donors and to obtain a high-quality components that is as safe as possible for the recipient. Some specific steps that are taken to ensure that blood is as safe as possible are the use of only volunteer donors; questioning of donors about their general health is scheduled; obtaining a medical history, including specific risk factors, before donation; conducting a brief physical examination before donation; laboratory testing of donated blood; checking the donor’s identity against a donor referral registry (Grossman et al., 1992; McCullough, 2005); and providing a method by which the donor can confidently designate the unit as unsuitable for transfusion after the donation is completed (McCullough, 2005).
2.1.6.1 Screening of Blood.
In an effort to ensure the blood supply is as safe as possible, all donors must meet specific eligibility criteria outlined by the Food and Drug Administration, accrediting organizations such as American Association for Blood Bank, and individual donation centers. To donate, individuals must be at least 16 years old (or the age specified by state law), healthy and feeling well on the donation day. In addition, donors must meet weight and hemoglobin level requirements. Specific criteria exist for donors of human cells, tissue, and cellular and tissue-based products as well. Although the criteria are very similar to that applied to blood donors, there are differences due to the unique patient needs for these products.
Donors also are screened for disease risk factors using a health history questionnaire. Through this confidential questionnaire, donors are asked specific and direct questions regarding lifestyle, health, medical history and travel to assure their own health will not be compromised by a blood donation and that patients receive safe blood products. Donors can be deferred for a variety of reasons: signs and symptoms of relevant transfusion-transmitted infections, such as HIV, viral hepatitis, HTLV, syphilis or West Nile virus; social behaviors that increase their risk of exposure to infectious diseases, including men who have sex with other men, intravenous drug use and exchanging sex for drugs or money; travel to certain countries where the risk of exposure to a particular infectious disease is of concern; medical procedures that involve receipt of dura mater graft, transfusion of blood or blood components within the previous 12 months, or human-derived clotting factors within the previous 12 months; incarceration under certain circumstances; obtaining a piercing or tattoo using non sterile materials within the previous 12 months; certain medications; and pregnancy.
Donors also may be deferred because of reactive test results to infectious diseases, such as syphilis, HIV, hepatitis, HTLV and WNV. In some cases, if it is determined that these results were false positives, an individual may be re-entered into the donor pool by following the requalification methods outlined by the FDA. Because donor eligibility requirements are considered to be an important step in assuring a safe donation process for the donor and reducing the risk of transfusion transmission of a disease to a patient, AABB works with the FDA to ensure appropriate eligibility requirements are in place. AABB also works with the FDA to streamline processes for re-entry of donors deferred for false-positive test results.
Laboratory screening of donated blood is the step that determines whether or not a donation is non-reactive for specific markers of infection and is therefore safe to release for clinical or manufacturing use. Each country decides on the TTIs to be screened for as part of the blood screening programme and develop a screening strategy appropriate to its specific situation. This will be influenced by the incidence and prevalence of infection, the capacity and infrastructure of the blood transfusion service (BTS), the costs of screening and the available resources. The critical factor is that whichever strategy is selected, it is implemented effectively, consistently and within a well-managed quality system. The national screening strategy provides an overall decision-making process on how tests are to be used and interpreted and defines the outcomes of screening with regard to whether a blood unit will be released or discarded. (WHO, 2010).
2.1.7. Transfusion Related Reactions
Transfusion related complications can be classed as acute or delayed, which can further be divided into the categories of noninfectious and infectious. Complications that occur within minutes to 24 hours of the transfusion are termed acute, whilst, complications may develop days, months, or even years later, in this case it is called delayed. The American Association of Blood Banks uses the term “noninfectious serious hazards of transfusion” to classify noninfectious complications (Hendrickson and Hillyer, 2009). Transfusion-related infections are less common because of advances in the blood screening process; the risk of contracting an infection from transfusion has decreased 10,000-fold since the 1980s (Vamvakas and Blajchman, 2009). Noninfectious serious hazards of transfusion are up to one thousand times more likely than an infectious complication (Hendrickson and Hillyer, 2009). However, there has been no progress in preventing noninfectious serious hazards of transfusion, despite improvements in blood screening tests and other related medical advances. Therefore, patients are far more likely to experience a noninfectious serious hazard of transfusion than an infectious complication (Vamvakas and Blajchman, 2009).
2.1.8. Acute Transfusion Reaction
2.1.8.1. Acute Hemolytic Reactions.
Haemolytic transfusion reactions are caused by immune destruction of transfused Red Blood Cells, which are attacked by the recipient's antibodies. The antibodies to the antigens of the ABO blood group or alloantibodies to other Red Blood Cell antigens are produced after immunization through a previous transfusion or pregnancy. There are two aspects of hemolytic transfusion reactions: acute and delayed. Nonimmune causes of acute reactions include, improper storing, bacterial overgrowth, infusion of blood through lines containing hypotonic solutions or small-bore intravenous tubes and infusion with incompatible medications (Lichtiger and Perry-Thornton, 1984; Hendrickson and Hillyer, 2009; Gaines et al., 2009).
In acute hemolytic transfusion reactions, there is a destruction of the donor's Red Blood Cells within 24 hours of transfusion. Hemolysis may be extravascular or intravascular. The most common type is extravascular hemolysis, which occurs when donor Red Blood Cells coated with immunoglobulin G or complement are attacked in the liver or spleen (Vamvakas and Blajchman, 2009). Intravascular hemolysis is a severe form of hemolysis caused by ABO antibodies. Clinical manifestations include chills, diffuse bleeding, fever, nausea rigors, vomiting, dyspnea, hypotension, , hemoglobinuria, oliguria, anuria, pain at the infusion site; and chest, back, and abdominal pain (Lichtiger and Perry-Thornton, 1984). Associated complications are clinically significant anemia, acute or exacerbated kidney failure, disseminated intravascular coagulation (DIC), need for dialysis, and death secondary to complications (Gaines et al., 2009).
The incidence of acute hemolytic reactions is approximately 1-5 per 50,000 transfusions (Lichtiger and Perry-Thornton, 1984). From 1996 to 2007, there were 213 ABO-incompatible RBC transfusions with 24 deaths. Systems using bar codes for blood and patient identification have decreased errors (Vamvakas and Blajchman, 2009).
2.1.8.2. Allergic Reactions.
Allergic reactions range from mild to life threatening. Examples of the latter and former manifestations are urticarial and anaphylactic reactions respectively. Urticarial allergic reactions are defined by hives or pruritus (Reutter et al., 2001). Patients that experience allergic transfusion reactions have been sensitized to the antigens in the unit of the donated blood. These antigens are soluble, and the associated reaction is always dependent on the dose. Allergic transfusion reactions occur in one to three percent of transfusions (Hendrickson and Hillyer, 2009).
Recipients having anaphylactic transfusion reactions, like those with urticarial reactions, may present with hives, albeit, they are different in that they also develop stridor, bronchospasm gastrointestinal symptoms and hypotension (Hendrickson and Hillyer, 2009). Anaphylaxis occurs in response to a recipient's presensitization to a variety of proteins in donor plasma. For example, anaphylaxis occurs because of donor IgA being infused into a recipient who is IgA deficient and has preexisting circulating anti-IgA (Vamvakas and Blajchman, 2009). In addition, anti–human leukocyte antigen (HLA) antibodies and anticomplement antibodies have been linked to anaphylactic reactions, which are estimated to occur in one in 20,000 to 50,000 transfusions (Pineda and Taswell, 1975)
The prevention of anaphylactic transfusion reactions includes avoiding plasma transfusions with immunoglobulin A in patients known to be IgA deficient. Cellular products (for example, Red Blood Cells and platelets) may be washed to remove plasma in patients with an IgA deficiency (Hendrickson and Hillyer, 2009).
2.1.8.3. Transfusion-Related Acute Lung Injury.
Transfusion-related acute lung injury (TRALI) is noncardiogenic pulmonary edema that causes acute hypoxemia occurring within six hours of a transfusion and has a clear temporal relationship to the transfusion (Fiebig et al., 2007). Patients with TRALI do not always have any other risk factors for acute lung injury. Anti-HLA antibodies activate the recipient's immune system, and this may result in massive pulmonary edema (Vamvakas and Blajchman, 2009; Engelfriet et al., 2011). Activated neutrophils present in the lungs may also release proteolytic enzymes, leading to more tissue damage (Stack and Tormey, 2008). Optimal methods for detecting these antibodies in already donated products are yet to be determined (Hendrickson and Hillyer, 2009).
Products from donors that contain large amounts of liquid plasma from multiparous women are associated with Transfusion Related Acute Lung Injury. Mortality in the UK decreased significantly after plasma from men was used exclusively (Vamvakas and Blajchman, 2009). In 2006, Transfusion Related Acute Lung Injury was the leading cause of transfusion-related mortality, it contributed about 50.7 percent of transfusion-related deaths (Hendrickson and Hillyer, 2009). The Transfusion Related Acute Lung Injury working group of the American Association Blood Bank recommends the use of male-predominant plasma for transfusions. Because this policy excludes a large number of female donors, maintaining an adequate supply of plasma and platelets became a concern.
2.1.8.4. Febrile Nonhemolytic Transfusion Reactions (FNHTR).
An FNHTR could be defined as a rise in body temperature of at least 1°C or above 37°C within 24 hours post transfusion. It may involve chills, rigors and discomfort (British Committee for Standards in Haematology 2003). The fever may occur more often in patients who have previously been transfused repeatedly and in patients who have been gravid (Addas-Carvalho et al., 2006). Leukoreduction, which is the removal of WBCs from donor blood, has appreciably decreased the rate FNHTR (King et al., 2004). FNHTRs are caused by platelet transfusions more often than Red Blood Cells transfusions and have an incidence that ranges from less than 1% to more than 35% (Hendrickson and Hillyer, 2009).
Two mechanisms have been proposed to explain FNHTRs: a release of antibody-mediated endogenous pyrogen, and a release of certain cytokines. Common cytokines that may be associated with FNHTRs include interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor (TNF) (Addas-Carvalho et al., 2006). FNHTR became a diagnosis of exclusion that can be made only after ruling out other causes of fever (for example, sepsis and hemolysis).
2.1.8.5. Transfusion-Associated Circulatory Overload.
Transfusion-associated circulatory overload is the clinical outcome of a rapid transfusion of a blood volume that is more than what the recipient's circulatory system can handle. It is not associated with an antibody-mediated reaction. Those at highest risk are patients with underlying kidney failure, chronic anemia or cardiopulmonary compromise and infants or older patients (Vamvakas and Blajchman, 2009). Clinical manifestations may include hypertension, tachycardia, cough, dyspnea, elevated pulmonary wedge pressure, elevated central venous pressure and widened pulse pressure. Cardiomegaly and pulmonary edema are often seen on chest radiography (Popovsky, 2009).
Diagnosis is made clinically, but may be assisted by measuring brain natriuretic peptide levels, which are somewhat elevated in response to an increase in filling pressure (Zhou et al., 2008). A study comparing patients who have transfusion-associated circulatory overload with patients who have TRALI found significantly greater levels of brain natriuretic peptide in those with the former condition. (Zhou et al., 2008). Transfusion of lower volumes or at a slower rate may be helpful (Hendrickson and Hillyer, 2009). Treatment is diuresis to decrease volume overload.
2.1.9. Delayed Transfusion Reactions
2.1.9.1. Transfusion-Associated Graft-Versus-Host Disease.
Transfusion-associated graft-versus-host disease is a consequence of a donor's lymphocytes proliferating which causes an immune attack against the recipient's tissues and organs. It is fatal in more than 90% of cases (Hendrickson and Hillyer, 2009). Patients vulnerable to this condition are those who are immunocompromised or immunocompetent and those who are receiving transfusion with shared Human Leukocyte Antigen haplotypes (that is., donor is a relative) (Vamvakas and Blajchman, 2009). Clinical manifestations may include fever, rash, liver dysfunction, diarrhea and pancytopenia occurring 1-6 weeks after transfusion (Hendrickson and Hillyer, 2009).
Risk factors include a history of fludarabine (Oforta) treatment, stem cell transplant, Hodgkin disease, intensive chemotherapy, erythroblastosis fetalis and intrauterine transfusion. Other probable risk factors may include a history of solid tumors treated with cytotoxic drugs, recipient-donor pairs from homogenous populations and transfusion in premature infants (Webb and Anderson, 2000). Gamma irradiation of blood products may help in keeping the donor lymphocytes from proliferating and can prevent transfusion-associated graft-versus-host disease (Hendrickson and Hillyer, 2009).
2.1.10. Whole blood Collection Storage and Processing
European and American guidelines recommend that the volume of whole blood collected is between 450 and 500 mL ± 10% (AABB, 2003; James, 2005; Council of Europe, 2007). Blood is collected into an anticoagulant comprised of citrate, phosphate and dextrose designed to prevent blood from clotting and maintain cellular function during storage. Adenine may also be added to the anticoagulant to improve the quality of red cells during storage if other solutions are not added during later processing steps. It is generally accepted that there are very few clinical indications for transfusion of whole blood, and the vast majority of blood is therefore processed into its basic components: red cells, platelets and plasma. This is achieved by centrifugation of whole blood in the primary collection pack, followed by manual or automated extraction of the components into satellite packs.
