European Journal of Cardiovascular Medicine

Blood transfusion remains a cornerstone of modern medical care, supporting a wide range of clinical conditions, from acute hemorrhage to chronic anemias. ^1,2 To ensure both availability and safety, donated blood is commonly preserved using anticoagulant-preservative solutions, among which Citrate-Phosphate-Dextrose-Adenine-1 (CPDA-1) has been widely employed. CPDA-1 facilitates the storage of whole blood for up to 35 days by maintaining red cell viability and providing a biochemical environment that slows cellular degradation.^3-5

Despite the advantages of CPDA-1, stored whole blood undergoes progressive hematological and biochemical alterations—commonly referred to as "storage lesions." These changes include shifts in red blood cell (RBC) morphology, reductions in cell membrane integrity, metabolic derangements, and decreased oxygen-carrying capacity. Such modifications can impact the clinical efficacy of transfused blood, especially in critically ill patients or those requiring massive transfusions.^6-9

Given the clinical significance of these storage-induced alterations, it becomes imperative to study the hematological dynamics associated with CPDA-1 preserved whole blood under real-world conditions. While several international studies have documented these changes, regional data from Indian healthcare settings remain limited, especially in resource-constrained environments.

This study, conducted at Relace Anugrah Narayan Magadh Medical College, Gaya aims to evaluate and document the hematological changes occurring in whole blood stored with CPDA-1 over the standard storage period. By understanding the extent and nature of these changes, this research seeks to contribute valuable insights toward optimizing transfusion practices and ensuring better patient outcomes in our clinical settings.

Study Design and Duration

This observational, prospective study was conducted in the Department of Pathology, Anugrah Narayan Magadh Medical College, Gaya, over a period of 20 months—from August 2018 to March 2020. The study was approved by the Institutional Ethics Committee, and all procedures adhered to standard ethical guidelines.

Sample Collection

A total of 300 units of whole blood were collected from healthy voluntary donors who met the eligibility criteria as per the standards set by the National Blood Transfusion Council (NBTC). Each unit of blood (approximately 450 mL ± 10%) was collected aseptically into standard blood bags containing Citrate-Phosphate-Dextrose-Adenine-1 (CPDA-1) as the anticoagulant-preservative solution. Each blood bag contained 63 mL of CPDA-1 solution, ensuring an optimal ratio for preservation.

Storage Conditions

After collection, all blood units were stored at a controlled temperature of 2–6°C in a monitored blood bank refrigerator. Each unit was labeled and tracked to ensure proper identification and maintenance of storage records.

Sampling Intervals

Hematological parameters were analyzed at five predefined intervals:

• Day 0 (baseline)
• Day 7
• Day 14
• Day 21
• Day 28

At each interval, 5 mL of blood was aseptically withdrawn from the blood bag using a sterile sampling port, following strict aseptic precautions to avoid contamination or compromise of the blood unit.

Hematological Analysis

The following hematological parameters were evaluated at each time point using an automated hematology analyzer (make and model, if available):

• Hemoglobin concentration (Hb)
• Hematocrit (Hct)
• Total red blood cell (RBC) count
• Mean corpuscular volume (MCV)
• Mean corpuscular hemoglobin (MCH)
• Mean corpuscular hemoglobin concentration (MCHC)
• White blood cell (WBC) count
• Platelet count

In addition to the automated analysis, peripheral blood smears were prepared and stained using Leishman stain for morphological evaluation of red blood cells.

Statistical Analysis

Data were entered into Microsoft Excel and analyzed using SPSS software (version 25). Descriptive statistics were used to summarize the data. Repeated measures ANOVA and paired t-tests were used to assess statistically significant changes in hematological parameters over the storage period. A p-value of less than 0.05 was considered statistically significant.

The following tables present the results of the hematological analysis conducted on 300 units of CPDA-1 preserved whole blood stored at 2–6°C over a 28-day period. The parameters were measured at five intervals (Day 0, Day 7, Day 14, Day 21, and Day 28). All values are expressed as mean ± standard deviation (SD). Statistical significance was assessed using repeated measures ANOVA, with a p-value < 0.05 considered significant. Morphological observations from peripheral blood smears are also summarized.

