Effect of cryopreservation on a rare McLeod donor red blood cell concentrate

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, 2070g, ACE 2.88 × 10⁷, 18–24°C, Sorvall RC 3BP+; Thermo Fisher, Langenselbold, Germany) to pack the cells followed by supernatant removal using a manual plasma extractor (4R4414; Fenwal, Lake Zurich, IL). The unit was sterile docked (CompoDock; Fresenius Kabi, Bad Homburg, Germany) to a processing set (LN235; Haemonetics), which was pre-loaded into the closed-system cell processor (Haemoentics ACP 215), and automated deglycerolization was initiated using two different concentrations of saline solutions (12% sodium chloride 4B7874Q, and 0.2% dextrose and 0.9% sodium chloride processing solution 4B7878; Baxter, Mississauga, ON, Canada). Finally, the deglycerolized RBCs were suspended in AS-3 (462A; Haemonetics) before storage in a 1–6°C refrigerator for 14 days.

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 2200g for 10 minutes at room temperature. The resulting supernatant was removed and approximately 2 mL of sample from the buffy coat region (cell interface) where white blood cells should be present was extracted. Samples were frozen at less than –65°C until DNA extraction could be completed, which was performed using a commercial kit (QIAamp DNA Blood Mini Kit [51104]; Qiagen, Hilden, Germany). Extracted genomic DNA were shipped frozen to the Diagnostic Laboratories at Versiti BloodCenter of Wisconsin for molecular analysis. Real-time polymerase chain reaction (PCR)-fluorogenic 5´ nuclease TaqMan chemistry was used in a mass-scale nanofluidic open-array format (Smart-Chip Real-Time PCR System; Takara Bio USA, Mountain View, CA) to determine the following blood group antigens: M, N, S, s, U; C, E, c, e, V, Vs, hr^B, hr^S, Crawford; K, k, Kp(a/b), Js(a/b); Fy(a/b); Jk(a/b); Lu(a/b); Do(a/b), Hy, Jo(a); Di(a/b); Co(a/b); Cr(a); Yt(a); and Vel. ¹² To confirm the McLeod phenotype, a custom comparative genomic hybridization array analysis was performed that included overlapping exon hybridization probes in the region of XK (Oxford Gene Technology, Tarrytown, NY). Hybridization was analyzed on a scanner (Innopsys, Carbonne, France) using software (Mapix, version 7.3.0, and CytoSure, version 4.7.13; Oxford Gene Technology, Begbroke, UK). Healthy male and female genomic DNA served as normal controls.

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-Js^b by the IAT.

Descriptive statistics were calculated for the control group using spreadsheet software (Excel 2016; Microsoft, Redmond, WA). The in vitro results obtained from the McLeod unit were considered either increased or decreased if the result was greater or less than the control group by 2 standard deviations (SDs).

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, n = 3]), indicating that the McLeod donor RBC unit was not more susceptible to cellular damage because of mechanical stresses of glycerolization, freezing, and thawing than RBC units with a typical RBC antigen profile. At 24 hours post-deglycerolization, all units, including the McLeod RBC, met the Canadian Standards Association (CSA) standard for hemolysis (<0.8%). ¹ Hemolysis for all units tested consistently met the required standard after units underwent extended hypothermic storage up to 14 days, the maximum allowable storage time at CBS for deglycerolized RBCs, indicating that McLeod RBCs withstood the deglycerolization process and post-thaw storage in AS-3 as well as the control group.

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. ¹ The McLeod RBC unit had an Hct of 48 percent, which was similar to the control group at 48 ± 0.04 percent (1 SD, n = 3). Total Hgb content (a dose indicating parameter) for the McLeod RBC unit was 54.0 g/RBC component, which was also comparable to the 56.1 ± 5.2 g/RBC component (1 SD, n = 3) achieved for the control group.

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.

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 K_EI, 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.

Fig. 1.

Fig. 2.

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, Kp^b, Js^b). ^2–5 The donor tested in this study also demonstrated weak phenotype reactions for k (1+), Kp^b (1+), and Js^b (3+) post-deglycerolization. In addition, RBC genotyping confirmed that the donor had the genes to express k, Kp^b, and Js^b. 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 XK and CYBB. This donor was confirmed as having a McLeod phenotype, previously identified at CBS through serology and the presence of corresponding anti-Kx and anti-Km before the donation of at least 6 autologous units for cryopreservation to ensure supply in case of transfusion need.

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 (n = 3), the insufficient or fragmented DNA collected did not amplify, so no molecular results were obtained.