The initial storage temperature of whole blood determines which components can be produced from it. Because platelet function rapidly deteriorates at 4°C, whole blood must be processed on the day of blood collection or stored overnight at 22°C for platelet production. However, for the production of red cells, whole blood can be stored at 4°C for 48-72 hours prior to separation. Plasma is generally separated from whole blood on the day of collection or from blood that has been stored at 22°C for up to 24 hours, as these methods have been shown to preserve plasma quality. In the USA, 'liquid plasma' (which has not been frozen) and thawed plasma are also available for use when transfusion of labile clotting factors (e.g. factors V and VIII) is not required. The storage of temperature, media and shelf life of blood components is tailored to each type of component, so that there is preservation of component quality while affording the maximal usable shelf life.
2.1.11. Collection of Blood Components by Apheresis
The use of blood components rather than whole blood for replacement therapy has become a common practice in transfusion medicine. Separation of a unit of whole blood into packed red blood cells (pRBCs) and fresh-frozen or platelet-rich plasma facilitates storage and increases the utility of donated blood. Specific component hemotherapy avoids hypervolemia in anaemic, normo-proteinemic patients and polycythemia in non-anaemic patients with coagulation factor deficiencies, hypo-albuminernia, or thrombocytopenia. The methodology currently recommended to prepare and store blood components has largely been extrapolated from human transfusion medicine techniques. One of the major techniques used in separation of blood components is by apheresis.
Apheresis, from a Greek word meaning 'to take away', is an alternative to producing blood components from whole blood donations by selectively collecting one or more components directly from donors and returning the rest to the circulation. Automated apheresis can be used to collect platelets, plasma, red cells or granulocytes, and more specialized products, such as stem cells. The main emphasis in the past has been the collection of platelets and plasma components, with red cells being returned to the donor. The size and complexity of the equipment, as well as welfare of the donor, has previously necessitated this activity to take place in static clinics. However, smaller portable machines are now available that can be used on mobile sessions to collect red cells, platelets and plasma. The main advantage of apheresis collections are that more than one dose of platelets or red cells can be collected from one donor per donation, thus reducing patient exposure to multiple donors. In addition, the hematocrit and hemoglobin content of red cells is much more consistent than those produced from whole blood donations, which vary considerably because of the variation in hematocrit of whole blood in different donors.
2.2. EFFECT OF STORAGE ON WHOLE BLOOD
Since the First World War (1914–1918), technology to store red cells under refrigerated conditions for short periods of time using sodium citrate as an anticoagulant has been enhanced. The advent of the Second World War (1939 – 1945) and the development of an anticoagulant containing an acid-citrate-dextrose (ACD) solution which significantly decreased the volume of anticoagulant that is required for storage led to refrigerated blood being stored for 21 days and blood banking becoming a reality (Hess, 2006). This allowed for increased volumes of blood to be available for transfusion, a longer period of storage, and a reduction in post transfusion citrate toxicity. Subsequently, further advances in the storage of red blood cell donations were made possible with the introduction of phosphates and adenine which allowed for a longer storage period of whole blood units. These advances encouraged the scientific community to develop additive solutions which would not only extend the storage period but also preserve the quality of the product. Introducing component therapy where red cells are separated from plasma by centrifugation and the development of more preservative solutions containing saline, glucose, mannitol and adenine (for example, SAGM) which were added to the separated red blood cells, increased the duration of red cell storage to 42 days when stored at 1∘C to 6 ∘C (Zubair, 2010). The addition of saline and mannitol decreases the hemolysis rate, and glucose provides an energy pathway substrate while adenine maintains the ATP levels. The standard Red Blood Cell additive solution used in Europe is SAGM. In South Africa, the western coastal region uses SAGM, whereas the inland areas use ADSOL (a solution consisting of adenine, dextrose, sodium chloride, and mannitol).The blood transfusion establishments in the USA use AS-1and AS-5 as additive solutions while the third additive solution yet to be licensed is AS-3. Although the additive solution AS-3, is a saline-adenine – glucose solution, it also contains the ATP producing phosphate and citrate since it is a SAGM variant. It should be worthy of note that neither of the additive solutions mentioned has a major advantage over the other as fragmentation or vesiculation of the RBCs still occurs in both solutions, although it has been reported that the membrane protein profile of Red Blood Cell Concentrate stored in AS-3 seems to be of more advantage than that stored in SAGM (D’Amici et al., 2012). Most customary blood bank practices in several countries involve the collection of approximately 450–575millilitres of whole blood into a collection bag containing citrate-phosphate-dextrose (CPD) solution as the anticoagulant. In modern practice, whole blood unit is not a commonly transfused product as the clotting factors and platelets depreciate within hours of donation and therefore the expiry date is 35 days compared to the more prolonged 42-day expiry date of other red blood cellular products. Whole blood is mainly used for patients suffering with massive hemorrhage or for the purpose of neonatal exchange transfusions. (Hillyer et al., 2001). Adult massive hemorrhage is defined as transfusion of more than 10 Red Blood Cell Concentrate units within 24 hours which is approximately the total blood volume or replacement of more than 50 percent of total blood volume using blood products within a three-hour period.
Due to the gradual decomposition of Red Blood Cells and as a result of the accumulation of products of cellular metabolism, i.e. anaerobic glycolysis, the biochemical composition of RBC concentrates changes. In particular, there is an increase in K+ and lactate levels and a simultaneous decrease in pH, glucose and Na+ levels. The storage time has no impact on Ca++ levels in the RBC concentrate. The changes are proportional to the storage time (Larsen, 2004). Large-volume RBC transfusion may contribute to changes in the patients’ plasma biochemical parameters (hyperkalemia) and may therefore be related not only to the volume of RBC products but also to storage duration (Brecher, 2005). Other changes include a reduction in red blood cell deformability, altered red blood cell adhesiveness and aggregability, and a reduction in 2,3-diphosphoglycerate and ATP. Bioactive compounds with proinflammatory effects also accumulate in the storage medium. These changes reduce post transfusion viability of red blood cells. The clinical effects beyond post transfusion viability are uncertain, but a growing body of evidence suggests that the storage lesion may reduce tissue oxygen availability, have proinflammatory and immunomodulatory effects, and influence morbidity and mortality (Ho et al., 2003).
2.2.1. CHANGES IN BIOCHEMICAL PARAMETERS
It is well documented that certain biochemical changes occur during the 35 to 42 days of blood cells storage at temperatures between 1∘C and 6 ∘C. The biochemical structure of the red blood cell is being altered due to anaerobic glycolysis and these changes are relative to the storage period and temperature of storage.
2.2.1.1. pH.
There are a number of body systems which all have their own specifically preferred pH. Overall, the body's internal chemical environment normally changes from a weak acid to a weak base within a 24-hour period, usually more acid at dawn and most base at sunset. These physiolical changes occur on a sine curve during this period. The slightly acid time period early morning: pH < 7.0 is optimal for the activity of the nerves, hormones and neurotransmitters such as adrenaline, thyroxine, histamine, acetylcholine and other biogenic amines. In this pH, the acidic connective tissue substances (stored acidic wastes) are dissolved by the hyaluronidase into liquid form and thereafter excreted from the body as waste. The bloodstream is the most critically buffered system of the entire body, far more sensitive than any other. Arterial and venous blood must maintain a slightly alkaline pH: arterial blood pH = 7.41 and venous blood pH = 7.36. Because the normal pH of arterial blood is 7.41, a person is considered to have acidosis when the pH of blood falls below this value and to have alkalosis when the pH rises above 7.41.
Clinical problems of pH are all related to pH of the plasma of whole blood. pH in extracellular fluid is always close to that of blood. pH inside cells differs from that of blood but it is not recognised as being an important clinical problem apart from blood pH changes.
In the clinical situation if the actual pH of the blood is lowered, one can usually assume that the primary disturbance has been the addition to the blood of acid or the removal of base and vice versa. Blood pH may be changed if acids or bases are added to or removed from the blood. Secretion of an acid (for example, gastric juice) implies that the acid involved (HCl in this case) is removed from the blood. A rise in concentration of any of these acids in the blood causes a fall in the pH of the blood. Loss of acid from the blood (e.g. into gastric juice) causes a rise in the pH. Only HCl and H2CO3 can be lost from the blood in appreciable quantities.
Administration of base by mouth or parenterally may cause blood pH to rise if rate of excretion does not match rate of administration. Loss of alkaline fluid from bowel (diarrhoea, intestinal obstruction or intestinal fistulae), or urine (after acetoazolamide) will cause blood pH to fall. pH levels of stored blood have been reported to decrease due to anerobic utilization of glucose through the glycolytic pathway to produce lactic acid which as a result reduces the pH of the stored blood.
Glycolysis occurs when blood is stored in a plastic bag. Adenosine deaminase causes the breakdown of adenosine resulting in the formation of inosine and ammonia but is considered not to be clinically significant. An increase in protons causes the pH level of the blood to decrease and subsequently changes glycolytic metabolism. The decrease in pH causes the 2, 3-diphosphoglycerate levels to decline with a simultaneous surge in ATP production. Glycolysis is slowed down and, as acid accumulates, the levels of ATP falls and the shape of the red cell is gradually altered from discoid to echinocytic formations. This alteration in erythrocyte formation fades when stored blood is rejuvenated and is reversed when blood is warmed. The process of rejuvenation is when red blood cells are stored in a nutrient solution having a neutral pH (Koch et al., 2008). The accumulation of lactic acid and proteins appear in the red cells after 14 days of storage due to glycolytic metabolism. It has been reported that a decrease in pH level and increases in lactate and potassium concentrations may occur within a few hours of storage while other changes may take weeks to appear (Leal-Noval et al., 2008).
2.2.1.2. 2, 3 DPG.
2,3-Diphosphoglyceric acid (2,3-Bisphosphoglycerate or 2,3-BPG, also known as 2,3- diphosphoglycerate or 2,3-DPG) is a three-carbon isomer of the glycolytic intermediate 1,3- bisphosphoglyceric acid (1,3-BPG). 2, 3-BPG is present in human red blood cells (RBC; erythrocyte) at approximately 5 mmol/L. It binds with greater affinity to deoxygenated hemoglobin (for example, when the red cell is near respiring tissue) than it does to oxygenated hemoglobin (for example, in the lungs) due to spatial changes: 2,3- BPG (with an estimated size of about 9 angstroms) fits in the deoxygenated hemoglobin configuration (11 angstroms), but not as well in the oxygenated (5 angstroms). It interacts with deoxygenated hemoglobin beta subunits by decreasing their affinity for oxygen, so it allosterically promotes the release of the remaining oxygen molecules bound to the hemoglobin, thus enhancing the ability of RBCs to release oxygen near tissues that need it most. 2, 3-BPG is thus an allosteric effector. It is the enzyme regulator of hemoglobin and aids in oxygen transportation to all tissues. The reduction in pH levels leads to an increase in the degradation of 2, 3 DPG. This causes an increase in the affinity of oxygen for hemoglobin eventually leading to the shifting to the left of oxygen dissociation curve, resulting in a reduction in the supply of oxygen to the peripheral tissues. In hypoxia, the oxygen dissociation curve shifts the delivery to the right, thereby increasing the transport of oxygen to the tissues. After the 42-day storage period, a RBC unit may lose more than 90 percent of its 2,3 DPG concentration (Basran et al., 2006; Weiskopf et al., 2006; Aboudara et al., 2008). While 2,3 DPG levels may not be detectable within 2 weeks of storage, levels normalize within 72 hours after transfusion without any irreversible outcome observed and it is not regarded to be clinically significant (Hesset al., 2000; Leal-Noval et al., 2008).
2.2.1.2. ATP.
The progressive loss of adenosine triphosphate (ATP) is well documented regarding morphological changes and RBC deformability during the storage period. ATP is not only an intracellular energy source but when ATP is released from the erythrocyte, it stimulates the production of nitric oxide leading to vasodilation during hypoxic conditions. The decrease of ATP concentration during storage causes the cellular reactions requiring energy, for example, phospholipid membrane distribution, active transport, and antioxidant reactions, to also decrease. It has been indicated that there is a 60% decrease in ATP levels after more than 5 weeks of storage (Basran et al., 2006). The continuous reduction in ATP concentrations and acidification results in irreversible shape alteration of the RBC as echinocytic surface protrusions appear. The phospholipid bilayer loses its asymmetry and the shedding of microvesicles occur (Koch et al., 2008).
2.2.1.2. Potassium and Sodium Ions.
Sodium is the major positive ion (cation) in fluid outside of cells. The chemical notation for sodium is Na+. When combined with chloride, the resulting substance is table salt. Excess sodium (such as that obtained from dietary sources) is excreted in the urine. Sodium regulates the total amount of water in the body and the transmission of sodium into and out of individual cells also plays a role in critical body functions. Many processes in the body, especially in the brain, nervous system, and muscles, require electrical signals for communication. The movement of sodium is critical in generation of these electrical signals. Therefore, too much or too little sodium can cause cells to malfunction, and extremes in the blood sodium levels (too much or too little) can be fatal.
Increased sodium (hypernatremia) in the blood occurs whenever there is excess sodium in relation to water. There are numerous causes of hypernatremia; these may include kidney disease too little water intake, and loss of water due to diarrhea and/or vomiting. A decreased concentration of sodium (hyponatremia) occurs whenever there is a relative increase in the amount of body water relative to sodium. This happens with some diseases of the liver and kidney, in patients with congestive heart failure, in burn victims, and in numerous other conditions. A Normal blood sodium level is 135 – 145 milliEquivalents/liter (mEq/L), or in international units, 135 – 145 millimoles/liter (mmol/L).
