Cryopreservation of red blood cell (RBC) concentrates is used for long-term storage of rare phenotyped RBCs to provide life-saving therapies to patients. To produce these components in Canada, blood manufacturers must demonstrate that the production method they use yields RBC units that meet regulatory guidelines for hemolysis, hemoglobin (Hgb) content, and RBC recovery. 1 The Canadian Blood Services (CBS) Rare Blood Program maintains a carefully selected inventory of cryopreserved RBCs with rare phenotypes, although these units are not represented in method validation or quality control data sets presently used to establish standard cryopreservation methods. This finding is problematic, since rare donor RBCs may have abnormal morphology and membrane structure that can affect component quality associated with the mechanical stresses of cryoprotectant addition/removal, freezing, thawing, and cell processing. Considering there is little evidence of the effect that rare phenotypes associated with membrane alterations have on deglycerolized RBC component quality, investigations to evaluate the downstream implications to patient care and cell banking practices would be useful.
An example of a rare RBC phenotype that should be cryogenically banked is the McLeod phenotype. This phenotype results from the absence of the Kx protein on the RBC membrane due to various missense and small to major nucleotide deletions of the
In addition to the challenge of understanding how RBCs having rare blood phenotypes can tolerate the cryopreservation process, blood manufacturers are also faced with the implications of long-term storage of these components. Cryopreserved RBCs can be stored for up to 10 years (30 years in Canada and other jurisdictions with medical approval), ensuring the availability of rare RBCs in acute clinical scenarios. However, given the pace of technological change, evaluating the impact of manufacturing changes must be ongoing to confirm that components frozen using older methods are compatible with new processing equipment and quality is maintained. The study presented herein evaluated the quality of cryopreserved donor RBC units stored for more than 20 years, including a previously identified McLeod donor unit. The RBC units were originally glycerolized and frozen using a manual method intended for deglycerolization on a semi-automated cell processor (COBE 2991; Terumo BCT, Lakewood, CO). CBS now uses a closed-system automated processor (ACP 215; Haemonetics, Boston, MA) for glycerolization and deglycerolization of RBCs requiring certain adaptations to be made to process the thawed RBCs evaluated in this study. Furthermore, because diagnostic testing has also improved since this rare McLeod donor unit was cryopreserved, this study also aims to evaluate whether samples from non-leukocyte–reduced cryopreserved RBCs can be used for phenotype confirmation and genotyping analysis. 3
To evaluate the component quality of the McLeod donor RBC unit, 3 additional cryopreserved RBC units were selected as controls. These units lacked RBC antigen phenotypes associated with known membrane abnormalities and had been cryopreserved in the same year (1993) and at the same site using the same method (high-glycerol method [40%] in Charter Medical [Winston-Salem, NC] containers) as that for the McLeod donor unit.
Twenty-four years after cryopreservation, each unit was thawed individually in a circulating 37°C water bath for up to 15 minutes. Before deglycerolization, the contents of the units were transferred to a 1-liter bag (OMAVSE6000XU; Macopharma, Mouvaux, France) followed by supernatant reduction. This reduction was performed by centrifuging the RBCs (HBB-6 Rotor, 2070
Each RBC unit was sampled at five different time points: 10 mL immediately post-thaw; 1 mL immediately post-deglycerolization; and 5 mL at 1, 7, and 14 days post-deglycerolization. Briefly, the RBC unit was mixed by gentle massage while inverting five times. A sampling site coupler (R4R1401; Fenwal) was inserted into one of the ports on the RBC bag. Using aseptic technique, an 18-gauge needle attached to a syringe was inserted through the coupler, and the desired sample volume was drawn slowly into the syringe. After removal of the needle, the sample was aliquoted dropwise into sample tubes for analysis.
