Most donor blood units for transfusion purposes are stored in Citrate Phosphate Dextrose Adenine-1 (CPDA-1) at blood-banks until used. Despite improvements made in blood storage attempts, CPDA-1 may still cause morphological and degenerative changes in red blood cells (RBCs) if storage is prolonged. The purpose of the study was to assess the viability of RBCs through characteristic changes resulting from prolonged storage in CPDA-1. A total of fifteen whole blood stored in CPDA-1 were analyzed for changes in erythrocytes morphology, Packed Cell Volume (PCV) and osmotic fragility in a seven day interval over a thirty-five day period. On day 1 (control), samples were analyzed within 6 hours of collection, and subsequently taken to the blood bank to be stored and re-analyzed from days 7 to 35. Mean Cell Fragility (MCF) which is suggestive of RBC membrane instability showed changes that were statistically significant between days 1 and 35. Morphological changes observed over the storage period included spherocytosis, echinocytosis, and marked sphero-echinocytosis with increased rouleaux formation. PCV value decreased marginally over the period. The author concluded that donor blood units stored for longer periods may result in RBC defects, affecting their viability and consequently interfere with expected therapeutic outcomes.
Blood transfusion is generally the process of infusing blood components into one’s circulation intravenously. Transfusion therefore is used in a variety of medical conditions to replace lost blood as discussed by Chessbrough [
The idea of blood storage has in recent times witnessed increased scrutiny regarding efficacy and risk of transfusion associated reaction or events as discussed elsewhere [
Conditions under which blood is stored, in most storage facilities (blood banks) invariably, produces changes in RBCs irrespective of the preservative or anticoagulant used as discussed by Gupta [
One of the commonly used anticoagulant for blood storage is Citrate Phosphate Dextrose Adenine-1 (CPDA- 1). CPDA-1 solution contains 2 g Dextrose (monohydrate), 1.66 g Sodium Citrate (dihydrate), 188 mg Citric Acid (anhydrous), 140 mg Monobasic Sodium Phosphate (monohydrate) and 17.3 mg Adenine. CPDA-1 is the only anticoagulant-nutrient solution in regular use. Citrate CPDA-1 is used as an anticoagulant for whole blood and is supplied as a high quality, convenient, 0.2 µm-filtered ready to use solution. Recommended anticoagulant: whole blood ratio is 1:9. The function of CPDA-1 is whole blood preservation to provide viable and functional blood components for patients requiring blood transfusion. More than 70% of red cells are expected to remain viable in circulation 24 hours after transfusion of blood stored at 2˚C - 6˚C in CPDA-1 for 35 days. Knowledge on prolonged storage effects on RBCs by certain anticoagulants will help improve RBC storage by reducing storage alterations (lesions) that will hitherto affect RBC viability and recovery, resulting in improved therapeutic efficacy of allogeneic blood transfusion
This study was an experimental study conducted from the month of June to July 2014.
Ethical clearance for this research was sought from the Ethics and Protocol Review Committee at the School of Biomedical and Allied Health Sciences, University of Ghana, Legon. All the participants gave their informed consent before their samples were collected.
The samples were collected from the Accra Area Blood Collection site of the Southern Area Blood Centre (SABC) located within the premises of Korle-bu Teaching Hospital (KBTH), Accra. Sample analysis was carried out at the haematology research laboratory of the University of Ghana Medical School (UGMS), College of Health Sciences, Korle-bu. A total of 15 samples from consented individuals were used in the study.
Material used include the following: Saline of varied concentrations (Sigma-Aldrich), Colorimeter, Light microscope & Haematocrit (Olympus Life and Material Science, Europa GMBH―Germany), Leishman stain-Pre- prepared according to manufacturer’s protocol, Sigma-Aldrich, Buffer (Sigma-Aldrich), CPDA-1 anticoagulant (Fenwal Inc.―USA), Centrifuge (Eppendorf 5810R, Rotor number A-4-62), slide rack, test tubes and automatic pipettes (Thomas scientific), were used.
Participants were between the ages of 18 to 47 years and have met all the criteria required to qualify as blood donors by the National Blood Transfusion Service (NBTS). A total of fifteen (15) apparently healthy participants were enrolled.