Storage of red cells for three weeks at 4oC under blood bank conditions may result in a rise in intracellular Na+ and a fall in intracellular K+ with concomitant opposite changes in Na+ and K+ levels in the suspending plasma. A decline in red blood cell ATP during the storage period did not appear to be contributing to the changes. Increasing red blood cell ATP to levels 2 to 3 times normal did not prevent the cation changes from occurring. When assayed at 37 oC in the presence of added Mg2+, ouabain-sensitive membrane ATPase activity and kinetics of activation by Na+ were unaffected by the three week period of cold storage. However, when assayed at 4 oC without added Mg2+, simulating the conditions of storage, ATPase activity was negligible. Sodium and potassium did not change when red blood cells with normal ATP content were stored at 20 to 24 oC even in the absence of added Mg2+. Thus, a major cause for the development of cation changes in the red blood cell during blood bank storage in the temperature which inhibits membrane ATPase, allowing cations to leak unopposed into and out of the red blood cells (Wallas, 2009)
Potassium
Potassium is the major positive ion (cation) found inside of cells. The chemical notation for potassium is K+. The proper level of potassium is essential for normal cell function. Among the many functions of potassium in the body are regulation of the heartbeat and the function of the muscles. A seriously abnormal increase in potassium (hyperkalemia) or decrease in potassium (hypokalemia) can profoundly affect the nervous system and increases the chance of irregular heartbeats (arrhythmias), which, when extreme, can be fatal.
Increased potassium is known as hyperkalemia. Potassium is normally excreted by the kidneys, so disorders that decrease the function of the kidneys can result in hyperkalemia. Certain medications may also predispose an individual to hyperkalemia. Hypokalemia, or decreased potassium, can arise due to kidney diseases; excessive loss due to heavy sweating, vomiting, or diarrhea, eating disorders, certain medications, or other causes. The normal blood potassium level is 3.5 – 5.0 milliEquivalents/liter (mEq/L), or in international units, 3.5 – 5.0 millimoles/liter (mmol/L). In stored whole blood, it has been reported by Clement et al that there was relatively higher increment in potassium concentrations on day 10 and 15 by 21.4% and 60.7% respectively. The increased in potassium concentrations may probably be due to leakage from cells to the plasma (Clement et al., 2015).
Blood stored at 1∘ to 6∘C decreases the rate of cellular metabolism and energy demand which allows blood to be stored for 35 to 42 days. This makes the sodium–potassium pump inoperative and consequently allows potassium ions to exit the cell and sodium ions to enter via the semipermeable membrane. It was demonstrated in critically ill patients that the sodium levels will revert to their normal levels within 24 hours after transfusion, whereas the potassium levels take about 4 days to stabilize (Weiskopf et al., 2006; Leal-Noval et al., 2008). The extracellular potassium levels of stored blood increase daily at approximately 1mEq/L. with the higher concentrations observed during the early days of storage (Koch et al., 2008). Increased potassium levels in red blood cells may lead to arrhythmia when neonates or infants are transfused with large volumes of stored blood (Walsh et al., 2004; Weiskopf et al., 2006).
2.2.1.3. Plasma Haemolysis.
In protracted storage period, the red cell membrane experiences both morphological and biochemical alterations. These changes are generally referred to as storage lesions. Haemolysis of red blood cells may occur during phlebotomy due to bacterial contamination, transportation, storage, donor red cell membrane deficiencies, and mechanical injury during filtration process, presence of leucocytes in unfiltered red blood cell units or because of increased levels of vitamin C or penicillin in the donor (Kaplan et al., 2002). The interaction of plasma haemoglobin with nitric oxide has been shown to cause endothelial dysfunction and is a risk factor for vasoconstriction, leucocyte adhesion, and intravascular thrombosis (Raghavan and Marik, 2005). The release of H2O2 and proteases by white blood cells present in unfiltered blood may cause lysis of RBCs storage. Signs of haemolysis may suggest that the RBCs have been either ruptured or it may be due to the loss of membrane-bound haemoglobin in microvesicles found on the cell’s surface of intact cells. The inclusion of membrane stabilizers, for example, citrate and mannitol, may lower the rate of haemolysis. It has been reported that, although the mean percentage haemolysis of Red Blood Cell Concentrate stored in ADSOL (AS-1) was lower than its counterparts stored in SAGM, the difference between both was found not to be statistically significant (Toy et al., 2005). One of the best ways to assess the presence of haemolysis in a red blood cell unit before the unit is issued from the blood bank or prior to transfusion is by physical observation, but this visual inspection is often deceptive as it leads to an overestimation of haemolysis levels (Consensus conference, 1988). It has also been reported that pink/red discoloration due to haemolysis observed in either plasma or suspending fluid may often be due to plasma haemolysis levels being as low as 25g/dL (±0, 09% plasma haemolysis) and under normal conditions these units are discarded unnecessarily. It is therefore advisable to incorporate a measure of quality control to determine plasma haemolysis accuracy by using either photometric or spectrophotometric methods on random units or prior to discarding the RBC unit (Expert Working Group, 2008). The clinical implication of Red Blood Cell haemolysis for the transfused individual is a very serious issue and may lead to redox injury of the endothelium, tissues, or the proximal tubules of the kidneys while proinflammatory and procoagulant surfaces appear mainly due to the infusion of microvesicles which affects the microcirculation and consequently impacts systemic haemodynamics (Marik and Corwin, 2008). Some studies have shown that patients with circulatory or cardiovascular pathologies should carefully consider using rheologically compromised Red Blood Cell due to the haemodynamic risk involved (Zallen et al., 1999; Marik and Corwin, 2008). Since the presence of haemolysis is a cause for concern, the guidelines proposed by the Council of Europe stipulate that the mean haemolysis level should be less than 0, 8 percent. The Food and Drud Administration has amended their standard regarding the mean haemolysis concentration by adding the “95/95 rule.” This rule which states that, in addition to attaining the standard plasma haemolysis concentration of less than one percent, blood transfusion establishments must now demonstrate that 95% of their red blood cellular products meet the standard, statistically achieving 95 percent of the time (Leal-Noval et al., 2008). It is well documented that the concentration of haemolysis aggravates during the storage, but due to rigid quality control standards before, during, and after processing, together with trained personnel, the percentage haemolysis levels of most Red Blood Cell Concentrates do not exceed the prescribed limits (Expert Working Group, 2008; Marik and Corwin, 2008).
2.2.1.4. Nitric Oxide (NO).
Among the observed adverse effects of blood transfusions, reduced oxygen delivery and reduced vasodilatory capabilities of stored RBCs are considered especially critical factors (Ho et al., 2003; Tinmouth et al., 2006; Klein et al., 2007). It is now known that one of the primary vasodilators and regulators of blood flow is the endothelium-derived relaxing factor, nitric oxide (NO) (Ignarro et al., 2003). Substantial production of NO occurs within tissues via several mechanisms. Initially, conversion of L-arginine to NO was thought to be primarily via endothelial nitric oxide synthase (eNOS) (Moncada et al., 1991; Ignarro et al., 2004; Kleinbongard et al., 2006) and to a lesser extent, via neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) enzymes (Cokic and Schechter, 2008). It has recently been realized that, in addition to NOS synthesis, nitrite reduction to NO may be catalyzed by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin in blood and by other heme-proteins in various tissues (Zweier et al., 1995; Cosby et al., 2003; Dejam et al., 2004; Dejam et al., 2005; Dejam et al., 2007; Sibmooh et al., 2008). Indeed, nitrite ions may be the major storage pool of NO bioactivity. On the other hand, erythrocytic hemoglobin is a major sink for the destruction of NO, and cell-free hemoglobin is an even more effective sink for NO (Rother et al., 2005). Clearly the physiological and potentially pathological effects of red cell transfusions will be affected by any changes in these NO synthetic and destructive processes prior to, during, and immediately after red cell administration. Currently, there is interest in the investigation of potential clinical consequences of changes in NO derivatives during storage, especially with respect to oxygen delivery and vasodilatory capabilities of transfused blood, as well as any association with transfusion-related complications. Two approaches to this have surfaced from our understanding of the metabolism of NO. In one, the nitrite/NO pathway is implicated–nitrite is a major storage pool of NO that can interconvert directly or indirectly with NO. In fact, studies on platelets have elucidated a functional role of nitrite as a modulator of platelet aggregation under hypoxic conditions (Srihirun et al., 2012; Park et al., 2013). In another approach, S-nitrosylated hemoglobin (SNOHb) is implicated indeed, it was proposed that the amount of SNOHb is responsible for the quality of stored red cells, and that replenishing SNOHb would be therapeutically helpful, supposedly restoring the oxygen-transport and vasodilatory capabilities of RBCs. However, this theory has been questioned on several grounds (Gladwin et al., 2002; Rassaf et al., 2002; Dejam et al., 2004; Winslow and Intaglietta, 2008). Evaluation of NO availability with respect to hemoglobin-mediated reductive mechanisms thus appears warranted.
2.2.2.1.5 Chloride
Chloride is the major anion (negatively charged ion) found in the fluid outside of cells and in the blood. Sea water has almost the same concentration of chloride ion as human body fluids. Chloride also plays a role in helping the body maintain a normal balance of fluids. The balance of chloride ion (Cl-) is closely regulated by the body. Significant increases or decreases in chloride can have deleterious or even fatal consequences.
Elevations in chloride (hyperchloremia) may be seen in diarrhea, certain kidney diseases, and sometimes in over activity of the parathyroid glands. Decreased chloride (hypochloremia) may be due to lost in the urine, sweat, and stomach secretions. Excessive loss can occur from heavy sweating, vomiting, and adrenal gland and kidney disease. The normal serum range for chloride is 98 – 108 mmol/L. In stored whole blood, chloride levels were observed to decrease after two days of storage (Michael et al., 2005).
2.2.1.6 Bicarbonate
The bicarbonate ion acts as a buffer to maintain the normal levels of acidity (pH) in blood and other fluids in the body. Bicarbonate levels are measured to monitor the acidity of the blood and body fluids. The acidity is affected by foods or medications that we ingest and the function of the kidneys and lungs. The chemical notation for bicarbonate on most laboratories reports is HCO3- or represented as the concentration of carbon dioxide (CO2). The normal serum range for bicarbonate is 22-30 mmol/L. The bicarbonate test is usually performed along with tests for other blood electrolytes. Disruptions in the normal bicarbonate level may be due to diseases that interfere with respiratory function, kidney diseases, metabolic conditions, or other causes. A fall in bicarbonate was observed in stored blood using CPDA-1 anticoagulant preservative (Latham et al., 2002).
2.2.1.7 Total Protein
The serum total proteins represent the sum of numerous different proteins, many of which vary independent of each other. Since measurement of the serum total protein concentration in blood is useful to evaluate, diagnose, and monitor a variety of diseases and conditions, it is one of the most frequent routine analyses done to investigate electrolyte disorders, inflammatory or infectious diseases, colostrum intake, and tumors (Colahan et al., 2009). Its routine determination is also a prerequisite of serum protein electrophoresis (SPE) (Kaneko, 2007), the most common means of fractionating serum proteins. If the results are properly interpreted, SPE could be considered one of the most useful diagnostic aids available to the clinician. This technique is used in equine medicine for diagnosis, monitoring, and prognosis of many diseases that cause changes in albumin and globulin concentrations (Colahan et al., 2009). Poor performance, depression, fever, diarrhea, abdominal pain, and polyuria are clinical signs for which evaluation of the serum protein fractions is recommended (Colahan et al., 2009). For diagnostic value, the measurements for all fractions must be reliable and correctly interpreted. To maximize the diagnostic value of SPE for clinical laboratories it is essential to have access to well-established relative and absolute reference values (Riond et al., 2009). Ideally reference intervals for serum or plasma parameters should be established in each laboratory; however, especially in veterinary laboratories, the establishment of reference intervals is expensive, and the ideal may not be feasible for all laboratories.
The traditional method for measuring total protein uses the biuret reagent, but other chemical methods such as Kjeldahl method, dye-binding and refractometry are now available. The measurement is usually performed on automated analysers along with other laboratory tests.
The reference range for total protein is typically 60-80g/L. (It is also sometimes reported as "6.0-8.0g/dl"), but this may vary depending on the method of analysis. Concentrations below the reference range usually reflect low albumin concentration, for instance in liver disease or acute infection. Rarely, low total protein may be a sign of immunodeficiency. Concentrations above the reference range are found in paraproteinaemia, Hodgkin's lymphoma, leukaemia or any condition causing an increase in immunoglobulins. Total protein is also commonly elevated in dehydration and C677T gene mutation.
2.2.1.8 Serum Albumin
Albumin is the most abundant protein in serum (3.5–5.0 g/dL, which constitutes approximately half of all serum proteins), it has several important characteristics. It is an anionic protein, containing an abundance of aspartate and glutamate residues; it is not functionally modified with carbohydrates; among all serum proteins, albumin has a midrange molecular weight of 67 kDa; and it has a longer-than-average t1/2 of approximately 20 days. It helps maintain osmotic balance between intravascular and interstitial spaces; therefore, a deficiency ordinarily results in oedema as water is redistributed to tissues. Albumin also functions as a transport protein for calcium, unconjugated bilirubin, thyroid hormones, and many drugs.
