Changes in hematological and biochemical parameters and rate of hemolysis during the storage of packed RBC units: A prospective study

INTRODUCTION

The preservation of human blood began during the First World War. The first anticoagulant preservative citrate-dextrose was introduced by Rous and Turner in 1916.^[1] The next significant development was the introduction of acid citrate dextrose (ACD) solution by Loutit and Mollison in 1943. This was followed by the development of citrate-phosphate dextrose (CPD) by Gibson in 1957 and CPD with adenine (CPDA) in 1978. The adenine content in CPDA is used for the synthesis of adenosine triphosphate (ATP) in stored blood, which helps enhance the viability of red blood cells (RBCs). Additive solutions are preservative solutions that are added to the RBC after removal of the plasma with or without platelet. The first RBC additive solution, saline-adenine-glucose (SAG) was developed by European researchers in the late 1970’s. Soon after, in 1981, the same researchers added mannitol to help protect RBC membrane and reduce hemolysis, enabling refrigerated storage for up to 6 weeks. At present, RBCs stored at 2–6°C in ACD, CPD, or citrate -phosphate-double dextrose (CP2D) can be preserved for up to 21 days, while those stored in CPDA can last for 35 days. However, with additive solutions such as SAGmannitol (SAGM), additive solution (AS)-1, AS-3, and AS-5, RBCs can be stored for up to 42 days.

The role of blood preservation is to maintain the viability and functional capacity of blood components under storage conditions. Because blood must be stored from the time of donation until the time of transfusion, the viability of RBCs must be maintained during storage time as well. The cold storage of packed RBCs (PRBCs) is associated with a gradual increase in the percentage of cells with impaired functionality.^[2-4] Recent studies have demonstrated that there may be a correlation between the number of days of cold storage of PRBCs and adverse outcomes, including mortality among transfused patients, presumably due to structural or functional changes in RBCs that occur during storage. This phenomenon has been referred to as storage lesions. These alterations can be extensive and are primarily classified into two broad categories: Biochemical and HematologicalHematological. Biochemical parameters such as pH, lactic acid, glucose consumption, ATP level, 2,3-DPG level, sodium, and potassium level were affected in cold storage. The structural and functional change of RBC affects the hematologicalhematological parameters during storage. The hemoglobinhemoglobin and hematocrit parameters are mostly affected and a variation in other parameters such as mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW) is also noticed in certain studies. Storage of red cells causes a progressive increase in hemolysis. Hemolysis during blood collection and storage is the most severe manifestation of the RBC storage lesion. The extent of hemolysishemolysis in blood components is an important indicator of cellular integrity and a quality parameter.

In India, the blood is stored in blood centers governed and regulated by the Central Drugs Standard Control Organization which is headed by the Drugs Controller General. The rules and regulations related to the storage of blood are followed as per the Drugs and Cosmetics Act of 1940 and its Rules of 1945, along with amendments made from time to time. The licensing and monitoring of blood banks are the responsibility of the Drug Controller General of India.^[5]

The study aimed to evaluate the changes in hematological and biochemical parameters in PRBC units stored in Blood Bank refrigerator conditions. The objectives were as follows:

1. To compare the hematological and biochemical parameters among PRBC units stored in CPDA anticoagulant and PRBC units stored in SAGM preservative solution

2. To measure and compare the rate of hemolysis of blood units under successive days of storage

3. To measure and compare the effect of pH in storage conditions Objective.

MATERIALS AND METHODS

This observational prospective study was conducted for a period of 2 years from October 2018 to September 2020 in the Department of Transfusion Medicine, Srirama Chandra Bhanja Medical College and Hospital, Cuttack, a tertiary care center of Odisha, India. The study was approved by the Institutional Ethical Committee (444/dt. October 14, 2020).

RESULTS

The packed red cell units were divided into two groups upon the anticoagulants used – CPDA and SAGM. The majority of donors were from the age group 25–34 years. The mean age group of donors of CPDA units and SAGM units collected from donors were 30.34 ± 6.12 and 29.28 ± 5.5, respectively. Among blood donors, 84 units were from male donors and 16 units from female donors. Among blood group distribution, the highest number of donors was found from Group O (48%) and the number of donors from other blood groups was B (29%), A (16%), and AB (7%). All donors were from Rh-positive groups.

The mean hemoglobin concentration levels were 17.0 ± 0.77 in CPDA units whereas 17.6 ± 0.92 (g/dL) in SAGM units on the day of preparation. There was a decrease in the mean hemoglobin levels in both groups which was statistically significant (P < 0.05) from day 0 to day 35. The observed mean hematocrit (%) value on different days of storage, i.e., on days 0, 7, 14, 21, 28, and 35 for CPDA units and SAGM units is shown in the Table 1. The decrease was statistically significant from day 7 to day 35 (P < 0.05).