Whole blood samples were collected from each randomly selected replacement donor into a labeled blood bag containing CPDA-1 as an anticoagulant. The samples were gently mixed to avoid clotting and kept in a vaccine carrier before being taken to the haematology research laboratory for analysis. An aliquot of the samples were analyzed within four hours of collection for determination of Packed Cell Volume (PCV), Osmotic Fragility and Thin Film for red cell general film comment to obtain baseline values (Day 1). The samples were then sent to the blood bank, where they were stored in the blood bank refrigerator. The storage temperature was monitored twice daily and aliquots were taken and analyzed in a weekly (7 days) interval for 35 days duration. The osmotic fragility test, PCV, and Leishman staining technique was used to determine changes in red blood cell membrane and morphological integrity.
Data from the study was analysed using Statistical Package for Social Sciences (SPSS) version 20 statistical software. It was checked for completeness and correctness and was double entered into data sheet. Data cleaning and verification was also done regularly. Data collected was transcribed immediately after each session. Anova test was used to determine whether there was any statistical significance between storage periods (Day: 1, 7, 14, 21, 28 and 35). Post Hoc analysis was used to determine the changes obtained between the chosen duration intervals of sample re-analysis throughout the 35 days period of storage. Other descriptive statistics such as mean, median, standard deviation and graph was also done. A p-value of <0.05 was taken as being statically significant.
The study used fifteen (15) samples from replacement blood donors. The minimum and maximum ages of the participants were 18 and 47 years (
It was seen that the mean cell fragility (MCF) increased with the duration of storage. Days1 and 7 recorded the least mean value of 0.45 while Day 35 recorded the highest mean value of 0.56 ± 0.11 (
The MCFs extrapolated from
The PCVs decreased with increased storage duration. Day 1 recorded the highest mean PCV value of 38. 47 ± 3.11, while Day 35 recorded the least mean PCV value of 35.80 ± 3.23 (
Variables | Participants |
---|---|
Males | 13 (86.7%) |
Females | 2 (13.3%) |
Age (Mean) | (31.87 ± 6.99) |
Total | 15 |
Period of Storage | N | Mean | S.D. | 95% C.I. | Minimum | Maximum | |
---|---|---|---|---|---|---|---|
Day 1 | 15 | 0.45 | 0.04 | 0.43 | 0.47 | 0.36 | 0.5 |
Day 7 | 15 | 0.45 | 0.03 | 0.44 | 0.47 | 0.37 | 0.49 |
Day 14 | 15 | 0.50 | 0.05 | 0.47 | 0.52 | 0.37 | 0.54 |
Day 21 | 15 | 0.49 | 0.04 | 0.47 | 0.52 | 0.38 | 0.53 |
Day 28 | 15 | 0.51 | 0.06 | 0.48 | 0.55 | 0.38 | 0.68 |
Day 35 | 15 | 0.56 | 0.11 | 0.50 | 0.62 | 0.38 | 0.72 |
Total | 90 | 0.49 | 0.07 | 0.48 | 0.51 | 0.36 | 0.72 |
Duration of Storage | N | Mean | S.D. | F-Value | p-Value |
---|---|---|---|---|---|
Day 1 | 15 | 0.45 | 0.04 | ||
Day 7 | 15 | 0.45 | 0.03 | ||
Day 14 | 15 | 0.50 | 0.05 | ||
Day 21 | 15 | 0.49 | 0.04 | 6.682 | 0.001** |
Day 28 | 15 | 0.51 | 0.06 | ||
Day 35 | 15 | 0.56 | 0.11 |
**Significant (p-value ˂ 0.05).
Intervention | N | Mean | Std. Dev | 95% C.I. | Minimum | Maximum | |
---|---|---|---|---|---|---|---|
1 | 15 | 38.47 | 3.11 | 36.74 | 40.19 | 33 | 44 |
7 | 15 | 38.20 | 3.12 | 36.47 | 39.93 | 33 | 44 |
14 | 15 | 37.40 | 2.97 | 35.75 | 39.05 | 32 | 43 |
21 | 15 | 36.80 | 3.10 | 35.08 | 38.52 | 31 | 43 |
28 | 15 | 36.40 | 3.07 | 34.70 | 38.10 | 31 | 42 |
35 | 15 | 35.80 | 3.23 | 34.01 | 37.59 | 30 | 42 |
Total | 90 | 37.18 | 3.16 | 36.52 | 37.84 | 30 | 44 |
Intervention | N | Mean | Std. Deviation | F-Value | p-Value |
---|---|---|---|---|---|
1 | 15 | 38.47 | 3.11 | ||
7 | 15 | 38.20 | 3.12 | ||
14 | 15 | 37.40 | 2.97 | ||
21 | 15 | 36.80 | 3.10 | 1.684 | 0.147* |
28 | 15 | 36.40 | 3.07 | ||
35 | 15 | 35.80 | 3.23 | ||
Total | 90 | 37.18 | 3.16 |
*Not Significant (p-value > 0.05).
