METHODS AND COMPOSITIONS TO DETERMINE THE QUALITY OF RED BLOOD CELL UNITS

Abstract

The present invention relates to a method for determining the quality of haemoglobin (Hb) during the storage period of red blood cell (RBC) units comprising a step of detecting soluble alpha-haemoglobin (α-Hb) pool in RBC lysates and concluding that the presence of α-Hb pool indicates a conservation of quality of Hb during the storage RBCs. Inventors have determined the impact of RBC units aging on the quality of Hb and on the soluble α-Hb pool level in RBCs. For this purpose, 21 RBC units were collected, stored at +4 to 6° C. and samples were taken at two different storage times (D3-D8 and D38-D42) to evaluate spectral characteristics of Hb and soluble α-Hb pool in RBCs. Two additional samples were collected from 16 RBC units, at intermediate time points during storage (D13-D17 and D24-D29; n=16). The α-Hb dosing assay uses the specific character of the interaction between the α-Hb and the AHSP, the α chaperone, to trap the α-Hb present in the RBC lysates of RBC units. They also investigated the effect of a short cryopreservation period at −80° C. for 15 days on the α-Hb pool for 4 different RBC units.

Claims

1. A method for determining the quality of haemoglobin (Hb) during a storage period of red blood cell (RBC) units comprising measuring, at least at the beginning and end of the storage period, the value of a soluble alpha-haemoglobin (α-Hb) pool in the RBC units and determining that the quality of Hb is conserved during the storage period when the value of the soluble α-Hb pool remains stable.

2. The method according to claim 1 further comprising: i) evaluating the value of the soluble α-Hb pool in RBC lysates at the beginning of storage; ii) evaluating the value of the soluble α-Hb pool in RBC lysates at the end of storage; iii) and determining that the quality of Hb is conserved during the storage period when the value of soluble α-Hb pool is maintained during the storage period; or determining that the quality of Hb during the storage period is not conserved during the storage period when the value of soluble α-Hb pool is increased during the storage period.

3. The method according to claim 1, wherein the method is performed at days 3 to 8 (D3-D8) and days 38 to 42 (D38-D42) after collection.

4. The method according to claim 1, wherein the value of the soluble α-Hb pool is measured by mass spectrometry.

5. The method according to claim 4, wherein the mass spectrometry is gas-chromatography/mass spectrometry (GC/MS) or liquid chromatography-tandem mass spectrometry (LC/MS/MS).

6. The method according to claim 1, wherein the value of the soluble α-Hb pool is measured by ELISA.

7. The method according to claim 1, wherein the value of soluble α-Hb pool is measured by affinity chromatography.

8. The method according to claim 7, wherein the affinity chromatography is performed with glutathione S-transferase-α-haemoglobin stabilizing protein (GST-AHSP)-coupled to a glutathione Sepharose 4B coated to 96-well filter plates.

9. The method according to claim 1, wherein said method determines whether the RBC units are suitable for transfusion.

10. A method of determining the suitability of RBC units for transfusion comprising i) measuring, at least at the beginning and the end of a storage period of the RBC units, the value of a soluble α-Hb pool in the RBC units; ii) determining that the stored RBC units are suitable to be used in a transfusion when the value of α-Hb remains stable or is not increased during the storage period.

11. The method according to claim 10, wherein the value of the soluble α-Hb pool in the RBC units is measured at days 3 to 8 (D3-D8) of the storage period.

12. The method according to claim 11, wherein the stored RBC units are suitable to be used in a transfusion when the value of the soluble α-Hb pool between day 8 and day 18 of the storage period remains stable or is not increased.

13. The method according to claim 10, wherein the stored RBC units are not suitable to be used in a transfusion when the value of the soluble α-Hb pool is increased during the storage period.

14. A kit for use in the method according to claim 1, comprising as separate components: a solid support, and an α-Hb-specific binding partner.

Description

FIGURES

[0088] FIG. 1: UV-visible absorbance spectra of lysates in stored blood units. The UV-visible absorbance spectra for soluble Hb in cytosols were obtained in RBC lysates from blood units from 250 to 700 nm, at the beginning (D3-D8, black line) and end (D38-D42, black dotted line) of the 42-day storage period. The spectra show a visible absorbance band typical of haem with a Soret band at 415 nm; the ratio of absorbance intensities at the Soret band and the UV peak at 280 nm were about 3.70±0.06 for D3-D8 and 3.79±0.04 for D38-D42. The spectra obtained were similar to that of native oxygenated Hb.sup.16 and in the ferrous form required for oxygen transport. So, the characteristics of soluble Hb were correctly conserved during a long-refrigerated storage and do not lead to significant methaemoglobin formation (data not shown), the latter would be at the origin of denatured Hb bound to the membrane. Representative values for five lysates are shown. Absorbance spectra were measured with an Eon™ microplate spectrophotometer.