Table 2 shows the mean value of MCV, mean concentration of hemoglobin (MCH), and MCHC. In SAGM units, the mean MCV showed a gradual increase from day 0 to day 35 but in CPDA units, the mean MCV increased from day 7 onward till day 35 and it was statistically significant from day 21 to day 35. There was a gradual decrease in mean MCH in both CPDA and SAGM units. In CPDA units, the mean MCV decreased from day 7 onward but in SAGM units, the mean MCV value decreased from day 0. The changes were statistically significant from day 0 to day 35 (P < 0.05). No significant change was observed in mean MCHC in both CPDA and SAGM units up to day 28 but there was a minimal decrease in mean MCHC level in CPDA units as compared to SAGM units in the 5^th week of storage. From day 0 to day 35, the mean MCHC value was statistically significant between the two groups (P < 0.05).

Table 3 shows the comparison of the mean value of RBC count and percentage of RDW among CPDA and SAGM groups on the day of preparation (day 0) to Storage day 35. The mean RBC count in CPDA groups markedly decreased in the 1^st week but increased in subsequent weeks till the end of storage but in SAGM groups, it increased continuously during the study period. At the end of storage, SAGM showed a higher RBC count in comparison to the CPDA group. The RDW percentage increased continuously in both in CPDA as well as SAGM groups during the study period. The mean percentage of RDW was nearly equal in both groups.

The biochemical parameters such as supernatant sodium and potassium are shown in Table 4. On the day of preparation, the mean supernatant sodium concentration was 143.98 ± 2.65 (mmol/L) in CPDA and 143.7 ± 2.13 in SAGM. During the progressive storage period, there was a continuous decrease in mean supernatant sodium levels in both CPDA and SAGM units. On day 35, SAGM units showed slightly higher value as compared to CPDA units. The changes were statistically significant from day 0 to day 35 (P = 0.000). In the CPDA group, the mean supernatant potassium on the day of preparation and the day 35 of storage were 4.33 ± 0.44 (mmol/L) and 33.05 ± 1.27 whereas in SAGM groups were 4.23 ± 0.49 and 32.57 ± 1.01, respectively. The mean supernatant potassium level increased markedly on day 35 as compared to day 0 in both CPDA and SAGM units. The changes were statistically significant from day 7 to day 35 (P = 0.000). The mean PH gradually declined but the hemolysis percentage increased progressive storage period as shown in Table 5.

DISCUSSION

Hemoglobin and hematocrit were two important parameters in the storage of blood. In our study, the hemoglobin level decreased in both groups, which is statistically significant from day 0 to day 35 (P < 0.05). The decrement in hemoglobin was started from the 1^st week onward in the CPDA group but well maintained in the SAGM group. Our study showed similar results to Aninagyei et al.^[6] who studied and found the consecutive reduction of hemoglobin levels in all successive weekly estimations compared to baseline values. Our study was contradictory to the study of Shirvastava and Dutta^[2] and Chhabra et al.,^[7] where Shirvastava and Dutta^[2] showed increased mean hemoglobin from day 1 to day 35, and Chhabra et al.^[7] observed that their study was statistically non-significant in hemoglobin level. This indicated that RBC was well preserved in SAGM as compared to CPDA.

The mean hematocrit level showed a gradual and steady fall during the storage period in both groups. On the 1^st week of storage, the hematocrit level fell more in both groups as compared to other weeks of storage. The results were similar to Bensinger et al.^[8] The cause of the decrease in Hematocrit can be attributed to erythrocyte hemolysis as per Bailey and Bove.^[9] At the end of storage, SAGM units showed higher hematocrit levels than CPDA units. This may be due to more adenine levels in SAGM units which generated more ATP in our study. The decrease in hematocrit level in our study was statistically insignificant on the day of preparation but significant on the rest of the days of storage. Our results were in accordance with the study by Shirvastava and Dutta^[2] but contradicted the result of Chhabra et al.,^[7] who observed that the result was statistically insignificant.

In complete blood count, MCV is an important parameter to identify the underlying cause of anemia. Although there was a gradual increase in mean MCV value in both groups throughout the study period, it was within the normal range. The result of our study was statistically insignificant. No correlation was observed between MCV and storage period or additive solution by Nakao et al.^[10] but Batham and Nayak^[11] observed that the mean MCV value was highly statistically significant. Although the mean MCH value decreased in both groups, it is maintained the normal range throughout the storage period and also statistically significant. Both MCH and MCHC values did not change in both groups during the storage period. In SAGM units, these were slightly better as compared to CPDA units in our study. Our study was similar to the results and statistical analysis of Adias et al.^[12] but contradictory to the study by Shirvastava and Dutta,^[2] who observed an increased MCH value at 35 days from baseline. The mean RBC count in CPDA units markedly decreased on day 7, but no significant difference was noted on day 14, 21, 28, and 35 as compared to day 0, whereas it increased continuously from day 0 to day 35 in SAGM units. The gradual increase of mean RBC count on storage was also found in other studies conducted by Tayer et al.^[13] During the storage period, the mean RBC counts were within the normal range in both groups, and the differences between the two groups were found to be statistically significant from day 0 to day 35. The mean value of RDW (%) on day 0 for the CPDA blood bag was 13.10 ± 0.48 and for the SAGM blood bag was 13.37 ± 0.47, which increased gradually, and finally, at 35 days, the value was 16.10 ± 0.99 in CPDA units and 15.90 ± 0.71in SAGM units. The progressive increase in RDW was noticed in our study, which agreed with the study of Cohl et al.^[14] The change in RDW between the two groups was statistically significant from day 0 to 35 (P < 0.05).