Table 1: Hemoglobin Concentration (Hb) and Hematocrit (Hct)

Table 2 presents the changes in red blood cell (RBC) count and mean corpuscular volume (MCV). The RBC count demonstrated a steady but statistically significant decrease starting from Day 14, corresponding to gradual red cell loss during storage. In contrast, MCV showed a consistent and significant increase over time, indicating cellular swelling. This increase in volume is often attributed to sodium and water influx resulting from impaired membrane pump function due to ATP depletion. These morphological and volumetric changes are classic features of storage lesions, suggesting that even structurally intact RBCs may undergo physiological alterations that compromise their post-transfusion performance.

Table 2: Red Blood Cell (RBC) Count and Mean Corpuscular Volume (MCV)

Table 3 details alterations in mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Both indices declined progressively, with significant changes beginning on Day 14 and becoming more pronounced by Day 28. The fall in MCH signifies a reduction in the average hemoglobin content per red cell, while the decline in MCHC indicates a relative dilution of hemoglobin within the cell volume, often due to cell swelling or partial hemolysis. These shifts point toward a deterioration in red cell quality, reducing their functional capacity to transport and deliver oxygen efficiently once transfused.

Table 3: Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC)

Table 4 highlights a marked reduction in white blood cell (WBC) count across the 28-day period, with statistically significant decreases observed from as early as Day 7 (p < 0.001 at all subsequent intervals). WBCs are particularly susceptible to apoptosis and enzymatic breakdown during storage, especially in the absence of leukoreduction. Their degradation can release bioactive substances and pro-inflammatory mediators, potentially increasing the risk of transfusion-related adverse effects such as febrile non-hemolytic transfusion reactions. The sharp decline in WBC count reflects the fragility of these cells in cold storage and underlines the importance of leukocyte filtration when clinically indicated.

Table 4: White Blood Cell (WBC) Count

Table 5 reveals a rapid and significant decline in platelet count, starting from Day 7 and continuing steeply through Day 28. By the end of the storage period, platelet counts had decreased by nearly 70% from the baseline value. Platelets are highly sensitive to cold temperatures and mechanical stress, both of which contribute to irreversible activation, clumping, and eventual destruction during storage. These findings emphasize that stored whole blood should not be used as a source of viable platelets beyond a few days post-collection, and platelet transfusion therapy should rely on specially prepared platelet concentrates.

Table 5: Platelet Count

Table 6 summarizes the morphological changes observed in red blood cells through peripheral smear analysis over time. On Day 0, cells appeared normal with a predominant discoid shape and minimal variation. However, morphological deterioration began as early as Day 7, with increasing numbers of echinocytes and signs of anisocytosis. By Day 14, spherocytes became evident, and by Day 28, there was extensive morphological distortion with severe anisocytosis, a high proportion of echinocytes (up to 40%), and a significant number of spherocytes (up to 20%). These structural changes are indicative of irreversible membrane damage and decreased red cell deformability, both of which negatively impact the survival and functionality of transfused RBCs in the recipient's circulation.

Table 6: Morphological Changes in Red Blood Cells (Peripheral Smear Observations)

The present study meticulously evaluated the hematological changes in 300 units of CPDA-1 preserved whole blood stored under standard blood bank conditions (2–6°C) over a 28-day period. A comprehensive set of hematological parameters, including red cell indices, leukocyte and platelet counts, and peripheral smear morphology, were monitored at regular weekly intervals. The findings not only reaffirm the known concept of "storage lesions" but also provide region-specific data relevant to transfusion practices in Indian healthcare settings, particularly in resource-limited environments.