Because albumin has a longer t1/2 when compared to many other proteins, its concentration in serum is a poor indicator of nutritional deficiency or impaired synthesis; prealbumin proteins and coagulation factors are more sensitive measures of impaired protein synthesis because their t1/2 are much shorter. The reason for decreased serum albumin is usually due to renal loss. Glomerular membrane permeability is partially a function of size but also is related to charge. The negative charge on albumin inhibits its filtration because the membrane likewise is of like charge. Diseases that lead to severe damage to the glomerular membrane increase its permeability to all proteins; however, its permeability to albumin may be particularly affected if the negatively charged groups on the membrane surface are neutralized. This appears to be the principal mechanism involved that lead to albuminuria associated with diabetic nephropathy.
Although albumin is highly conserved across many species, there exist mostly benign polymorphisms in the specific genes that code for this protein. For instance, there are forms of albumin that have higher-than-normal affinity for thyroid hormones; these do not produce clinical manifestations but may cause errors in immunochemical methods that measure free hormone concentrations because the methods basically rely on competition between antibodies against thyroid hormones and endogenous hormone-binding proteins. Albumin with increased affinity for thyroxine will also result in elevated total tetra-iodothyronine levels in patients with healthy thyroid-gland function because only the free fraction of thyroid hormones is biologically in the active form. Another albumin variant results in a disease condition called bisalbuminemia, this is a benign disorder in which 2 distinct albumin peaks appear on an Serum Protein Electrophoresis gel.
A fascinating paradox surrounds albumin: it is a protein that is highly conserved across many species and has unique properties that seem functionally indispensable, such as the osmoregulation of plasma volume, solubilizing unconjugated bilirubin, buffering the serum ionized-calcium concentration and binding cationic drugs. The logic of evolutionary design would argue that such a protein must be essential for the sustenance of life. However, an extremely rare autosomal recessive genetic defect impairs albumin synthesis and produces analbuminemia, which is the absence of albumin in the blood. This condition is usualy benign and produces only mild edema. Hyperalbuminaemia are uncommon and not clinically significant.
2.2.1.9 Calcium
Calcium ions (Ca2+) play a pivotal role in the physiology and biochemistry of organisms and the cell. They play an important role in signal transduction pathways, where they act as a second messenger, in neurotransmitter release from neurons, in contraction of all muscle cell types, and in fertilization. Many enzymes require calcium ions as a cofactor, those of the blood-clotting cascade being notable examples. Extracellular calcium is also important for maintaining the potential difference across excitable cell membranes, as well as proper bone formation.
Calcium levels in mammals are tightly regulated, with bone acting as the major mineral storage site. Calcium ions, Ca2+, are released from bone into the bloodstream under controlled conditions. Calcium is transported through the bloodstream as dissolved ions or bound to proteins such as serum albumin. Parathyroid hormone secreted by the parathyroid gland regulates the resorption of Ca2+ from bone, reabsorption in the kidney back into circulation, and increases in the activation of vitamin D3 to Calcitriol. Calcitriol, the active form of vitamin D3, promotes absorption of calcium from the intestines and the mobilization of calcium ions from bone matrix. Calcitonin secreted from the parafollicular cells of the thyroid gland also affects calcium levels by opposing parathyroid hormone.
Calcium storages are intracellular organelles that constantly accumulate Ca2+ ions and release them during certain cellular events. Intracellular Ca2+ storages include mitochondria and the endoplasmic reticulum.
A unit of whole blood contains approximately 3g citrate, which binds ionized calcium. The healthy adult liver will metabolize 3g citrate every 5 minutes. Transfusion at rates higher than one unit every five minutes or impaired liver function may thus lead to citrate toxicity and hypocalcaemia. Hypocalcaemia does not have a clinically apparent effect on coagulation, but patients may exhibit transient tetany and hypotension. Calcium should only be given if there is biochemical, clinical or electrocardiographic evidence of hypocalcaemia.
2.2.1.10 Blood Glucose
The blood sugar concentration or blood glucose level is the amount of glucose (sugar) present in the blood of a human or animal. The body naturally tightly regulates blood glucose levels as a part of metabolic homeostasis. With some exceptions, (Daly et al., 2008) glucose is the primary source of energy for the body's cells, and blood lipids (in the form of fats and oils) are primarily a compact energy store. Glucose is transported from the intestines or liver to body cells via the bloodstream, and is made available for cell absorption via the hormone insulin, produced by the body primarily in the pancreas.
Glucose levels are usually lowest in the morning, before the first meal of the day (termed "the fasting level"), and rise after meals for an hour or two by a few millimolar. Blood sugar levels outside the normal range may be an indicator of a medical condition. A persistently high level is referred to as hyperglycemia; low levels are referred to as hypoglycemia. Diabetes mellitus is characterized by persistent hyperglycemia from any of several causes, and is the most prominent disease related to failure of blood sugar regulation. Intake of alcohol causes an initial surge in blood sugar, and later tends to cause levels to fall. Also, certain drugs can increase or decrease glucose levels.
2.2.1.11 Haemoglobin
In human haemoglobin there are four major units, each containing a smaller, but highly significant motif called a 'heme' group. This group contains a single iron atom, held in the centre of a square described by four nitrogen atoms and exists at the heart of an array of organic rings called a porphyrin. It is this iron that binds oxygen and allows haemoglobin to do its job.
The iron in haemoglobin is often thought to account for the distinctive red colouration of our blood, in the same way that iron oxide gives rust its colour. As it happens, here the iron content is just a coincidence – the red colouration in fact comes from the porphyrin (the word 'porphyrin' derives from a Greek word for a reddish purple). Just how red the haemoglobin is, depends on whether there is oxygen bound to it. When there is oxygen present, it changes the shape of the porphyrin, making the colour a brighter red. Haemoglobin levels rise during whole blood storage due to lysis of red blood cells.
2.2.2. Complications Resulting from Leukocytes
Leucocytes found in whole blood are seldom of therapeutic benefit to the patient but are known to escalate the rate of cellular damage and to cause adverse transfusion reactions in recipients. These adverse reactions include alloimmunization to human leucocyte antigens (HLA), nonhaemolytic febrile transfusion reaction (NHFTR), transfusion-associated lung injury (TRALI), and immunomodulatory changes which may include possible post-operation infection, postoperative mortality, or cancer recurrence (Pietersz et al., 1998; Zimmermann et al., 2006). White blood cells may also be regarded as the vector of infectious pathogens for instance cytomegalovirus, Epstein Barr virus and human T-lymphotropic virus I/II. There is the establishment that B-lymphocytes are vectors for the prions causing variant Creutzfeldt-Jakob disease (Zimmermann et al., 2006). There is the report also that using leucocyte reduced Red Blood Cell reduces the incidence of multi-organ failure in patients having vascular or oncological surgery and decreases hospital stay as well as mortality in patients having gastrointestinal oncological surgery. The average reduction of 2, 4 days per patient would significantly cut costs of a national hospital (Bux, 2005). British haemovigilance evidence demonstrates that the use of filtered Red Blood Cell components reduce the frequency of transfusion-associated graft versus host disease. It should be noted however, that only using leucoreduced Red Blood Cell to prevent TA-GvHD is not recommended as the Red Blood Cell used for transfusion should be filtered and irradiated to prevent this serious and often lethal disease condition (Kumar et al., 2006). The filters used for leucocyte depletion are readily available and filtration of RBCC may be prepared at the patient’s bedside during transfusion, before storage (in-line filtration) or after the buffy-coat layer and plasma have been removed (pre-storage or 24 hour expiry product). Leucocyte depletion by filtration is best performed in the processing laboratory of the transfusion services as this maintains better quality assurance. It is advisable to filter the blood soon after collection and/or processing as granulocytes fragment and degranulate during storage, which may cause a NHFTR or the antigen presenting cells presenting major histocompatibility complex (MHC) classes I and II antigens, leading to alloimmunization. It has also been reported that white blood cell antibodies associated with TRALI are possibly targeted at Human Leukocyte antigens (particularly class II) and neutrophil alloantigens. In antibody-mediated TRALI, the particular antibody causing TRALI in a patient is usually recognized in multiparous female donors, but these donors cannot be excluded as this would reduce the donor-pool substantially (Dzik, 1995). The FDA recommends that a filtered unit of blood contains less than 5 × 106 of white blood cells (WBC) and a retention of approximately 85% of the original RBC. Patients are stimulated to produce antibodies against the transfused histocompatibility antigens when the WBC exceed the 5-log count and thus to prevent primary alloimmunization, the FDA has stipulated this rule. They also suggest that quality control testing be done on 1% of filtered units, of which 100% should not have more than 5× 106 WBC.
2.3. Impact of Blood Storage on Clinical Outcome
2.3.1. Impact of Transfusion on RBC.
There are reports that suggest RBC transfusions in generally are associated with increased mortality and morbidity. Several retrospective and prospective studies had evaluated the association of RBC transfusion and mortality (Salze et al., 2008; Willekens et al., 2003) risk for acquiring infection (Vamvakas and Carven, 1999; Leal-Noval et al., 2001) multiorgan failure ( Zallen et al., 1999; Basran et al., 2006) and length of stay (Leal-Noval et al., 2001; Keller et al., 2002; Basran et al., 2006). Several studies had examined the effect of stored pRBC on patient care outcome. These studies focus mainly on limited clinical areas that include cardiac surgery, trauma and critically ill patients. Most of the studies were reported in the last ten years. The studies were made of more retrospective than prospective designs. There was no prospective randomized double blind study in adults or pediatrics that specifically addresses the effect of pRBC storage duration on patient care outcome. Only a few of the studies indicated whether they use leukoreduction or not. Even though most of the studies have different cut offs for new and old RBC units, majority used 2 weeks cut off and reported that RBC units older than 2 weeks were associated with higher mortality and morbidity. Two large retrospective studies reported in 2008 appear to contradict each other. The relatively smaller study (N 5 670) by Yap et al. from Melbourne Australia involved nonemergent recipients of coronary artery bypass graft or aortic valve replacement and who received at least 2 units of RBC between 2001 and 2007 (Yap et al., 2008). Only 3.8% of the transfused blood units were leukoreduced. Thirty day cut off was used to define newer and old blood units. Age of transfused RBC was analyzed using logistic and linear regression models to determine an independent association with clinical outcome. The study findings show it was the quantity of RBC transfused and not the RBC age that independently correlated with clinical outcomes. The other relatively larger study (N 5 6,002) reported by Koch et al. from Cleveland Clinic Ohio involved similar patient profile as the Melbourne study who were transfused between 1998 and 2006 (Koch et al., 2008). About 33% of the newer RBC and 50% of old RBC units transfused were leukoreduced. Fourteen days was used as a cut off to define newer and old blood units. Multivariate logistic regression analysis was used to evaluate the effect of duration of RBC storage on clinical outcome. The study findings show transfusion of RBC units stored for more than 14 days was significantly associated with increased risk of postoperative complications and reduced short- and long-term survival. There are clear differences between the studies. The geographic location of the study site could potentially influence the study outcomes. This could be attributed to slight variation in how blood products are prepared and stored in different parts of the world. The practice of universal leukoreduction is not yet a common practice in Australia while it is in the United Stated and this may have contributed to the contradictory findings. In addition, the cut off used, 14 days versus 30 days might influence the clinical outcome. Finally, the differences in transfusion practice and general patient care between the two continents may have an impact on the study outcomes.
Both reports have limitations that are commonly associated with retrospective studies. Many potential confounders were not equally represented or not considered. For example, both studies failed to account for surgical blood loss, salvaged blood transfusion, surgeon/anesthesiologist involved and intraoperative medications administered such as aprotinin, plavix, aspirin etc. It is a common practice for a patient to receive both old and new blood. It is unclear from both studies whether old blood negates the possible benefit of the newer blood in patients that received both. Association of transfusing old blood and increased incidence of transfusion-related acute lung injury (TRALI). The incidence of transfusion-related acute lung injury (TRALI) was reported to increase with transfusion of older plasma containing blood products (Silliman et al., 2003b). In addition to prolonged storage of transfused products, the presence of an underlying condition such as recent surgery, massive blood transfusion, cytokine therapy and active infection have been implicated in some, but not all, studies (Toy and Gajic, 2004). These reports suggest that the accumulation of bioactive products in stored blood may be important in the development of TRALI. In experimental models, lipopolysaccharide (Silliman et al., 2003b) and soluble CD40 ligand (Khan et al., 2006) in stored blood were shown to play an important role in the pathogenesis of TRALI. Plasma from stored (but not fresh) platelets induced lung injury in rats pretreated with endotoxin (Silliman et al., 2003a). Using rat lung model, development of TRALI was demonstrated when endotoxin pretreatment, which causes increased neutrophil adherence, was followed by the infusion of plasma from stored packed red blood cells, but not with fresh plasma (Silliman et al., 2003a). Association of transfusing old blood and higher incidence of bacterial contamination and sepsis. Bacterial contamination of blood products, particularly platelets, is the most common cause of transfusion transmitted infection (Williamson et al., 1999; Kuehnert et al., 2001). In the United States, the frequency of bacterial contamination was reported to be 1 in 5,000 for platelet units and 1 in 30,000 for pRBC (Jacobs et al., 2001; Kleinman et al., 2006; Jacobs et al., 2008;). One important risk factor is duration of blood storage. In one series that involved over 3,000 random donor platelet pools, the incidence of contamination detected by a Gram’s stain was much lower in units stored for 4 days compared with units stored for 5 days. In addition, among the contaminated units, the magnitude of contamination was less in those stored for 4 days (Yomtovian et al., 1993). Routine bacterial detection assay for platelet units was instituted by the FDA in 2004 and the impact of this measure is yet to be ascertained. American Red Cross, the largest blood collection center in the USA and one of the earliest centers to institute bacterial detection system has reported that their bacterial detection assay had significantly reduced incidence of transfusion transmitted bacterial infection due to platelet units. However, residual risk of septic transfusion reaction due to platelet transfusion remained a significant safety concern (Fang et al., 2005; Eder et al., 2007).