[0089] FIG. 2: Detection and follow-up of the soluble α-Hb pool in RBC lysates. (A) Changes in the α-Hb pool at four different time points during storage. Measurements were made on lysates from sixteen leukoreduced RBC units, stored at +4 to 6° C. for 42 days, on D3-D8, D13-D17, D24-D29 and D38-D42. Each symbol represents a different RBC unit and each α-Hb value is the mean of two measurements. Statistical analyses were performed with Friedman's test followed by Dunn's multiple comparisons test. *p<0.05, ***p<0.001. (B) Effect of a freezing/thawing procedure on the α-Hb pool. Measurements were made on four RBC lysates from freshly prepared leukoreduced RBC units stored in SAGM for 3 to 8 days (D3-D8) and from the same units after freezing at D3-D8 and thawing 15 days after. Each symbol represents a different RBC unit; each α-Hb value is the mean of two measurements.

[0090] FIG. 3: Effect of storage time on the soluble α-Hb pool in RBC units. The α-Hb pool was detected in RBC lysates from leukoreduced RBC units (n=21) stored at +4 to 6° C. Measurements were made at the beginning and end of storage (D3-D8 versus D38-D42). Each α-Hb value is the mean of two measurements. Statistical analyses were performed with Wilcoxon matched-pairs signed-rank test. *p<0.05, ***p<0.001, ****p<0.0001. Results are shown as box-and-whisker plots with individual values indicated as dots; horizontal bars indicate the median.

EXAMPLE

[0091] Material & Methods

[0092] RBC Units, Storage and Sampling

[0093] This research was performed in accordance with the Helsinki Declaration and was approved by our institutional ethics committee (CPP no. 11-047). Blood from twenty-one healthy adult donors was collected into sterile blood bags containing citrate phosphate dextrose (CPD) as an anticoagulant, at the Etablissement Français du Sang (EFS). These fresh samples were maintained at a temperature of +18° C. to +24° C. for 2 to 24 hours before processing, in accordance with European guidelines.sup.19, at EFS Preparation Unit (Rungis, France). RBCs were isolated by plasma removal and leukoreduction at room temperature. The RBCs were then suspended in SAGM to constitute the RBC unit.

[0094] The RBC units arrived at our laboratory two to three days after blood collection and processing and were stored at +4 to 6° C. in a standard blood-bank refrigerator until day 42. We checked that none of the selected units displayed the sickle-cell trait but a deletion in one or two of the four α-globin genes cannot be totally excluded. Two samples were removed aseptically from the RBC units during the storage period with sampling couplers (Fenwal Inc., Lake Zurich, IL, USA), 3 to 8 days after collection (D3-D8; n=21) and 38 to 42 days after collection (D38-D42; n=21); two additional samples were collected from sixteen RBC units, at intermediate time points during storage (D13-D17 and D24-D29; n=16). Samples were also collected from four RBC units at D3-D8 for an alternative cryopreservation/thawing process involving the use of 57% glycerol (SpA Lab. Farmacologico, Bergamo, Italy) as a cryoprotective agent, as previously described.sup.20. The glycerol-treated RBCs were immediately frozen and stored for 15 days at −80° C. They were then thawed in a +40° C. water bath, processed for automatic deglycerolisation on a Cobe 2991 machine (Terumo BCT, Inc, Lakewood, CO, USA) and used for investigations.

[0095] Preparation of RBC Lysates and Preservative Solutions

[0096] We centrifuged aliquots of RBC units at 2,880×g for 10 min at +4° C., to separate the RBCs from the preservative solution. The preservative solution was centrifuged again at 2,880×g for 10 min at +4° C., to eliminate all traces of RBCs. The various RBC fractions and preservative solutions were then frozen at −80° C. until use. RBCs were thawed gently and lysed with four volumes of cold distilled water. The mixture was incubated for 30 min on ice, centrifuged at 16,000×g for 30 min at +4° C., and RBC lysates were recovered in the supernatant and immediately frozen at −80° C.