Immediately post-thaw and post-deglycerolization, RBCs were analyzed for RBC hemolysis to determine whether there was any noticeable damage due to the length of storage or processing methods. Component quality was tested at 1, 7, and 14 days post-deglycerolization—testing for RBC hemolysis, supernatant potassium, Hgb content, adenosine triphosphate (ATP) concentration, RBC deformability, RBC indices, osmotic fragility, and morphology, all as previously described. 9–11
To obtain genomic material for this study, a method to collect samples for analysis from a thawed RBC component was developed. Immediately post-thaw, samples were prepared by centrifuging 9 mL of glycerolized RBCs at 2200
Deglycerolized RBCs (1 mL) from the McLeod donor was used for serologic phenotyping using direct agglutination testing with monoclonal antisera (Anti-K, Ortho 13129; Ortho Clinical Diagnostics, Raritan, NJ) and the indirect antiglobulin test (IAT) with another antisera (Anti-k [Anti-Cellano], Ortho 721030; Ortho Clinical Diagnostics). The CBS National Immunohematology Reference Laboratory confirmed the weak Kell system reactions using additional commercial antisera and unlicensed polyclonal donor anti-Jsb by the IAT.
Descriptive statistics were calculated for the control group using spreadsheet software (Excel 2016; Microsoft, Redmond, WA). The
Immediately post-thaw and before deglycerolization, hemolysis for the McLeod RBC (still in glycerol) was 5.16 percent. RBC hemolysis was within 1 SD of the mean of the control group (5.94 ± 3.87% [1 SD,
The CSA requirements for hematocrit (Hct) (≤80%) and Hgb content (≥35 g/RBC component in 100% of units tested and ≥40 g/RBC component in 90% of units tested) were met by all study RBCs at the accepted 24-hour expiration date for open-system processed units.
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The McLeod RBC unit had an Hct of 48 percent, which was similar to the control group at 48 ± 0.04 percent (1 SD,
Osmotic fragility was represented by the mean corpuscular fragility (MCF) of the RBCs, indicating the saline concentration at which 50 percent RBC hemolysis is achieved. Although the McLeod unit appears to have had increased MCF, the values obtained were within the SD of the control group at all storage time points (Table 1). This result was also in line with data previously reported in the literature by Kuypers et al., 13 who found that non-cryopreserved McLeod RBCs had similar osmotic fragility to non-McLeod RBCs (although it has been shown that RBCs without Kx do have decreased osmotic water permeability). At 24 hours post-deglycerolization, the McLeod unit demonstrated increased ATP and supernatant K+, and decreased mean cell volume (MCV), which were all greater than 2 SDs when compared with the control group at that time point (Table 1). However, this observation did not hold true for these parameters for the additional extended storage at days 7 and 14 post-deglycerolization.
Length of hypothermic storage post-deglycerolization | ||||||
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McLeod ( |
Control group ( |
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Parameter | 1 day | 7 days | 14 days | 1 day | 7 days | 14 days |
RBC hemolysis (%) Mean (SD) | 0.23 | 0.48* | 0.55 | 0.19 (0.02) | 0.31 (0.02) | 0.52 (0.10) |
ATP (µmol/g Hgb) Mean (SD) | 4.196* | 2.150 | 1.432 | 3.735 (0.223) | 2.617 (0.512) | 1.816 (0.349) |
MCF (g/L NaCl) † Mean (SD) | 6.104 | 5.986 | 5.918 | 5.541 (0.688) | 5.504 (0.656) | 5.473 (0.653) |
Supernatant K+ (µmol/L) Mean (SD) | 5.2* | 15.0 | 20.6 | 3.7 (0.5) | 13.2 (1.1) | 18.8 (1.6) |
MCV (fL) Mean (SD) | 85.1* | 84.9 | 86.3 | 104.1 (9.3) | 98.4 (8.7) | 98.4 (8.4) |
Indicates when the unit of McLeod red blood cells (RBCs) is >2 standard deviations (SDs) from the control group at the same storage time point.
MCF = mean corpuscular fragility (derived from osmotic fragility); MCF is presented as the concentration of sodium chloride (NaCl) in which 50 percent hemolysis is calculated to be achieved.
ATP = adenosine triphosphate; Hgb = hemoglobin; K+ = potassium; MCV = mean cell volume.
RBC deformability (Fig. 1) and morphology (Fig. 2) demonstrate distinct differences between the McLeod unit and the control group, likely due to the observed acanthocytosis in the McLeod unit. The KEI, a measure of RBC rigidity, was increased in the McLeod RBCs more than 2 SDs (21-SD difference) from the control group at 24 hours post-deglycerolization. This result maintains greater than 2-SD differences when compared with the controls at 14 days of hypothermic storage. Figure 2A demonstrates a marked decrease in the morphology index of the McLeod RBCs, which is influenced by the presence of acanthocytes shown in Figure 2B and slightly microcytic RBCs, as indicated by decreased MCV.