2.3.2. Consequences of Transfusing New Blood
There is a lack of consistent cut off that defines RBC storage duration among the studies that investigated the clinical impact of RBC storage lesions. As a result, the clinical importance of transfusing old RBC versus new RBC remains to be established. Some studies suggested that the clinical impact of the storage lesions become significant after 2 weeks (Zallen et al., 1999; Offner et al., 2002; Koch et al., 2008). However, there is no clear definition of what is ‘‘fresh,’’ ‘‘new,’’ ‘‘young,’’ and ‘‘older’’ units of blood. If we were to shorten the shelf life of RBC to 14 days as some of the above studies recommended, more blood units may potentially be wasted. The median storage duration of RBC units in the USA is about 15 days and the maximum allowed shelf life of a unit of RBC is 42 days (Whitaker and Sullivan, 2006). The nation’s blood supply is already under duress because of a shrinking donor pool, increased regulatory requirements and increasing processing and infectious disease screening costs and a rising demand for blood. From 1991 to 2004 the number of RBC units transfused has increased by 10% (Whitaker and Sullivan, 2006). Because of more stringent criteria for donor selection and more sensitive serologic and nucleic acid testing, more donors are disqualified from blood donation. Donor deferral requirements due to travel history are also increasing. Increasing regulatory requirements that demand multiple donor screening tests are recommended or required by many voluntary and government agencies that monitor blood products and transfusion practice. All of these factors make blood components safer but shrink the donor pool. Additionally, even though the turn-around time for these tests continues to improve, it takes about 48– 72 hours before the blood products are ready to be released to the general inventory. Therefore, if RBC shelf life is to be reduced to 14 days, their availability for transfusion will only be about 10–12 days. The cost of producing and screening blood components is already very high and shortening the shelve life of RBC will significantly increase the cost of a unit of blood. The impact of this may be substantial. Indeed, according to a nationwide survey conducted in 2005 by American Association of Blood Banks the number of whole blood and RBC units transfused in 2004 was 14,182,000 units (Whitaker and Sullivan, 2006).
2.3.3. Solutions to Conservative approach to blood transfusion.
With the current demand for blood, it is almost impractical to shorten the shelf life of pRBC. To decrease the demand for blood to a level that we can afford to reduce the shelve life, a comprehensive blood management/conservation program will need to be set up in all healthcare centers. For a start, every healthcare institution should develop its own guidelines for RBC transfusion, indications and triggers. Moreover, institutions should develop measures to ensure the guide- lines are being followed. The goal is to minimize unnecessary transfusion and to decrease the overall number of RBC units transfused. It is only when the demand for RBC is less than the supply that the shelf life of RBC can be practically reduced. There are studies that suggest a restrictive approach of RBC transfusion is at least as effective as, and potentially superior to, a liberal transfusion approach in critically ill patients with exception of patients with acute myocardial infarction or unstable angina (Hebert et al., 1999). A more recent study by Foss et al. suggests there was no statistically significant differences in postoperative rehabilitation scores and length of stay between elderly patients with hip fracture who were managed with liberal or restrictive approach to blood transfusion. In addition, they observed liberal approach to transfusing patients with hip fracture did not result in increased ambulatory scores. However, there were fewer patients in the liberal transfusion group with cardiovascular complications. Therefore, extra caution should be given when restrictive blood transfusion approach is used in elderly patients (Foss et al., 2009). Limit transfusing new blood units to risky patients. Administration of less than 14-day-old blood can be limited to risky patients. As previously highlighted, the effective- ness of new RBC was only suggested in cardiothoracic and critically ill patients and even then it has not been proven. Detrimental effect of transfusion of old RBC units is yet to be demonstrated in patients with chronic illness. Therefore, the use of new blood can be limited to only cardiac and critically ill patients. However, those cases tend to require many units of blood all at one time. In most instances, enough new blood may not be available to meet the patients’ needs. The only option then is to limit the number of units transfused. However, it is not established whether the risks of limiting blood transfusion is lower than the risks of transfusing old RBC units. A policy of issuing new blood when available cannot be instituted until studies examining the risks/benefits of issuing old blood versus limited blood transfusion are performed. Avoid circumstances that require RBC transfusion. The best way to reduce or prevent RBC storage related ailments is to avoid circumstances that necessitate RBC transfusion. This can be achieved through aggressive management of anemia through iron supplements and erythropoietin therapy (Goldberg, 1995; Feagan et al., 2000). In addition, judicious use of cell savers whenever possible in the operating rooms will minimize RBC transfusion (Niranjan et al., 2006). In summary, recent studies suggests storage lesions are associated with increased morbidity and mortality. However, there are reports with opposite results. A prospective randomized double blind study is needed to determine if the previous reports are actually true. There is also a need to identify the patient population at risk. Transfusion of new blood units is only feasible after establishment of rigorous blood inventory management, blood conservation and blood utilization programs.
2.4 Blood Component Storage Temperature Range
Blood components and products must be stored within the temperature range specified for that particular component or product. Red cells, platelet concentrates, fresh frozen plasma and cryoprecipitate should be stored at temperatures in compliance with the storage temperature requirements, as detailed below.
Red cells leucocyte depleted with shelf-life of 42 days with the appropriate additives should be stored at temperature range of 2–6 ºC. All blood refrigerators, including theatre and other holding refrigerators, must comply with AS 3864.1, AS 3864.2 as amended from time to time. Red cells paediatric leucocyte depleted with shelf-life of 35 days should be stored at temperature range 2–6 ºC. Red cells washed leucocyte depleted with shelf-life of 28 days if re-suspended in additive solution should be stored at temperature range of 2–6 ºC. Fresh frozen plasma with shelf-life of 12 months additives should be stored at temperature range of at –25 ºC or below. Cryodepleted plasma Cryoprecipitate with shelf-life of 12 months should be stored at temperature range of at –25 ºC or below. Platelets with shelf-life of 5 days should be stored at temperature range of 20–24 ºC. Platelets components must be agitated gently and continuously in a single layer on a platelet agitator.
Key points
Keep handling of refrigerated components outside of refrigeration to a minimum to ensure that maximum temperature requirements are not exceeded.
Red cell components must not exceed 30 minutes at room temperature in each occasion according to the AABB Technical Manual (Roback, 2011)
Maintain components in a controlled temperature environment until administered
Handle and store components in a way that minimizes the possibility of product tampering
Alarm set points of components within 0.5 ºC of the storage temperature range
Fractionated plasma products must be stored according to the information on the product packaging or the product insert.
Details for storage and shelf-life of plasma-derived therapeutic products can be found in respective product information sheets.
2.4.1 The Blood Bank Refrigerator
The blood bank refrigerator is an essential piece of equipment in the immunohematology department and provides safe and convenient storage of whole blood, blood components (e.g., blood cells, plasma), and reagents. Blood bank refrigerators ensure freshness and integrity of blood and blood components.
Product description
The refrigeration system includes an electrically powered compressor, a condenser, a capillary tube or expansion valve, an evaporator, and interconnecting tubing. A thermostat regulates the refrigerator temperature. In many models, the compressor and motor are connected to the same shaft and sealed in a compact, airtight compartment, making more space available for storage. Systems are either cylindrical with rotating shelves or rectangular with pullout drawers or shelves. A temperature alarm is either included or optional. An emergency power system is necessary in the event of a power failure. Configurations include tabletop, or floor units.
Principles of operation
Refrigerant leaves the evaporator as gas at a low temperature and pressure. The compressor establishes a pressure difference in the system, drawing refrigerant gas through a suction valve, and circulating it to the condenser. Compressed gas enters the condenser at a higher-than-ambient temperature and is cooled to a liquid. As the liquid refrigerant leaves the condenser, a capillary tube or an expansion valve controls its flow to the evaporator. The capillary tube forms a heat exchanger to help further cool the refrigerant. As the refrigerant leaves, it enters a low-pressure area that permits it to expand rapidly and evaporate, absorbing heat from the refrigerator storage area, thereby cooling the storage area and its contents. Finally, the refrigerant gas is circulated from the evaporator back to the compressor and is drawn through the compressor suction valve to repeat the cycle.
Operating steps
• Refrigerator is installed in setting of use.
• Line power is plugged in.
• When refrigerator reaches desired temperature, it is safe to use.
• Laboratory technician should continuously monitor the temperature of the refrigerator. The technician should also check backup power systems periodically.
Reported problems
The most common problems involve the temperature-alarm system and the monitoring of refrigerator temperature. The alarm should be tested at monthly intervals to ensure proper operation. Because backup power sources have been known to fail, written instructions should be readily available to explain how to determine the cause of any temperature problem and how to handle temporary and prolonged power failures, (WHO, 2012).
CHAPTER 3
MATERIALS AND METHODS
3.1 Description of Study Area
This study was conducted in Braithwaite Memorial Specialist Hospital (BMSH). Braithwaite Memorial Specialist Hospital is a government owned hospital, named after Eldred Curwen Braithwaite, an Australian doctor and a pioneer of surgery. It is located in Old GRA, Port Harcourt, Rivers State and is operated by Rivers State Hospital Management Board. The geographical location of Rivers State is Latitude 4°31′ – 5°31′ and Longitude 6°30′- 7°21′. The temperature in this area ranges from 21oC to 27o C.
3.2 Study Subjects and Enrolment Criteria
The study subjects comprised of adult males (aged 19 to 30). They included a total of 37 apparently healthy volunteer donor subjects. 20 of these donors donated 450mls of whole blood each into Citrate Phosphate Dextrose Adenine (CPDA-1) anticoagulant blood bag. These units were stored in a Standard Blood Bank (SBB) at BMSH. The remaining 17 subjects also donated 450mls of whole blood each into bags with the same anticoagulant. These latter units were instead stored in a Traditional Refrigerator (TR).
3.2.1 Inclusion Criteria
The following were included in the study:
Male donors, aged between 19-30 years.
Apparently Healthy Subjects.
3.2.2 Exclusion Criteria
Subjects with Hb levels lower than 12g/dl.
Subjects that test positive to HCV, HbsAg, syphilis and HIV 1 and 2
Female subjects
3.3 Ethical Consideration:
Considering the nature of the study, ethical clearance from the ethical committee of the hospital is not required. However, a formal approval has been sought and permission granted by the H.O.D., Blood Bank Department, BMSH Port Harcourt. Volunteer donors were duly informed of the purpose for their being recruited for the study (see APPENDIX 5).
3.4 Study Design:
The study is a Prospective Observational Study stratified into two groups: Standard Blood Bank (SBB) refrigeration and Traditional Refrigerator refrigeration.
3.4.1 Sampling Method:
Subjects that fulfilled the inclusion criteria were recruited earlier before the commencement of analysis. It took approximately one month to recruit donors for this study. A total of 37 apparently healthy donor subjects were used for this study. They had their ages ranging from 19 to 30 years (mean age 23.62 yrs.) with corresponding blood groups of:
10 A Rh “D” Positive subjects
5 A Rh “D” Negative subjects
10 O Rh “D” Positive subjects
2 O Rh “D” Negative subjects
10 B Rh “D” Positive subjects
3 B Rh “D” Negative subjects
The donors were all male and tested negative for: HCV, HbsAg, Syphilis and HIV 1 & 2. Donors were divided into two groups. Twenty (20) donors donated for storage at the Blood bank of BMSH, while the remaining 17 donated 17 pints that were stored in a Traditional refrigerator. Both were subjected to the same relatively stable power supply and other physical conditions. The experimental phase of the study spanned through a period of thirty five (35) days. At the time of donation samples were collected for both groups and analyzed to form the baseline values. Subsequent samples were taken at intervals of seven (7) days for the rest of the experiment. Invariably, samples were taken at Day 1, Day 7, Day 14, Day 21, Day 28 and Day 35.
Materials Used
Screening kits for HCV, HbsAg, Syphilis and HIV 1 and 2.
Plain bottles, measuring cylinders, cuvette, test-tubes, sterile syringes, test-tube racks, Pasteur and automatic pipettes, pipette tips, cotton wool
Adult Citrate Phosphate Dextrose Adenine (CPDA-1) anticoagulant/preservative blood bags. It has the composition as cited in APPENDIX 2.
Reagents Used
Drapkin Solution
Total Protein Reagent by Randox
Glucose Reagent by Randox
Albumin Reagents by Randox
Calcium Reagent by Randox
pH, and electrolytes reagents were housed in the Automated Biochemistry Analyzer.
