Several changes were observed during storage of whole blood in blood bank. Some parameters were found to be decreased while some showed increased values. In some analytes no change was observed. RBC stored for a period of time at 4°C loose viability. Some may undergo spontaneous hemolysis while in storage; others lose the ability to survive in the recipient’s circulation following transfusion. Whole blood was stored in CPDA-1 bags. This anticoagulant
present in the collection bag is composed of Citrate (chelates ionized calcium that prevents coagulation), Dextrose (a source of energy for the red blood cells), phosphate containing anticoagulants (lower acidity than other anticoagulants without phosphate and have a higher concentration of 2,3 DPG and red cell phosphate) and Adenine (ATP content and post-transfusion viability of red cells regenerated by addition of adenine) [4
Prolonged contact of plasma with RBCs results in exchange of contents between plasma and red cells which leads to changes in analyte concentrations as well as dilution. Potassium levels increased within the period of 3 days and the increase continued subsequently. There was a significant rise in K+ concentration (p<0.001) from day 1 to day 21 of storage. Latham et al. and Bailey et al. also observed decline in concentration of plasma glucose
and bicarbonate and increase in potassium, lactate, LDH, ammonia, and hemoglobin concentration with storage [7
In blood bags the glucose concentration is limited and as glucose is utilized, there is a concomitant ATP (adenosine triphosphate) depletion and decrease in red cell viability [9
]. So, energy required for operating ATPase pump in RBCs decreases with time. Under physiological conditions, it pumps three sodium ions out of the cell for every two potassium ions pumped in. Inhibition of sodium pump leads to hyperkalemia
as observed in present study. Adias et al. also observed hyperkalemia in their study but they didn’t find any significant change in Na+ [10
]. Hemolysis also leads to hyperkalemia. During storage there is slow and constant release of potassium ions from cells into surrounding plasma
which may be responsible for dramatic increase in potassium in this study. At low temperature there occurs cold-induced blockade of ATP. This leads to extracellular release of K+ and entry of Na+ ions into RBCs [10
results in increased concentration of lactic acid
which may have caused a decrease in pH as observed in this study. Significant increase in LDH may also be because of this reason. Hemolysis results in release of LDH in plasma. LDH best reflects the degree of hemolysis by its increased activity [2
]. Castro et al. has also identified total hemoglobin concentration, bilirubin level, lactate dehydrogenase, and the arginine:ornithine ratio to be markers of hemolysis [12
At 0 day pH was within normal range which decreased to 6.75 at the end of 21st
day. Each 0.1 unit of pH change results in a 0.4 mmol/L change in the serum potassium level. Potassium levels are increased by acidosis and decreased by alkalosis [13
]. So, this also leads to hyperkalemia. This dramatic increase in potassium levels is dangerous for recipient’s body and more dangerous if transfused to a patient with severe kidney disease. This can be prevented by using potassium adsorption filters [11
] prior to transfusion or by transfusing blood with 0-3 day’s storage only.
Ionized calcium levels were found to be significantly increased during storage duration from 0 to 21st day, although the levels were very low (below normal range). This low level of ionized calcium can be explained because of citrate present in CPDA-1 bags, the role of which is to chelate calcium ions to prevent coagulation. Ionized calcium binds to negatively charged sites on protein molecules, competing with hydrogen ions for the same binding sites on albumin and other calcium-binding proteins
. This binding is pH dependent and alters the level of ionized calcium in the blood. Alkalosis, promotes increased protein binding, which decreases free calcium levels. Acidosis, on the other hand, decreases protein binding, resulting in increased free calcium levels [9
]. So, increase in levels with storage time may be because of acidosis.
Chloride levels increased initially up to 7 days thereafter a significant decrease was seen. Chloride, calcium and sodium are low molecular compounds and on storage, these enter erythrocytes
under the influence of their concentration gradients, namely 19:1 for sodium, 128:1 for calcium and 1.5:1 for chloride resulting in decreased levels in plasma [14
]. Phosphorus levels were found to be significantly increased (p<0.001) with storage duration, from 11.86 ± 0.62 mg/dL at 0 day to 16 ± 0.88 mg/dL at 21st day. Whole blood contains many phosphatases which cause hydrolysis of phosphate esters resulting in increase in inorganic phosphate levels [14
]. In addition CPDA also has phosphates in it resulting in increased levels [4
Bicarbonate levels were found to be significantly reduced at the end of study (p<0.001). This is usually due to buffering of excess lactate production by cells with anaerobic metabolism. Chloride-bicarbonate shift results in inward movement of chloride and outward movement of bicarbonate ions with subsequent buffering of H+
ion (from lactic acid) by bicarbonate with production of CO2
gas leading to increased chloride and decreased bicarbonate levels [15
]. The blood bags used for storage are made up of polyvinyl chloride (PVC) with plasticizer, di-(2-ethylhexyl) phthalate (DEHP). These bags are easily permeable to CO2
. Moderate hemolysis also decreases bicarbonate levels.
Significant changes were observed in AST levels (p<0.001) which can be because of hemolysis as observed in other studies. Koseoglu et al. studied the effects of hemolysis interferences on routine biochemistry parameters. Hemolysis interference affected LDH and AST almost at undetectable hemolysis by visual inspection (plasma hemoglobin <0.5 g/L) while clinically meaningful variations of potassium and total bilirubin were observed in moderately hemolyzed samples (hemoglobin >1 g/L). ALT, cholesterol, gamma glutamyltransferase (GGT), and inorganic phosphate concentrations were not interfered up to severely hemolyzed levels (hemoglobin: 2.5-4.5 g/L) [16
]. RBCs contain 20 fold as high concentration of AST as plasma, so, even mild hemolysis produces significant alterations in AST. We found 51% increase from day 0 to day 21 in AST levels. The disproportionate correlation with AST, and not ALT, is consistent with higher concentrations of AST than ALT in red blood cells released during intravascular hemolysis [17
Hemoglobin (Hb) strongly absorbs light at 540 nm. Hemolysis therefore increases absorption in this wavelength range and causes an apparent increase in the concentration of analytes measured in this range. Significant increase in protein concentration is because of addition and optical interference. Additional interference is because of intracellular leakage of total proteins. False elevated protein levels were also observed in study by Roman et al. [18
]. Quantitative estimation of total protein was done by biuret method in which absorbance is measured at 546 nm. Hb itself is a protein with same absorbance range [19
] but the method was said to be not significantly affected till Hb concentration of 250 mg/dL and we did not measure Hb concentration. So we cannot say whether the increase in total protein was because of addition or optical interference but both can account for this increase.