Photomicrographs from Leishman stained thin films indicate significant degenerative changes in the samples as the storage duration increased. Days 1 and 7 showed generally normocytic normochromic films, Day 14 showed fairly normochromatic with scanty echinocytic cells on the film. Day 21 showed few sphero-echinocytic cells with days 28 and 35 showing hypochromasia with rouleaux formation and marked sphero-echinocytic cells (
In order to achieve efficient therapeutic red cell transfusion with decreased risk of transfusion-associated reactions or events, the concept of blood storage merits scrutiny. This study, revealed that donor blood stored in a preservative (CPDA-1), at the prevailing blood bank conditions over a 35 day period at a temperature of 2˚C - 8˚C, produced significant changes in erythrocytes osmotic fragility, morphological integrity and marginally decreased changes in PCV.
Results obtained from osmotic fragility test, showed increased MCFs as storage duration progressed from day 1 to 35 as shown in
The trend observed in the study with respect to the RBCs membrane stability is consistent with studies by Tinmouth and Chin-Yee (2001) which showed that, during RBCs storage, metabolism slows down and, as RBCs
“sit” on the refrigerator shelf, metabolic by-products and cellular breakdown products accumulate in the enclosed system. This results in RBC shape change, haemolysis and loss of viability.
From the morphological findings, the film analysis on day 1 to 21 indicated varying but generally non-signi- ficant morphological changes (Normocytic Normochromic) as compared to the marked morphological chan- ges―(marked sphero-echnocytic with increased rouleaux formation) seen on films from days 28 and 35. These findings were consistent with our earlier study which posited that, EDTA anticoagulated blood samples stored over time could lead to marked changes in erythrocytes morphology as well as their osmotic fragility, as discussed by Antwi-Baffour et al. [
A limitation to this study was the fact that experiments were done in-vitro as compared with an in-vivo study where the stored RBCs would have been radio labeled and returned to circulation before measurements are taken. In an experiment by Hess and Thomas (2003), RBCs stored for six weeks had a mean recovery of 77% after 24 hours, when transfused into the same donor: This was described as an acceptable measure to determine RBCs functional ability upon prolonged storage. However, although the early stage of the process of RBC storage lesions may be reversible by metabolic rejuvenation of lost components like ATP, 2, 3-DPG and pH, the advanced stage characterized by shedding of membrane cannot be undone irrespective of the preservative used as discussed by Hess [
CPDA-1 is a universally accepted combined anticoagulant-nutrition solution capable of preserving blood for a maximum duration of 35 days. However the findings of this study showed progressive loss of membrane seen as increasing MCF as storage duration increased with most significant change seen on day 35. The authors therefore conclude that donor blood units stored for 28 days and beyond in CPDA-1 is likely to cause RBC defects that can affect their viability and may prevent practitioners from achieving the expected therapeutic benefit. Rather, it may increase patients risk to transfusion-associated adverse effects.
We are grateful to the management and staff of the Accra Area Blood Collection site of the Southern Area Blood Centre (SABC), Korle-Bu Teaching Hospital, Accra. Also, the haematology research laboratory of the University of Ghana Medical School (UGMS), College of Health Sciences, Korle-Bu.
We the author(s) declare(s) that there is no conflict of interest regarding the publication of this paper.
Samuel Antwi-Baffour,Samuel Appiah Danso,Jonathan Adjei,Ransford Kyeremeh,Michael Mark Addae, (2015) Prolonged Storage of Red Blood Cells for Transfusion in Citrate Phosphate Dextrose Adenine-1 Affects Their Viability. Open Access Library Journal,02,1-7. doi: 10.4236/oalib.1101908