Equipment Used
Automated Biochemistry Analyzer (Olympus AU400 Automated Chemistry Analyzer)
Spectrophotometer (Model 6305)
Swing Angle Bucket Centrifuge
Refractometer Model- (SPR-T2 Model, Fischer Bioblock, France)
3.4.2 Collection of Blood Samples (Bleeding of Donors)
A total of 37 pints of blood were collected from forty donors. 20 pints were collected at the Blood Bank Unit of BMSH while 17 pints were collected at same location but stored in a traditional refrigerator. Donor blood for both groups were screened for HCV, HbsAg, Syphilis and HIV 1 and 2. None of the donors reacted to the aforementioned kits. Donors' medical history was also taken as part of the screening. 20 pints collected at the BMSH Blood Bank Unit were stored in a Standard Blood Bank and Temperature readings were observed throughout the period of the study. The standard blood bank in this case is a blood bank refrigerator armed with a refrigerator alarm with model number WDB-220. The blood bank refrigerator is in perfect condition and meets the AABB requirements for a standard blood bank. Whereas, 17 pints collected at another location of the blood bank were stored but in a Thermocool Traditional Refrigerator procured by the researcher five days before the commencement of the experimental phase of the study. A thermometer was inserted to measure the working temperature of the traditional refrigerator. Both the blood bank refrigerator and the traditional refrigerator were allowed to have equal time of relatively stable power supply.
Procedures for Collection of blood into CPDA-1 blood bags involved the adherence to instructions and procedures provided by the manufacturer. (Cited in APPENDIX 2)
General Precautions
Aseptic Technique was observed.
Bags with turbid solution were discarded.
Sealing was done in a manner that avoided fluid splatter.
Blood-contaminated products were disposed in a manner consistent with established biohazard safety procedures.
3.4.3 Blood Sample collection for analysis.
Blood collection
Blood was collected from voluntary donors following guidelines based on recommendations from the World Health Organization, International Society of Blood Transfusion, American Association of Blood Banks and the International Federation of Red Cross and Red Crescent Societies (WHO, 2010).
Whole blood collection
The donations were collected into a closed set of sterile blood packs with Citrate Phosphate Dextrose Adenine anticoagulant/preservative. Approximately 450 ± 10% mL of donor blood was collected via aseptic venipuncture into the CPDA-1 blood bag. The donation was collected over 5–10 minutes. The appointment took around 40 minutes for each donor including registration and interview time, as well as rest and refreshment time after the donation has finished.
Sample collection was done at intervals of seven (7) days after the baseline for both groups. Sample collection was done according to the schedule below:
Day 1 (13/8/15)
On the first day, five (5) mls of whole blood was taken from each CPDA-1 anticoagulanted blood and put into a 5ml plain bottle before storage for both groups.
Day 2 (20/8/15)
On the second day, 5mls of whole blood was also taken.
Same thing was done for Day 3, Day 4, Day 5 and Day 6, which corresponded with 27th August, 3rd, 10th and 17th of September respectively.
Sample collection was done by gently swirling the pint of blood, draining the traces of blood in the line and then dispensing 5mls of blood into the plain bottle. The samples were spun using a fixed angle bucket centrifuge at 3500rpm for 5 minutes. Before then, about a 1 ml of the whole blood was turned into another plain bottle for the analysis of Haemoglobin concentration. After spinning, the plasma was separated from the cells and the former was used immediately for the estimation of Electrolytes (Na+, K+, HCO3-, and Cl-), pH, Total Protein, Albumin, Calcium, Glucose and Haemoglobin concentrations
3.4.4 Sample Analysis.
Electrolytes (Na+, K+, HCO3-, and Cl-), and pH were analyzed using an Automated Biochemistry Analyzer (Olympus AU400 Automated Chemistry Analyzer) while a Refractometer was used for Total Protein estimation. Albumin, Glucose, Calcium and Haemoglobin concentrations were analyzed manually using their respective reagents (stated above) from Randox and results read photometrically using a spectrophotometer with model number- Model 6305 Spectrophotometer.
3.4.4.1 Estimation of Total Protein:
Estimation of plasma total protein was done using an Atago (SPR-T2 Model, Fischer Bioblock, France) Refractometer. Total protein concentration in plasma samples without visible lipemia was analyzed. The instrument was blanked with distilled water before each series of measurement. All readings were made at room temperature (approximately 25oC).
Principle
The refractive index of an aqueous solution increases directly with an increase in solute concentration. Even though changes in concentration of different substances may affect the refractive index unequally, the concentrations of electrolytes and small organic molecules in serum usually do not vary greatly; hence, a change in refractive index is indicative of a change in protein concentration. A rapid and direct way of measuring the plasma total proteins requiring only one drop of plasma. Values obtained by refractive index correlate well with those found by the Biuret method.
Procedure
A drop of the plasma was placed on the glass plate of the refractometer and the instrument was closed. On closing the instrument, the plasma spreads across the surface, it was closed such that no air bubbles was formed. While holding the plastic cover in place, the refractometer was pointed toward a strong light source. The horizontal line between the bright and dark area was used to read the scale. The values were recorded in g/dl (Cheesbrough, 2005).
To calibrate the instrument, a drop of distilled water was applied to the glass plate just as if it were a sample and allowed for about 30 seconds for the temperature of the sample to adjust to the temperature of the refractometer. The scale was read to be zero.
3.4.4.2 Determination of plasma Albumin
Plasma albumin was estimated using Bromocresol Green method as modified by Randox Laboratories (United Kingdom).
Principle
The measurement of plasma albumin is based on its quantitative binding to 3,3,5,5-tetrabromocresol sulphonephthalein (Bromocresol Green, BCG). The albumin-BCG complex absolves maximally at 578nm, the absorbance being directly proportional to the concentration of albumin in the sample
Most clinical laboratories assay albumin by automated dye-binding methods, using bromocresol green (BCG) or purple (BCP) dyes. These dyes have great affinity for albumin; therefore, the initial rate of binding usually is measured and related to the concentration of albumin in the sample.
Procedure
Test tubes were labelled (B) for blank, (S) for standard (C1, C2 and C3) for quality control samples one, two and three respectively, (T1, T2……Tn) for all the test samples. This was followed by the introduction of 0.1ml of distilled water into test tube (B), same volume of standard solution into test tube (S), same volume of the three control samples into their corresponding test tubes (C1,C2 and C3), and finally same volume of the different plasma sample into their corresponding test tubes, (T1, T2……Tn). Next was the addition of 1ml of BCG working reagent into all the test tubes. The solutions were mixed properly and incubated for 10 minutes at room temperature and read at 540nm using a spectrophotometer with model number SR-6305. The concentrations of albumin in the various test samples were calculated using Beer-Lambert’s Law (Ochei and Kolhatkar, 2000).
3.4.4.3 Estimation of Calcium
Calcium is the fifth most abundant element in the body. Most of the calcium in the human adult is extracellular and 99% of it exists as crystalline hydroxyapatite in bones and teeth where it confers rigidity. Calcium exists in the serum in three forms: protein-bound (45%); ionized (45%) and 10% is complexed with small diffusible ligands such as citrate, lactate, phosphate and bicarbonate.
Principle
The calcium method is a modification of the calcium o-cresolphthalein complexone (OCPC) reaction originally reported by Schwartzenbach, et al. The procedure does not require protein precipitation. This modified Calcium OCPC procedure is based on calcium ions (Ca2+) reacting with o-cresolphthalein complexone in an alkaline solution to form an intense violet colored complex which maximally absorbs at 577 nm. 8-Hydroxyquinoline is added to remove interference by magnesium and iron and glycine buffer for pH control. In this method the absorbance of the Ca-oCPC complex is measured spectrophotometrically at 570nm. The resulting increase in absorbance of the reaction mixture is directly proportional to the calcium concentration in the sample (Tiezt et al., 2012). Procedure is cited in the APPENDIX 2
3.4.4.4 Estimation of Glucose
The glucose oxidase enzymatic method was used because it is specific for glucose.
Principle
Colorimetric method without deproteinisation. Glucose was determined after enzymatic oxidation in the presence of glucose oxidase. The hydrogen peroxide produced, reacts with phenol and 4-aminophenazone to form a red – violet quinoneimine dye. The intensity of the final color (pink) is directly proportional to the glucose concentration and is measured at 505 nm. Procedure used was as described by Monica Cheesbrough (Cheesbrough, 2005).
3.4.4.5 Estimation of Haemoglobin
Hemoglobin levels were measured photometrically using the Haemiglobincyanide Technique. It is the most widely used method for the estimation of hemoglobin.
Principle
Whole blood is diluted in modified Drabkin's solution which contains potassium ferricyanide and potassium cyanide. The red cells are hemolyzed and the hemoglobin is oxidized by the ferricyanide to methaemoglobin. This is converted by the cyanide to stable haemiglobicyanide (HiCN). Absorbance of the HiCN was read in spectrophotometer at wavelength 540nm. The absorbance obtained was compared to that of a reference HiCN standard solution. Procedures and details of Drabkin solution preparation are cited in the APPENDIX 2.
3.4.4.6 Determination of Electrolytes (Na+, K+, Cl- and HCO3-) and pH
Estimation of Sodium, Potassium, Chloride, Bicarbonate and pH was done using the Ion Selective Electrode Analyzer incorporated inside a Clinical Biochemistry Analyzer. Procedures used were as described by Tiezt et al, (Tiezt et al., 2012).
Principle of Test
The development of sodium and pH selective glass and selective organic compounds for potassium, calcium, and chloride has permitted the development of sensors capable of measuring biological fluids directly, throughout the physiological range. These sensors are known as ISE. An ISE is structured to function as an electrochemical cell which measures (senses) the electrochemical activities of ions. It develops a voltage that varies with the concentration of the ion to which it responds. The relationship between the voltage developed and the concentration of the sensed ion is logarithmic as stated in the Nernst Equation: E = Eº + R/nF · T · Log (gC)
The potential of each electrode is measured relative to a fixed, stable voltage established by the silver / silver chloride reference electrode. The flow through sodium and pH contain glass tubing specially formulated to be sensitive to sodium ions. The flow through potassium and calcium electrodes employs plastic tubing, incorporating neutral carrier ionophores. The flow through chloride and lithium electrode includes a plastic tube, specially formulated to be selective to chloride or lithium ions.
Statistical Analysis
Graphpad Prism version 5.05 and SPSS version 22.0 of windows statistical package were used to analyze the data generated. The mean ± standard deviation was determined. T-test and ANOVA, bar charts and line graphs were also done using the same statistical packages. From the values obtained statistical decision and inferential evaluation were made. A probability (p) value of less than 0.05 was considered statistically significant.
CHAPTER 4
RESULTS
4.1 Demographic Characteristics of Enrolled Subjects
The demographic characteristics of subjects are shown in detainls in table 4.1. A total of 37 pints of blood donated by thirty seven voluntary donors were used for the study. All subjects comprised of adult males aged between 19-30 years, mean age of 23.6 years. All subjects were resident in the city of Port Harcourt as at the time of donation. The table shows that subjects weighed between 68 – 89kg, with mean weight of 71.5kg. Of the 37 subjects, 31 (83.8%) were indigenes of Rivers State, 2 (5.4%) hail from Imo State, 3 (8.1%) were indigenes of Akwa Ibom state, and 1 (2.7%) was an indigene of Ondo State.
The ABO and Rhesus blood groups of the subjects are as follows; 10 (27%) are of A Rh “D” Positive, 10 (27%) are of O Rh “D” Positive, 10, (27%) are B Rh “D” Positive, while 5 (13.5%), 3 (8.1%) and 2 (5.4%) are respectively A Rh “D” Negative, B Rh “D” Negative and O Rh “D” Negative.
Table 4.1 Demographic Characteristics of Enrolled Subjects
Subjects Number
Total Number of Subjects 37
Total Number used for SBB 20
Total Number used for TR 17
Overall Weight Range 68-89kg
Mean Weight 71.5kg
States of Origin of Subjects
Rivers 31 (83.8%)
Imo 2 (5.4%)
Akwa Ibom 3 (8.1%)
Ondo 2 (5.4%)
Blood Groups of Subjects
A Rh “D” Positive 10 (27%)
O Rh “D” Positive 10 (27%)
B Rh “D” Positive 10 (27%)
A Rh “D” Negative 5 (13.5%)
B Rh “D” Negative 3 (8.1%)
O Rh “D” Positive 2 (5.4%)
SBB = Standard Blood Bank
TR = Traditional Refrigerator
4.2 Mean Values and Significance Levels of SBB Refrigeration for all Parameters
Table 4.2 shows Mean values and significance levels of all parameters when compared with the baseline values (values for Day 1).
Mean values for Na+ levels from Day 1 to Day 35 were observed to be 138.2 ± 0.22 mmol/L, 136.8 ± 0.97 mmol/L, 136.2 ± 0.82 mmol/L, 135.4 ± 1.18 mmol/L, 134.7 ±1.29 mmol/L and 132.0 ± 1.04mmol/L respectively. There were significant decrease (at p<0.001) when Na+ values for Day 1 were compared using paired t test with the other groups. K+ levels recorded significant increase at p<0.001 at all levels (Day 1 vs Days 7, 14, 21, 28 and 35). Chloride levels recorded the highest mean value of 82.15 ± 1.67 mmol/L on Day 1 and least value (75.95 ± 2.14 mmol/L) on Day 35. Values significantly decreased at all levels (p<0.001) when compared with the baseline values (Day 1 values).
