Impact of long-term deep freeze storage on human leukocytes viability & DNA extraction: Insights from a resource-limited biorepository

INTRODUCTION

The term ‘biobank’ introduced in 1996, refers to a specialized repository that systematically collects, processes, preserves, and distributes a variety of biospecimens under controlled conditions (e.g. Deoxyribonucleic acid [DNA], ribonucleic acid [RNA], body fluids, cells, tissues, etc.) with detailed clinical and demographic information, primarily for biomedical research.¹ Biobanks can be categorized as population-based or disease-oriented biobanks and may focus on specific biospecimens. There is a variety of biobanks, such as tissues, cells, organoids, liquids, imaging, or digital biobanks. To maintain sample integrity, specimens are often stored at extremely low temperatures, ranging from -20°C to -80°C, or cryopreserved in liquid nitrogen at -196°C.²

Blood is the most preferred biospecimen due to its ease of processing, wide availability, and extensive applications. Laboratory assessments of circulatory blood cell size and quantity are typically conducted via complete blood count (CBC) tests, which evaluate various parameters, including red blood cells (RBC), mean corpuscular volume (MCV), red cell distribution width (RDW), hematocrit, platelets, mean platelet volume (MPV), platelet distribution width (PDW), platelet crit, large-platelet cell ratio (LPCR), white blood cells (WBC), hemoglobin, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), lymphocytes, granulocytes, and mid-sized white blood cells (MID).³

A recent study found that at 4°C, hematological indices remain stable for up to eight days.⁴

WBCs or leukocytes, including basophils, eosinophils, lymphocytes, monocytes, and neutrophils, play a crucial role in the immune system. Their nuclei contain genetic material, making their viability essential during long-term storage. However, cryopreservation can reduce viability and functional integrity and trigger senescence over time.⁵ While numerous studies have evaluated the effects of low temperatures and long-term storage – with or without cryoprotectants - limited research exists on incubations exceeding one year, particularly in resource-limited settings like India.^6-9

MATERIAL AND METHODS

RESULTS

Univariate logistic regression of leukocyte counts with Other CBC indices

The univariate logistic regression revealed a strong positive correlation for granulocytes with r values of 0.99, 0.92, 0.85, and 0.91 at 0, 2, 4, and 18 months, respectively; lymphocytes with r values of 0.93, 0.98, and 0.99 at 2, 4, and 18 months, respectively; and MID cells with r values of 0.93, 0.97 and 0.96 at 1, 2 and 4 months, respectively. MPV was negatively associated with leukocyte counts at 0 months (r = -0.89) [Figure 1].

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Figure 1:

Correlation of leukocytes with other hematological parameters at different intervals. MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, MCV: Mean corpuscular volume, MPV: Mean platelet volume, PDW: Platelet distribution width, RBC: Red blood cells, RDW: Red cell distribution width, MID: Mid-sized white blood cells, LPCR: Large-platelet cell ratio.

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Analysis of extracted DNA from stored blood

The isolated DNA exhibited good quality (O.D. _260/280 = 1.83 ± 0.02) across all periods (p = 0.07) [Figure 2]. Although the quantity of DNA was satisfactory (202.82 ± 51.11 ng/μL), it significantly decreased with longer storage (p = 0.005). The variability in DNA quality across different time-periods can likely be attributed to the manual method of isolation, even though the same reagents were used in each set.

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Figure 2:

Quality estimation of isolated DNA at different intervals. DNA: Deoxyribonucleic acid.

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DISCUSSION

A meta-analysis indicated that blood samples can be reliably incubated at 4°C for metabolomics study.⁶ However, the viability of RBCs and their associated parameters – hemoglobin, hematocrit, RDW, MCV, MCH, and MCHC diminish due to altered RBC metabolism, increased oxidative stress, and membrane damage and has a shelf life of up to 42 days at 4°C.¹² In our study, RBC remained stable for 4 months and significantly decreased over time (17.6% vs 22.2%), while hemoglobin & hematocrit showed no change.

Platelets stored in 6% dimethyl sulfoxide can last 3 years at -80°C but in our research, platelet-related parameters such as platelet counts, MPV, and plateletcrit didn’t significantly change over time, although PDW increased by ∼16.6% without the cryoprotectant.⁹ LPCR remained stable for one month but showed a non-significant increase in long-term storage (p = 0.083) [Table 1].

A recent study from low-resource settings in Sri Lanka and Turkey found that most CBC parameters remained stable at 4°C, 25°C, and 33°C over 6, 24, and 48 hours, with platelets remaining unaffected for up to 4 days at 4°C.^7,8 However, WBC counts, including granulocytes, decreased drastically (54.6% and 91.6%, respectively), while lymphocytes increased by 22% non-significantly. This leukopenia and lymphocytosis may explain the lower DNA yield from long-term stored blood compared to fresh samples (p = 0.005). Lymphocytes, granulocytes, and MID cells significantly dropped, with a decrease in leukocyte counts after 2 months of storage (r > 0.9; p < 0.05) [Figure 1].

Factors such as slow freeze-thaw cycles, decreased phagocytic activity, reduced cytokine production, and the choice of cryoprotectants may lead to cell senescence and functional loss during prolonged storage of leukocytes.¹³

CONCLUSION

We conclude that for molecular biology research, blood samples can be stored for up to one year at -80°C without compromising the quantity of nucleic acid. Utilizing appropriate cryoprotectants at optimal concentrations could further enhance viability. Future studies involving larger cohorts, varying incubation temperatures, and extended storage durations will provide deeper insights and validate these findings.