The present study showed that the percentage of hemolysis was 0.53 ± 0.08 in SAGM units and 0.68 ± 0.13 in CPDA units during the 5^th week of storage. This was statistically significant on days 0, 14, 21, 28, and 35 (P < 0.05). This result followed the guidelines dictated by the Council of Europe^[15] and the US food and drugs administration (FDA).^[16] As per the Council of Europe, the hemolysis percentage in all RBC units should be below 0.8%. Moreover, US FDA guidelines suggested that hemolysis concentration should be <1% at 95% of RBC units. The hemolysis percentage suggests that both units were safely transfused for up to 5 weeks. Compared to the methods of detection of hemolysis, our study was in accordance with the findings of Sawant et al.^[17] studies, who evaluated hemolysis using the tetramethylbenzidine method. Again, SAGM units had a lower hemolysis percentage than CPDA units at 35 days of storage which may be due to higher concentrations of adenine, glucose, and mannitol that prevent hemolysis. This study was very similar to Zimmermann et al.^[18] study, which studied the effect of adenine, mannitol, and glucose on hemolysis of stored blood and found lower hemolysis in ADSOL additives than those stored in SAGM.

The sodium concentration was nearly equal in both CPDA and SAGM units on day 1 of storage and gradually decreased up to 35 days. The loss of sodium was slightly higher in CPDA units as compared to SAGM units in the 5^th week of storage. The differences in the value of mean sodium are found to be statistically significant. A study by Jobes et al.^[19] showed a similar finding to the sodium value found in our study, where a declining level of sodium was reported during the storage of blood. The average weekly changes were likely to move sodium concentration in stored blood to a level outside the normal reference range. During storage, suppression of the Na-K ATPase pump caused leakage of potassium into the extracellular fluid and pulling of sodium into intracellular fluid, as a result of which sodium level decreases. The decreased sodium level caused possibly adverse clinical effects on recipients of such units, which increased the chance of edema in those recipients, as collaborated by Metheny,^[20] especially in patients with low sodium intake and those with previous diarrhea.

Our study showed that the mean supernatant potassium level was equal in both groups (4.33 ± 0.44 in CPDA vs. 4.23 ± 0.49 in SAGM). There was nearly an eight-fold increase in the potassium level from the baseline value in both preservative groups. This indicates that the preservative had no role in preventing the leakage of potassium during storage. A similar result was observed by Adias et al.,^[12] who found that the potassium level increased within seven days and continued subsequently. Mukherjee et al.^[21] observed a significant rise in potassium from day 0 to day 21, which was in accordance with our study. A study by Opoku-Okrah et al.^[22] and Murthy et al.^[23] demonstrated that there was an increase in potassium concentration from day 10 onward. At the end of the study period, our study showed a lesser rise in the potassium level as compared to D’Alessandro et al.^[24] where the potassium level rose to 50 meq/L. Our study was also similar to Verma et al.^[25] study in statistical analysis, where the rise of potassium showed a statistically significant (P < 0.05) value from day 7 onward. This dramatic increase in potassium level may be due to the failure of the Na-K ATPase pump during the storage period, as hypothesized by Wallas et al.^[1] This increase was dangerous for the recipient’s body and more dangerous if transfused to a patient with severe renal disease. This can be prevented using potassium adsorption filters before transfusion or by transfusing blood with early days of storage only.^[26]

The mean pH value dropped to 6.54 ± 0.06 in 35 days of storage from a baseline value of 7.2 ± 0 in CPDA units and 6.51 ± 0.04 from 7.19 ± 0.02 in SAGM units. The proper pH level is maintained in the blood units to maintain the physiological function of RBC units. A decrease in pH hampers the oxygen-carrying and oxygen-delivering capacity of hemoglobin at the tissue level. A similar result by Almoshary et al.^[27] observed on day 0 and at the end of day 28. In standard blood bank refrigerators, the pH level declines in stored PRBC units due to a rise in lactate level from the anerobic metabolism of glucose. Gradually fall of pH of RBC units stored in both preservatives in our study period. This result was in accordance with Achor and Brown,^[28] who compared pH in CPDA blood among traditional refrigerators and standard blood bank refrigerators and also similar to studies done by Huisman et al.^[29] and Burger et al.^[30]

CONCLUSIONS

Of the two types of preservatives used, CPDA and SAGM, the hematological and biochemical changes that were observed are similar without any marked differences, which suggests that both preservatives are useful. Therefore, PRBCs stored in CPDA and SAGM can be kept till the end of the storage period with better blood bank inventory control and using special techniques such as leukofilter and potassium adsorption filter before transfusion.