[0097] Absorbance Spectra and Determination of Hb Concentration

[0098] Hb concentrations were determined with an Eon™ microplate spectrophotometer (BioTek Instruments Inc, Winooski, VE, USA) in the Soret band at 415 nm, for all RBC lysates obtained at the beginning (D3-D8) and end (D38-D42) of the storage period. For five RBC units, UV-visible absorbance spectra were obtained for wavelengths of 250 to 700 nm at the beginning and end of the storage period. For preservative solutions, Hb concentrations were measured by determining absorbance at 415 nm with an extinction coefficient of 125 mM.sup.−1cm.sup.−1, and at 540 nm by the cyanmethaemoglobin method (Drabkin's method), with an extinction coefficient of 11 mM.sup.−1cm.sup.−1. All the Hb concentrations are expressed on a haem basis.

[0099] Preparation of GST-AHSP Protein

[0100] Assessments of the α-Hb pool required the upstream preparation of recombinant AHSP. Recombinant AHSP was produced as a fusion protein with glutathione S-transferase (GST-AHSP) in E.coli and purified by affinity chromatography with glutathione-Sepharose 4B beads (GE Healthcare, Lifescience, Uppsala, Sweden) as previously described..sup.21 The purified GST-AHSP was stored at −80° C. in phosphate buffered saline (150 mM NaCl, 10 mM Na.sub.2HPO.sub.4, pH 7.4) containing 1% bovine serum albumin and 10% glycerol.

[0101] Assessment of the in Vitro Soluble α-Hb pool in RBC Lysates

[0102] The α-Hb assay makes use of the specific nature of the interaction between the α-Hb and the AHSP, the a chaperone, to trap the α-Hb present in RBC lysates..sup.17 It was performed as previously described..sup.22 Briefly, 500 μL of RBC lysate were applied to 96-well filter plates (GST MultiTrap 4B, GE Healthcare, Lifescience, Uppsala, Sweden) coated with the GST-AHSP—coupled to glutathione Sepharose 4B. The plates were washed with phosphate buffer saline and the bound proteins (GST-AHSP and GST-AHSP/α-Hb complexes) were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl buffer at pH 8.0. The quantity of α-Hb in the eluted fraction was determined by spectrophotometry at 414 nm (on a haem basis) with an Eon™ microplate reader and the data were analysed with Gen5 software. The best analytical wavelength for α-Hb pool detection was 414 nm, at which proteins other than haemoprotein were not detected; in parallel, the total Hb concentration of the RBC lysates was determined. The α-Hb value was expressed in ppm, equivalent to ng of α-Hb per mg total Hb subunits per mL of RBC lysate, to take RBC Hb concentration into account. The α-Hb pool values reported are the means of two independent measurements.

[0103] Statistical Analysis

[0104] Quantitative variables are expressed as arithmetic mean±standard deviation (SD). Data were analyzed with Prism 6.0 software (GraphPad, La Jolla, CA). We performed non-parametric tests, Friedman tests followed by Dunn's multiple comparisons test, Wilcoxon matched-pairs signed-rank tests and Mann Whitney tests. P values <0.05 were considered statistically significant.

[0105] Results

[0106] Hb in Stored RBC Lysates and Supernatants

[0107] We evaluated the UV and visible absorbance spectra of RBC lysates from RBC units at the beginning (D3-D8) and end (D38-D42) of the period of storage (42 days). All RBC lysate spectra had a visible absorbance band typical of haem, with a Soret band at 415 nm (FIG. 1). The ratio of absorbance intensities at the Soret band and the UV peak at 280 nm was about 3.70±0.06 for D3-D8 and 3.79±0.04 for D38-D42 (n=5). These ratios are not significantly different from that for oxygenated native Hb A..sup.16 We determined the Hb concentrations of lysates from the twenty-one RBC units. No significant difference was observed between the beginning (D3-D8; 3.79±0.67 mM on a haem basis) and end (D38-D42; 3.85±0.65 mM) of storage (FIG. 1). Hb was also determined in the preservative solutions (i.e. supernatants) of the twenty-one RBC units analysed (data not shown), by two spectroscopic methods, at 540 nm after cyanmet Hb transformation and directly at 415 nm. The supernatant Hb concentrations based on absorbance at 540 and 415 nm were similar, at 0.020±0.012 mM at D3-D8 versus 0.049±0.022 mM at D38-D42, corresponding to a small but significant difference between these time points (p=0.0019).