The observed decline in hemoglobin concentration (Hb) and hematocrit (Hct) values, particularly after Day 14 (Table 1), aligns with the established pathophysiology of red cell degradation during storage. This decline, though initially minimal, becomes statistically and clinically significant by Day 21 and Day 28. The reduction can be attributed to both hemolysis—caused by oxidative damage and loss of membrane integrity—and microvesiculation, which reduces red cell volume and surface area. These changes lower the oxygen-carrying capacity of stored blood, potentially affecting its effectiveness in critically ill or hypoxic patients. Additionally, the release of free hemoglobin into plasma, though not quantified in this study, may contribute to pro-inflammatory responses and nitric oxide scavenging, both of which can impact vascular tone and immune response post-transfusion.^10,11

The progressive fall in RBC count and concurrent rise in mean corpuscular volume (MCV) (Table 2) suggest an intriguing interplay between red cell destruction and morphological adaptation during storage. While red cells gradually lyse due to mechanical fragility and metabolic exhaustion, surviving cells tend to swell due to intracellular accumulation of sodium and water—a direct result of the failure of ATP-dependent membrane pumps. This rise in MCV, although seemingly compensatory, indicates declining membrane integrity and cytoskeletal dysfunction. From a clinical perspective, such enlarged and rigid erythrocytes are less deformable and more prone to splenic sequestration and clearance, reducing their in vivo survival after transfusion.

Mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), both of which exhibited a downward trend from Day 14 onwards (Table 3), further illustrate the internal changes in red cell composition during storage. The reduction in MCH reflects a decreased hemoglobin load per red cell, potentially due to hemoglobin oxidation or leakage. Simultaneously, the drop in MCHC points toward intracellular water retention and dilutional effects. These indices, while often underemphasized in routine blood bank settings, are crucial indicators of red cell functionality, and their alteration implies that stored red cells progressively lose their optimal oxygen-delivery efficiency.

The findings related to white blood cell (WBC) count (Table 4) are particularly noteworthy. A steep and statistically significant reduction in WBCs was observed as early as Day 7, continuing sharply through Day 28. Leukocytes, particularly granulocytes, are metabolically active and prone to apoptosis and autolysis during storage. The degradation of WBCs can release reactive oxygen species (ROS), cytokines, and other bioactive molecules into the plasma, increasing the risk of febrile non-hemolytic transfusion reactions and contributing to transfusion-related immunomodulation (TRIM). In the absence of leukoreduction—which was not employed in this study—these risks remain clinically relevant, particularly for multiply transfused patients, immunosuppressed individuals, and neonates. This underscores the importance of considering pre-storage leukoreduction, especially in institutions with high transfusion volumes.

Platelet counts, as shown in Table 5, declined rapidly and consistently from Day 7 onwards, with a drastic reduction by Day 28. Platelets are extremely sensitive to storage conditions and undergo irreversible shape changes, degranulation, and membrane damage when stored in cold temperatures. This renders them non-functional within a few days. Despite their presence in whole blood, platelets stored in CPDA-1 bags at 2–6°C rapidly lose hemostatic capability. Hence, stored whole blood beyond the first few days should not be considered a viable source of platelet support. Clinical management of thrombocytopenic patients must instead rely on dedicated platelet concentrates stored at 20–24°C with agitation, which preserve platelet function more effectively.

Morphological changes observed on peripheral blood smears (Table 6) provide visual confirmation of the underlying cellular alterations associated with storage lesions. The gradual emergence and dominance of echinocytes, followed by the appearance of spherocytes and increasing anisocytosis, reflect the cumulative effects of membrane lipid peroxidation, protein denaturation, and cytoskeletal disorganization. Echinocyte formation is an early, often reversible, indicator of membrane stress, while spherocyte formation indicates irreversible membrane loss and cytoskeletal collapse. By Day 28, the predominance of these aberrant forms highlights the diminished functional quality of stored red cells, particularly their impaired deformability, which directly affects their ability to traverse the microcirculation. This in turn may reduce the overall efficacy of transfusion, particularly in patients with high perfusion demands or compromised vascular systems.