Mean HCO3- level for SBB refrigeration for the first day was 19.25 ± 0.97 mmol/L. When the values for Day 1 were compared with those of other days, significant decreases at p<0.01 were observed at Day 1 vs Day 7 and Day 1 vs Day 14, while the remaining three periods, Days 21, 28 and 35 also had significant decreases, but in the latter case, the significance level was at p<0.001. Mean Total Protein (TP) value for the first day was observed to be 79.80 ± 0.86g/dl and 79.27 ± 1.76 g/dl was recorded on the last day (Day 35). TP values for Day 1 when compared with others showed significant decrease at (p<0.01) for both Day 7 and Day 21 while Day 14 recorded decreases at p<0.001 level of significance and there was no statistical significance observed on the last day when compared with Day 1 using paired sample t-test.
Mean Albumin values were observed to reduce from 38.48 ± 1.37 g/dl for Day 1 to 35.70 ± 1.99 g/dl for Day 35. This registered a decrease at p<0.01 level of significance for Days 7 and 14 while a significant decreases at p<0.001 were also observed when values for Day 1 were compared with those of Days 21, 28 and 35. Mean pH values fell from 6.92 ± 0.21 (Day 1) to 6.07 ± 0.59 (Day 35). All the stages had statistically significant decreases at p<0.001. There was no significant change in the means of Ca2+ levels throughout.
14.79 ± 1.27mmol/L was observed to be the mean glucose value for Day 1 and 11.06 ± 2.16 mmol/L for Day 35. There were significant decrease in all groups (Day 1 vs Days 7, 14, 21, 28 and 35). Mean Hemoglobin (Hb) values across all the days were observed to be 14.09 ± 0.73 g/dl, 13.70 ± 0.75 g/dl, 14.01 ± 0.72 g/dl, 14.01 ± 0.72 g/dl, 14.35 ± 0.64 g/dl and 14.51 ± 0.77 g/dl for Days 1, 7, 14, 28 and 35 respectively. No significant difference was observed when Hb values for Day 1 were compared with those of Days 14, 21, 28 and 35. A significance decrease at p<0.05 was however recorded when the baseline values were compared with the values of Day 7.
4.3 Mean, SD and Significance Levels of TR Refrigeration for all Parameters
Table 4.3 shows mean ± SD and significance levels for TR refrigeration for all parameters. From the table, it shows that the mean values for TR on the first day (Day 1) for Na+ were recorded as 136.6 ± 1.14mmol/L. Subsequently, mean values for Days 7, 14, 21, 28 and 35 were recorded as follows 136.0 ± 1.20 mmol/L, 135.4 ± 1.05 mmol/L, 134.8 ± 1.06 mmol/L, 134.2 ± 1.09 mmol/L and 132.0 ± 1.32 mmol/L respectively. A paired sample t-test shows the various levels of significance for Na+ when values for Day 1 (baseline values) were compared with Days 7 to 35. Na+ levels for Day 1 when compared with those of Day 7 showed significant decrease at p<0.05, while a significance decrease at p<0.001 was recorded when Na+ values for Day 1 were compared with those of Days 14, 22, 28 and 35. The mean value 9.48 ± 0.98 mmol/L was observed as the highest mean K+ value (Day 35). Whereas, the least mean K+ value (2.52 ± 1.72 mmol/L) were observed on the first day. When compared with the baseline values, K+ levels for all the days were observed to be significant higher than those of the baseline at p<0.001. Mean chloride values were recorded to be significantly higher than the baseline values at all levels at p<0.001(Days 14 to 35) and p<0.01 (Day 7). Total protein levels significantly decreased at (p<0.05) for Day 1 vs Days 7 and 21, while an increased level of significance (p<0.01) was observed when total protein values for Day 1 were compared with those of Day 14. No significant difference was observed when total protein values of Day 1 were compared with those of Days 28 and 35. Albumin values showed a significantly decreased levels at p<0.05 all through from Day 7 to Day 35 when compared with the baseline.
Table 4.3 also shows mean values for pH from Day 1 to Day 35. Significant decreases were observed all through at p<0.001 when values for Day 1 were compared with others. Calcium values showed no significant difference all through. Levels of glucose showed a significant decrease at (p<0.001) throughout when compared with the baseline values. Mean Hb values from Day 1 to Day 35 are also recorded in the table. No significant difference was observed when the Hb baseline values were compared with values for Day 7, Day 14 and Day 21, while significant increase were observed when values for Day 1 were compared with those of Day 28 (p<0.05) and Day 35 (p<0.001).
4.4 Mean, p values and Levels of Significance for Day 1 for all Parameters Comparing SBB and TR Refrigeration
Table 4.4 shows the significance levels of all parameters and their mean values for the first day (Day 1) of analysis comparing values gotten from units stored in a Standard Blood Bank (SBB) with those stored in a Traditional Refrigerator (TR). Values for all parameters were compared and found to be non-significant.
4.5 Mean, p values and Levels of Significance for Day 7 for all Parameters Comparing SBB and TR Refrigeration
Table 4.5 shows that plasma K+, Cl-, HCO3- values for SBB were found to be significantly lower than those of TR while Na+ and TP values for SBB refrigeration were found to be statistically higher than those of TR. Mean values for albumin, pH, Ca2+, glucose and Hb were not significant statistically when compared.
4.6 Mean, p values and Levels of Significance for Day 14 for all Parameters Comparing SBB and TR Refrigeration
From table 4.6, plasma K+ and Cl- values for SBB were found to be significantly lower than those for TR refrigeration. Whereas, TP and Na+ values for SBB recorded significantly higher values when compared with those for TR refrigeration. However, mean HCO3-, albumin, pH, Ca2+, Glucose and Hb concentration were not significant.
4.7 Mean, p values and Levels of Significance for Day 21 for all Parameters Comparing SBB and TR Refrigeration
From table 4.7, it shows that K+ and Cl- levels for SBB were observed to be significantly lower while Na+ and TP levels where significantly higher than those of TR refrigeration. Values for Na+, HCO3-, albumin, pH, Ca2+, glucose and Hb when compared were not significant.
4.8 Mean, p values and Levels of Significance for Day 28 for all Parameters Comparing SBB and TR Refrigeration
Plasma K+ levels for SBB were significantly lower than those of TR, whereas, a rather significantly higher levels of TP were observed for SBB when compared with values for TR refrigeration. The remaining eight parameters were not significant.
4.9 Mean, p values and Levels of Significance for Day 35 for all Parameters Comparing SBB and TR Refrigeration
Table 4.9 shows that K+ values for SBB were significantly lower than those for TR refrigeration. Whilst, albumin and Glucose levels for SBB had significantly higher values than those of TR refrigeration. Mean values for Na+, Cl-, HCO3-, total protein, pH, Ca2+ and Hb concentration were observed to be non-significant.
4.10 Details of outcome of Questionnaire
Table 4.10 shows details of the outcome of a well-structured and adequately distributed questionnaire. From the table, 113 medical laboratories in Port Harcourt city were sampled. 29 (25.7%) did not operate a blood bank, while 84 (74.3%) did. Out of the 84 laboratories that operated a blood bank, 52 (61.9%) used traditional refrigerators as blood bank while 32 (38.1%) used blood bank refrigerators. Also, 61 (72.6%) were non-hospital laboratories while 23(27.4%) were hospital laboratories (medical laboratories domiciled in a hospital setting). Out of the 84 laboratories also, 15 (17.9%) were public owned laboratories while 69 (82.1%) were private owned laboratories.
Figure 4.1 shows a line graph of temperature fluctuations from Day 1 to Day 35 for both SBB and TR Refrigeration.
Bar charts showing Mean values plotted between SBB and TR from Day 1 to Day 35 for all parameters are shown in figures 4.2 to figure 4.11.
CHAPTER 5
DISCUSSION
Evaluation of changes in plasma levels of sodium (Na+), potassium (K+), chloride (Cl-), bicarbonate (HCO3-), total protein, albumin, pH, calcium (Ca2+), glucose and hemoglobin (Hb) levels in Citrate Phosphate Dextrose Adenine stored whole blood, comparing values from units stored in a Standard Blood Bank (SBB) and Traditional Refrigerator (TR) had not been reported.
The outcome of a well-structured and adequately distributed questionnaire prompted the need for this study. In the questionnaire (cited in the appendix 1), summarized in table 4.10, it is interesting to note that out of 84 sampled laboratories in the city of Port Harcourt that operate blood banks, 61.9% (52 laboratories) used traditional refrigerator as their blood bank. This obviously is not in line with the basic requirements of The Blood Bank Society of Nigeria as well as American Association of Blood Bank (AABB) and other regulatory bodies for the operation of a blood bank, prompting the need for an evaluation of likely changes that would occur in CPDA-1 stored whole blood using a traditional refrigerator and comparing values obtained with values that would be observed when units of blood contained in the same anticoagulant/preservative and subjected to the same power supply but stored in a standard blood bank (approved blood bank refrigerator). Recent reports suggest that transfusion of “old blood” and blood not stored in the approved facilities and condition was associated with the risk of postoperative complications and higher mortality rate in surgical patients as well as post transfusion complications and this has caught public attention, (Yap et al., 2008). Noncompliance with rules and regulation governing the storage of whole blood or any of its products can cause severe clinical consequences to the recipient, (Hamasaki and Yamamoto, 2000).
This study has shown changes in some biochemical parameters that occurred in CPDA-1 stored whole blood when subjected to both Standard Blood Bank and Traditional refrigeration, the latter being increasingly practiced in this part of the world (Port Harcourt city) according to a questionnaire conducted by the same study. In the 20 units of CPDA-1 anticoagulant/preservative packaged whole blood stored in a standard blood bank, the mean Na+ level on the first day was observed to be 138 ± 0.22mmol/L (table 4.3). This serves as the baseline value for Na+ levels and was used to compare with Na+ values of subsequent days (17, 14, 21, 28 and 35) to ascertain the level of statistical significance. The value is about one unit higher than the mean Na+ (137.38mmo/L) reported by Adias et al. Although they used ten samples and observed a non-significant relationship between the mean values for Day 1 and that of other days, (Adias et al., 2012). During refrigerated storage, Na+ and K+ leak through the red cell membrane rapidly. The cells lose and gain Na+, however, the K+ loss is greater than the Na+ gain during storage.
The lowest mean Na+ value (132.0 ± 1.32 mmol/L) was recorded on the last day (Day 35). This indicates that Na+ levels declined as the number of days of storage increased. Adias et al also recorded a decline in mean Na+ levels (137.38 – 129.44mmol/L). However, in this study a significant level (p<0.001) was observed when Na+ values for Day 1 were compared with those of other days. Also, mean Na+ value for units that were stored in the traditional refrigerator was observed to be 136 ± 1.14mmo/L for Day 1. This is one unit lower than that reported by by Adias et al. Meanwhile, when Na+ values for Day 1 were compared with those of other days for TR refrigeration, Day 7 was observed not to be significant, but all other days were significantly decreased at p<0.001. Radovan et al also recorded a decrease in Na+ levels when units that were stored for longer periods were transfused. They observed a mean in vivo Na+ value of 137.0mmo/L when whole blood collected on the first day were transfused (Radovan et al., 2011). It has been observed that following blood transfusion of stored blood, complications such as hyperkalemia, hyponatremia and citrate toxicity among other conditions do occur. Comparison between Na+ values for SBB with those of TR refrigeration showed that Na+ values for SBB were significantly higher than those of TR refrigeration at (p<0.001) for Days 7, 14 and 21, while Days 28 and 35 both recorded no significant difference. The difference may probably be due to changes in the rheology of red blood cells due to the accumulation of waste products, particularly in the storage that had the temperature fluctuating between 2 to 9oC. No study has reported changes in Na+ levels in CPDA-1 anticoagulant blood stored in a traditional refrigerator. However, Li et al reported extreme reduction in Na+ levels when the temperature for RBC storage was allowed to fluctuate between 5oC above the AABB designated temperature (2-6oC) for blood storage (Li et al., 2003). In severe kidney disease even small amount of K+ fluctuation can be dangerous and relatively fresh or washed RBCs are indicated.
This study has also shown that there was a tremendous increase in K+ levels from Day 1 to Day 35 for all groups. Mean K+ value of 5.42 ± 1.10mmo/L was recorded as the highest value and 2.89 ± 0.11 recorded as the lowest value for SBB refrigeration, while 9.48 ± 0.94 and 2.52 ± 1.72mmo/L were respectively observed for the last and first day for TR refrigeration. K+ values were observed to increase with the time of storage. This is in agreement with values obtained by Adias et al. In both groups, mean K+ values were significant at all levels. It was also observed that increases in K+ levels were more in the TR refrigeration. This may probably be due to excessive breakdown of RBC and leakage of K+ into the plasma, and more leakage when the temperature increased. Electrolyte, particularly K+ disturbances can be associated with a number of occurrences including drug usage (Buckley et al., 2010) but the kidney is expected to manage it. Hypokalaemia and hyperkalaemia has been seen as problem for some hospitalized patients (Hoskote et al., 2008) with hyperkalaemia being implicated for complications of massive blood transfusion (Murthy et al., 1999; Buntain and Pabari, 1999; Matthew et al., 2008). Assessment of K+ levels as an impact of transfused blood on biochemical parameters depending on the volume and age of administered product, as well as the biochemical changes occurring during the storage of these products in vitro were analyzed by Radavan et al. According to them, K+ values increased tremendously in both cases, recording a mean in vivo K+ values greater than 5.5mmol/l (Radavan et al., 2011).
