Hemolysis Pathways during Storage of Erythrocytes and Inter-Donor Variability in Erythrocyte Morphology

Abstract

Background: Red blood cells (RBCs) stored for transfusions can lyse over the course of the storage period. The lysis is traditionally assumed to occur via the formation of spiculated echinocyte forms, so that cells that appear smoother are assumed to have better storage quality. We investigate this hypothesis by comparing the morphological distribution to the hemolysis for samples from different donors.

Methods: Red cell concentrates were obtained from a regional blood bank quality control laboratory. Out of 636 units processed by the laboratory, we obtained 26 high hemolysis units and 24 low hemolysis units for assessment of RBC morphology. The association between the morphology and the hemolysis was tested with the Wilcoxon-Mann-Whitney U test.

Results: Samples with high stomatocyte counts (p = 0.0012) were associated with increased hemolysis, implying that cells can lyse via the formation of stomatocytes.

Conclusion: RBCs can lyse without significant echinocyte formation. Lower degrees of spiculation are not a good indicator of low hemolysis when RBCs from different donors are compared.

Keywords: Erythrocytes; Hemoglobin; Lipid bilayers; MALDI; Storage.

Fig. 1

Formation of stomatocytes and echinocytes in association with changes in the relative areas of the inner and outer leaflets of the RBC membrane, showing formation of a stomatocyte as the inner leaflet expands relative to the outer leaflet (a), a discocyte, when the two leaflets are at the preferred area difference to give the minimum membrane energy (b), and formation of the convex bumps of echinocytes when the outer leaflet expands relative to the inner leaflet (c).

Fig. 2

Cell shapes and the corresponding values used for the MI calculations: stomatocyte (S), −1 (a); discocyte (D), 0 (b); echinocyte I (E I), 0.5 (c); echinocyte II (E II), 1 (d); echinocyte III (E III), 3 (e–h). These values are compared to literature values in the Materials and Methods section. The images shown here are cropped selections from the larger fields of view used for the morphological analysis, as shown in online supplementary Figures S1–S3. The focal plane of the images is near the boundary of the cells in b and c. As the cells become rounder (e–h), features move out of the focal plane, and also become too fine to resolve (h). The stomatocytes are distinguished by the brighter ring surrounding the central spot, in addition to their smaller diameter.

Fig. 3

Distribution of cell shapes for the low hemolysis group (white boxes) and the high hemolysis group (shaded boxes). S, stomatocytes; D, discocytes; E I, II and III, echinocytes I, II and III (as defined in Fig. 2). The mid-line of the boxes indicates the median, the top and bottom ends indicate the quartiles, and the whiskers show the maximum and minimum values. The stomatocyte counts are significantly higher for the high hemolysis group (p = 0.0012); the type III echinocytes are also higher for this group, but at p = 0.049, with a difference that becomes insignificant after the correction for multiple testing (for discussion of the statistical analysis, see text). The type I echinocytes are lower for the high hemolysis group (p = 0.0019); the type II echinocytes are slightly lower, but not significantly so (p = 0.018); the discocytes are present at similar levels in both groups (p = 0.401).

Fig. 4

RBC morphology at the end of the storage period for a sample with high stomatocyte counts, low type III echinocytes, low MI, and high hemolysis (16.9, 13.2, 0.37, and 1.2%, respectively; a), and a sample with low stomatocyte counts, high echinocyte counts, high MI, and low hemolysis (1.0, 25.8, 1.11, and 0.09%, respectively; b). Scale bars, 5 µm. The full fields of view for a and b are shown in online supplementary Figures S1 and S2. Representative discocytes are circled on both images; representative stomatocytes on either side of the circled discocyte in a are indicated by asterisks.

Fig. 5

Samples can show a range of different RBC morphologies. a Stomatocytes from a single sample. b Echinocytes from a single sample. RBCs are initially discocytes, and can form increasingly rounded stomatocytes, or increasingly rounded echinocytes. Each set here was taken from a single field of view image, as illustrated in online supplementary Figures S2 and S3.

Fig. 6

MI for RBCs as a function of hemolysis at the end of the storage period. The low and high hemolysis groups of samples can be clearly distinguished by the hemolysis values. The average hemolysis ± SD was 0.11 ± 0.02% for the low hemolysis samples, and 0.76 ± 0.20% for the high hemolysis group. The MI values, however, are similar for both groups (0.87 ± 0.22 for the low hemolysis group and 0.91 ± 0.44 for the high hemolysis group).

Fig. 7

Relative amounts of hemoglobin and POPC measured by MALDI-ToF for the low hemolysis group (white boxes) and the high hemolysis group (shaded boxes). The mid-line, top and bottom ends of the boxes, and whiskers are as described for Figure 3. The Hb was determined using the peak for the Hbα monomer. Relative amounts of the Hb dimer, trimer, and doubly charged trimer are compared for the high and low hemolysis groups in online supplementary Figure S10.

Fig. 8

Comparison of cell composition and shape: amounts of Hb (a) and POPC (b), as measured by MALDI-ToF, as a function of the percentage of stomatocytes. The Pearson correlation coefficient (Pcc) is shown for each graph, indicating that there is no correlation between stomatocyte count and Hb, but that there is a slight negative correlation between POPC content and stomatocyte formation.