From a clinical perspective, the results of this study reinforce the concept that although CPDA-1 allows storage of whole blood for up to 35 days, the hematological and morphological quality of the blood begins to deteriorate significantly after the second week. The most pronounced changes occur between Day 14 and Day 28, marking this period as a critical window for quality reassessment.^11-12 For patients requiring optimal red cell function—such as those with cardiac, respiratory, or critical surgical conditions—fresher blood (ideally less than 14 days old) may be more beneficial. Additionally, these findings highlight the limitations of using unseparated whole blood in transfusion practice and emphasize the advantages of component therapy, wherein each blood component is stored and used under optimized conditions tailored to its stability and functional duration.

Finally, this study adds to the growing body of literature on blood storage lesions but is particularly relevant for Indian settings, where whole blood transfusion is still widely practiced, and resources for advanced component separation or leukoreduction may not always be available. The findings call for heightened awareness among clinicians and transfusion services regarding the temporal quality degradation of stored blood and the need for protocols to ensure appropriate blood unit selection based on patient needs.

This study comprehensively demonstrates that CPDA-1 preserved whole blood undergoes significant hematological and morphological alterations during storage at 2–6°C, with pronounced changes observed after the second week. Key hematological indices—including hemoglobin, hematocrit, RBC count, and platelet count—showed progressive declines, while mean corpuscular volume increased, indicating red cell swelling and reduced viability. Simultaneously, leukocyte degradation and substantial morphological abnormalities, such as echinocytosis and spherocytosis, were evident by Day 28, confirming the onset and progression of storage lesions. These changes collectively compromise the functional integrity of stored blood and highlight the reduced efficacy of transfusions involving older blood units. The findings emphasize the clinical importance of transfusing fresher blood—preferably within 14 days—particularly in high-risk patient populations, and support the adoption of component therapy and leukoreduction strategies to optimize transfusion safety and outcomes. Furthermore, this region-specific data provides valuable insights for improving transfusion practices in resource-limited Indian healthcare settings, where whole blood transfusion remains prevalent.

1. World Health Organization. Blood safety and availability [Internet]. Geneva: WHO; 2023 [cited 2025 Apr 16]. Available from: https://www.who.int/news-room/fact-sheets/detail/blood-safety-and-availability
2. World Health Organization. Blood transfusion safety [Internet]. Geneva: WHO; 2023 [cited 2025 Apr 16]. Available from: https://www.who.int/health-topics/blood-transfusion-safety#tab=tab_1
3. Beutler E, West C. The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1). Blood. 1979;54(1):280–4.
4. Antwi-Baffour S, Danso S, Adjei J, Kyeremeh R, Addae M. Prolonged storage of red blood cells for transfusion in citrate phosphate dextrose adenine-1 affects their viability. Open Access Libr J. 2015;2:1–7.
5. Latham JT Jr, Bove JR, Weirich FL. Chemical and hematologic changes in stored CPDA-1 blood. Transfusion. 1982;22(2):158–9.
6. Zubair AC. Clinical impact of blood storage lesions. Am J Hematol. 2009;85(2):117–22.
7. van de Watering LMG. Red cell storage and prognosis. Vox Sang. 2011;100(1):36–45.
8. Acker JP, Marks DC, Sheffield WP. Quality assessment of established and emerging blood components for transfusion. J Blood Transfus. 2016;2016:4860284.
9. Cabrales P, Intaglietta M. Blood substitutes: evolution from noncarrying to oxygen- and gas-carrying fluids. ASAIO J. 2013;59(4):337–54.
10. Schaer DJ, Buehler PW. Cell-free hemoglobin and its scavenger proteins: new disease models leading the way to targeted therapies. Cold Spring Harb Perspect Med. 2013;3(6):a013433.
11. Meledeo MA, Peltier GC, McIntosh CS, Bynum JA, Cap AP. Optimizing whole blood storage: hemostatic function of 35-day stored product in CPD, CP2D, and CPDA-1 anticoagulants. Transfusion. 2019;59(S2):1549–59.
12. Sivertsen J, Braathen H, Lunde THF, Kristoffersen EK, Hervig T, Strandenes G, et al. Cold-stored leukoreduced CPDA-1 whole blood: in vitro quality and hemostatic properties. Transfusion. 2020;60(5):1042–9.