This study shows an increase in plasma potassium levels which are also in accordance with those reported by Aboudara et al., 2008. Their study focused on hyperkalemia (>5.5mmol/L) in a group of 131 trauma patients undergoing cardiopulmonary regulation during the initial 12 hours after admission to a hospital. 96 (73.3%) of the patients received blood (a mean of 11.2 blood units/patient, range 1-55 whole blood units/patient). Interestingly, 38.5% of transfusion patients developed hyperkalemia, as compared with only 5.9% of patients without transfusion. The study documented a more dramatic rise in potassium levels in transfusion (from 3.7mmol/L to 5.3mmol/L) than in non-transfusion patients (from 3.6mmol/L to 4.0mmol/L). Blood stored at 1∘ to 6∘C decreases the rate of cellular metabolism and energy demand which allows blood to be stored for 35 days. This makes the sodium–potassium pump inoperative and consequently allows potassium ions to exit the cell and sodium ions to enter via the semipermeable membrane. It was demonstrated in critically ill patients that the sodium levels will revert to their normal levels within 24 hours after transfusion, whereas the potassium levels take about 4 days to stabilize, but such is not the case if the patient has developed hyperkalemic or hypernatremic condition before transfusion. The condition is exacerbated when the patient receives large volume of RBCs of whole blood. (Weiskopf et al., 2006; Leal-Noval et al., 2008). The plasma level of potassium may increase by 0.5-1.0mmol/L per day of refrigerator storage (Bailey and Bove, 2005). There is a notion that the total amount of extracellular potassium in a unit of blood stored for 35days falls within 7mmol/L to 25mmol/L (Ratcliffe et al., 2006).
Table 4.2 and 4.3 show mean chloride values for both SBB and TR refrigeration. Chloride values for the former decreased from 82.15 ± 1.67mmol/L for the first day to 75.97 ± 2.14mmol/L for Day 35. The latter group recorded a mean chloride level of 89.94 ± 3.42mmol/L on the first day and values decreased gradually across the period, eventually 76.82±3.80 was observed for the last day. In both cases, the chloride levels decreased from Day 1 to Day 35. Chloride values of both groups are in line with the work of Adias et al. who reported 75.93mmol/L and 72.19mmol/L as highest and lowest values for Days 1 and 35 respectively. Chloride is the major anion (negatively charged ion) found in the fluid outside of cells and in the blood. In stored whole blood, chloride levels were observed to decrease after two days of storage (Michael et al., 2005).
Tables 4.2 and 4.3 also show mean HCO3- values for both SBB and TR refrigeration. In the former, it was observed that the HCO3- values decreased from 19.86 + 0.96mmol/L to 17.38 + 0.60mmol/L. This is in line with the work of Letham et al who stated that a fall in bicarbonate was observed in stored blood using CPDA-1 anticoagulant preservative (Latham et al., 2002). Significant decrease was observed at p<0.01 when HCO3- values for Day 1 vs Days 7, 14 were compared, while comparison between HCO3- levels for Day 1 vs Days 21, 28 and 35 were also significantly decreased but at p<0.001 confidence level for SBB refrigeration. In TR refrigeration, significance decrease were observed when values for Day 1 were compared with all other days. Decrease in HCO3- may be due to reduction in CO2 levels due to leakage from the bag. CO2 produced form metabolism of glucose accumulate and is expected to diffuse through the containing material. The plastic material should be sufficiently permeable to CO2 in order to maintain higher pH during storage. Currently the blood is stored in plastic bags made of polyvinyl chloride (PVC) with plasticizer, di-(2-ethylhexyl) phthalate (DEHP). It is known that DEHP leaches from plastic into plasma and cell membrane during storage and may be harmful to the patient on transfusion.
Mean total protein values for Day 1 was observed to be 79.30 ± 0.86g/dl and 79.27 ± 1.76g/dl for Day 35. These values were for the SBB refrigeration, while for TR refrigeration, mean total protein levels of 77.24 ± 4.17g/dl and 77.55 ± 3.28g/dl were observed for Day 1 and Day 35 respectively. From the results, a significant decrease was observed at Days 7 and 21 (p<0.01) and Day 14 (p<0.001), while, Day 35 was not significant for SBB refrigeration. For TR refrigeration, Day 1 vs Days 28 and 35 were not significant while a significant decrease at (p<0.05) was observed for Day 7 and (p<0.01) for Day 14 respectively. Slight increase in total protein levels may be due to lysis resulting to leaking of haemoglobin into the plasma. The release of hydrogen peroxidase and proteases by the leucocytes present in unfiltered blood may cause lysis of red blood cells during the storage period. Signs of haemolysis in the plasma or suspending fluid may suggest that the red blood cells have been either ruptured or it may be due to the loss of membrane-bound haemoglobin in microvesicles found on the cell’s surface of intact cells.
Comparison between SBB and TR for total protein level for Days 1 and 35 were not significant, while at Days 7, 14, 21 and 28 TP values for SBB were significantly higher than those for TR refrigeration at p<0.05.
Mean albumin levels for Day 1 was observed to be 38.48 ± 1.37g/dl and 35.70 ± 1.99g/dl for Day 35 in SBB refrigeration, while TR refrigeration recorded a mean albumin levels of 38.27 ± 1.65g/dl and 34.42 ± 1.47g/dl for the first and last days respectively. Also, for SBB, and TR refrigeration, there was significant decrease between both groups in Day 35 while other days recorded no statistical significance.
Mean pH levels for SBB in the first day was 6.92 ± 0.21, as against 6.84 ± 0.17 for TR. The last day recorded a mean pH value of 6.07 ± 059 and 6.16 ± 0.28 for SBB and TR respectively. pH levels decreased in both groups from the first day to the last day. In tables 4.2 and 4.3, comparing pH values for Day 1 and other days showed significant decrease at p<0.001 for both SBB and TR refrigeration. Comparison between SBB and TR for pH shows no significant difference at all levels (Day 1 to Day 35).
Mean Ca2+ levels for Day 1 and Day 35 in the SBB refrigeration recorded values of 2.12 ± 0.33mmol/L and 2.31 ± 0.31mmol/L as against 2.08 ± 0.26 and 2.21 ± 0.28 for TR refrigeration in the first and last days respectively. No significant difference was observed throughout the days when Ca2+ values for Day 1 was compared with those of other days for both SBB and TR refrigeration. This means that storage probably does not have effect on the levels of Ca2+. A unit of whole blood contains approximately 3g citrate, which binds ionized Ca2+.
Table 4.2 and 4.3 show that mean glucose levels for Day 1 and 35 for SBB are 14.79 ± 1.27mmol/L and 11.06 ± 2.16mmol/L, while TR refrigeration recorded mean glucose values of 15.11 ± 1.26mmol/L and 9.88 ± 0.76mmol/L for Day 1 and Day 35. It was observed that the glucose levels for both SBB and TR in all the days decreased as the days of storage increased. Similarly, significant decreases were observed at p<0.001 when glucose values for Day 1 were compared with those of other Days for both groups. Comparison between glucose levels for SBB and TR showed that the glucose level for SBB were significantly higher than those of Tr refrigeration on Day 35, while in other days no significant difference was observed. The decrease of ATP concentration during storage causes the cellular reactions requiring energy, for example, phospholipid membrane distribution, active transport, and antioxidant reactions, to also decrease. The decrease in glucose level in this work is in line with the work of Basran et al that stated that it has been indicated that there is a 60% decrease in ATP levels after more than 5 weeks of storage (Basran et al., 2006). The continuous reduction in ATP concentrations and acidification results in irreversible shape alteration of the RBC as echinocytic surface protrusions appear. The phospholipid bilayer loses its asymmetry and the shedding of microvesicles occur (Koch, et al., 2008).
Mean Haemoglobin (Hb) levels for the first day was observed to be 14.09 ± 0.73g/dl as against 13.77 ± 0.45g/dl for TR refrigeration. Day 35 recorded mean Hb levels for both SBB and TR refrigeration as 14.5 ± 0.77g/dl and 14.65 + 065g/dl respectively. Hb levels were observed not to be significant when Hb values for Day 1 were compared with those of other days for both SBB and TR refrigeration save for Day 7 (for SBB) and Days 28 and 35 (for TR). When compared, Hb values for SBB and TR for all days were not significant. This shows that there is probably no significant storage effect on the levels of Hb in CPDA-1 stored whole blood.
CHAPTER 6
6.1 CONCLUSION
This study has recorded tremendous increase in plasma potassium levels in whole blood as the days of storage increased. The rate of increase being significantly higher in units stored in the traditional refrigerator. Transfusion of such blood into a patient who already has complications due to an underlying hyperkalemic condition may exacerbate the clinical condition. Death may result due to hyperkalemic shock, particularly if large volume of whole blood is transfused as commonly practiced in our hospitals. This study has also shown that there is a decrease in plasma sodium, chloride, bicarbonate, albumin and pH levels in both SBB and TR methods of refrigeration. The decrease in plasma sodium were observed to be more in the TR refrigeration. Decreases in plasma bicarbonate, total protein, glucose and albumin were also higher in the TR refrigeration, but at different weeks of storage. Transfusion of whole blood with storage induced increase/decrease in biochemical components could lead to severe clinical consequences. In all the lower temperature at standard condition keeps the rate of glycolysis and other cellular activities at lower limit and minimizes the proliferation of bacteria that might have entered the blood unit during venipuncture or from the atmosphere. The rate of diffusion of electrolytes (particularly sodium and potassium) across the cell membrane is also less at lower temperature. Increases in storage temperature can lead to rise in many metabolic activities leading to extreme leakage of substances from cells.
6.2 RECOMMENDATIONS
Following the outcome of this study, the following recommendations are warranted:
Medical Laboratory Science Council of Nigeria (MLSCN) in collaboration with The Blood Bank Society of Nigeria should as a matter of urgency perform a thorough assessment of all medical laboratories operating in Port Harcourt to ensure strict adherence to the code of practice of the professional body, particularly in the operation of blood bank. Disciplinary measures should be taken on defaulters.
Transfusion of large units of whole blood, as mostly practiced in our hospitals should be discouraged, as it may complicate the patient’s clinical condition. Medical procedures that will quickly arrest blood loss should be enhanced instead.
Transfusion should be component specific, blood component should be separated into their various components and transfused as such so as to avoid the interference of any blood component with another and subsequently preventing unwarranted clinical consequences.
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APPENDIX 2
TEST PROCEDURES AND REAGENTS
Composition of Citrate Phosphate Dextrose Adenine Anticoagulant/Preservative
Ingredient Name Basis of Strength Strength
Dextrose Monohydrate Dextrose Monohydrate 2.01g in 63 mL
Sodium Citrate Sodium Citrate 1.66g in 63 mL
Citric Acid Monohydrate Anhydrous Citric Acid 0.206g in 63 mL
Sodium Phosphate Sodium Phosphate 0.140g in 63 mL
Adenine Adenine 0.017g in 63 mL
Procedure for Calcium Estimation
The solution was properly mixed and incubated for 5 minutes at room temperature (25-30oC) and absorbance of standard and sample were read against reagent blank at 570 nm (530 to 580 nm or with GREEN filter).
Test Results: Calcium concentration (mmol/L) = Absorbance of test x 10
Absorbance of standard
Reagent Preparation: Reagents were prepared as per the daily requirements by mixing equal volumes of 1 calcium and 2 calcium.
Presentation:
No. of Bottles
All reagents to be stored at 2-80 oC. 100 ml
• 1 CALCIUM (COLOUR REAGENT) 1
• 2 CALCIUM (COLOUR DEVELOPING REAGENT) 1
• CALCIUM STANDARD (2.5mmol/L.) 1
Reaction Parameters :
• Type of Reaction: End Point
• Wavelength: 570 nm
• Flowcell Temperature: 30 0C
• Incubation: 5 min. at RT
• Std. Concentration: 10 mg/dL
• Sample Volume: 10 Microlitres (0.01 ml)
• Reagent Volume: 1.0 ml.
• Zero setting with: Reagent Blank
• Light Path: 1.0 cm.
Quality Control: During operation two levels of an appropriate quality control material were tested a minimum of once a day. In addition, controls were performed after calibration, and after specific maintenance or troubleshooting steps as described in the User’s Guide. Quality control testing were performed in accordance with regulatory requirements and the laboratory’s standard procedure.
Procedures for Estimation of Hemoglobin and preparation of Drabkin solution
Materials Amount
Drabkin’s solution reagents used are:
1. Potassium ferricyanide = 200 mg
2. Potassium cyanide = 50 mg
3. Potassium dihydrogen phosphate = 140 mg
4. Non-ionic detergent = 1 ml
5. Distal water = Make up to 1000 ml (1 L)
Procedure for Hb Estimation
1. 20 µL of blood was added to 4mL of Drabkin = 1: 200 dilution.
2. It was properly mixed.
3. It was read within 6 hours of mixing on filter 540.
4. It was read against blank of drabkin solution (Drabkin solution can be used as blank).
5. The standard solution was also read (12 g/dL) with the same dilution like test sample.
6. Calculation method = (OD of test /OD of STD) X conc.of stand. = Hb of test sample.
APPENDIX 3
RAW DATA
Demographic Details of Study Subjects for SBB Refrigeration
Demographic Details of Study Subjects for TR Refrigeration
Mean Values for all Parameters and Days for Standard Blood Bank
Mean Values for all Parameters and Days for Traditional Refrigerator
Temperature Fluctuation from Day 1 to Day 35 for both groups (SBB and TR)
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