Serology results for the McLeod RBCs confirmed the historical phenotype kept on record at CBS for that donor. Typically, McLeod patients’ RBCs demonstrate weak reactions for the common Kell blood group system antigens (k, Kpb, Jsb).
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The donor tested in this study also demonstrated weak phenotype reactions for k (1+), Kpb (1+), and Jsb (3+) post-deglycerolization. In addition, RBC genotyping confirmed that the donor had the genes to express k, Kpb, and Jsb. Comparative genomic hybridization array provided genomic evidence that the male donor would have a McLeod phenotype, identifying a 4.69-Mb arr[hg19] Xp21.1p11.1(33,341,040-38,030,042) × 0 hemizygous deletion resulting in the complete loss of
Genotyping was also performed on the control units, indicating that the method developed to collect and isolate DNA was successful in producing samples for RBC genotyping for non-leukocyte–reduced cryopreserved RBCs. However, when this method was tested on leukocyte-reduced cryopreserved RBCs (
From this investigation, it is apparent that McLeod RBCs can be cryopreserved using a manual glycerolization procedure and an automated deglycerolization method with process adaptations. Like all “open system” unit modifications, RBCs deglycerolized in this manner would have a 24-hour post-deglycerolization expiration date. Nevertheless, this study provides evidence that RBCs of this phenotype can be stored for up to 14 days post-thaw and still meet quality standards for Hgb, Hct, and hemolysis. This finding suggests that RBCs collected from McLeod donors can be cryopreserved using a closed-system cell processor and have the benefit of extended hypothermic storage using current methods at CBS.
Although quality standards were met in this study, there were differences observed between the McLeod RBCs and the control units for the additional quality parameters (osmotic fragility, MCV, RBC deformability, and morphology). These differences are likely attributed to donor factors (McLeod phenotype) and not to cell processing and cryopreservation methods. 14 The most prominent deviation from the control data for the McLeod RBC was detected in RBC rigidity. Typically, as hypothermically stored RBCs age, their morphology demonstrates crenation and eventually becomes more spheroid, but their ability to deform remains unaffected. 15,16 The McLeod donor unit demonstrated a substantial increase in membrane rigidity post-deglycerolization. Because the control units exhibit KEI values lower than typical hypothermically stored RBCs at CBS (indicating that their membranes are less rigid likely due to the effects of cryopreservation), it can be concluded that acanthocytosis typical of McLeod phenotype RBCs causes increased RBC membrane rigidity and reduced deformability. 16 Additionally, it is not surprising that although the McLeod RBCs demonstrate decreases in deformability, they are still stable during hypothermic storage, which agrees with findings previously reported by Ballas et al., 14 in which McLeod RBCs demonstrated mechanical stability but reduced deformability.
This study was also successful in demonstrating a method to obtain genetic material from non-leukocyte– reduced cryopreserved RBCs for genotyping and molecular investigations. Although the method described requires further development, it shows proof of concept that genomic DNA for molecular analyses on units cryopreserved for 20 years can be obtained. Having this capability allows molecular confirmation of (and potentially further elucidation of) the phenotype of previously banked rare units. Further improvements of this method are required to reduce the potential for bacterial contamination when obtaining samples and to develop an alternative method to obtain genetic material from leukocyte-reduced units. It is unknown whether sufficient DNA can be obtained from leukocyte-reduced units if the sample processing volume was increased or whether leukocyte reduction itself degrades DNA, making sufficient DNA for molecular analyses unattainable.
Finally, it is important to draw attention again to the fact that cryopreserved RBCs with exceedingly rare and unusual phenotypes can be retained in storage for at least 30 years. Management of these units will require a greater understanding of how the quality of these units with rare phenotypes are affected by the cryopreservation process and how changes in manufacturing will influence these characteristics. Future studies should evaluate the quality of cryopreserved RBCs with phenotypes with known RBC membrane abnormalities such as those found in the MNS (MkMk), Diego (southeast Asian ovalocytosis), Gerbich (Yus and Leach), and Gil blood group systems. Not only will this information allow us to put these components to better use, but it will also allow us to improve management policies for this patient population, ensuring that adequate components are available for use.