[0108] Soluble α-Hb Pool in the Lysates of Stored RBC Units

[0109] We investigated the presence of a soluble α-Hb pool in lysates from 16 RBC units after storage at +4° C. to +6° C. for various times (FIG. 2A). The “soluble α-Hb pool” corresponds to the α-Hb not bound to β-Hb in RBCs but that can be linked to AHSP..sup.17 An α-Hb pool was detected at D3-D8, at the beginning of storage, just after the preparation of RBC units from whole blood. At this time, values ranged from 72 to 165 ppm, with a mean value of 126±23 ppm (equivalent to a mean of 7.71 μg of α-Hb bound to resin-coupled GST-AHSP protein). A dispersion of the values of different RBC units was also observed (interquartile range=33). At the two intermediate times (D13-D17 and D24-D29; n=16), mean α-Hb pool amounts were 131±30 ppm and 134±34 ppm, respectively. At the end of storage, α-Hb pool was between 114 and 209 ppm, with a mean value of 152±29 ppm (corresponding to a mean of 8.95 μg α-Hb bound to GST-AHSP). A more pronounced dispersion of α-Hb values between RBC units was observed at D38-D42 (interquartile range=48.5); the values obtained at D38-D42 were also significantly higher than those obtained at D3-D8 (n=21, p <0.0001) (FIG. 3).

[0110] The freezing of RBC units with rare blood phenotypes may be required for transfusion in specific populations of recipients, such as the sickle-cell disease patients studied by our team. We therefore also investigated the effect of cryopreservation on the α-Hb pool of RBC units. We assessed the effect of a short period of freezing on soluble α-Hb pool amount in four RBC units, by comparing samples selected at D3-D8 before (97±24 ppm) and after freezing for 15 days at −80° C. (129±10 ppm). The α-Hb pool of these samples were slightly, but not significantly higher (n=4; p=0.25) after freezing for 15 days (FIG. 2B).

[0111] Discussion

[0112] Previous studies by our team.sup.20 on the same blood units stored for 42 days showed no significant changes in RBC volume, osmotic resistance, or mean corpuscular Hb concentration (MCHC) over time, providing evidence that conventional storage of RBC units did not modify RBC rheology. By contrast, and as expected, the pH of the unit supernatants decreased rapidly.sup.6, 20.

[0113] The presence of isolated Hb subunits during storage of RBCs units has never previously been studied. An α-Hb pool is detected in blood units from the beginning of storage (D3-D8) at temperatures from +4 ° C. to +6 ° C., increasing over the 42-day storage period. The term “soluble α-Hb pool” corresponds to the α-Hb not bound to β-Hb that can be linked to AHSP in RBCs. The detection of such an α-Hb pool in blood units is not surprising, given that the presence of α-Hb has already been reported in RBC lysates obtained from healthy volunteers with a normal Hb phenotype.sup.22; in that context, the mean value was 81±15 ppm, with a lesser degree of dispersion (54-115 ppm; interquartile range 21).

[0114] The differences observed in α-Hb pool values between that of RBC units and that of freshly prepared RBCs from healthy volunteers can be explained by the difference in storage temperatures after collection, in the use of different anticoagulants, or in the sample preparation. In fact, for the preparation of RBC units, whole peripheral blood is collected into CPD-anticoagulated bags and kept at room temperature for 2-20 hours before processing.sup.19. Leukoreduction is then performed by filtration before the transfer of the RBCs to a bag containing SAGM; all these steps are performed at room temperature, taking a mean time of 10-24 hours, before storage at temperatures from +4 ° C. to +6 ° C. in blood banks. By contrast, the preparation of fresh RBCs drawn from volunteers was processed at +4 ° C. within two hours of collection on EDTA and no leukoreduction step was carried out.sup.17. Furthermore, we have observed that α-Hb pool values tend to increase when the whole blood sample is stored for a period of time at room temperature (unpublished observations). It has been also reported that temperature influences the kinetics of dissociation of the αβ dimers into α and β monomers: an increase in temperature from +7 ° C. to +37 ° C. resulted in a 50-fold increase in the dissociation rate constant. All these data indicate that an increase in temperature, along with the duration of the procedure in processing the RBC unit, can have an impact on the α-Hb pool values detected and may explain the increase in the α-Hb pool in blood units.

[0115] An excess of α-Hb chains that, by precipitating on the RBC membrane and acting as active oxidants, leads to oxidant damage has previously been reported.sup.24,25. In our team, we initially detected the α-Hb pool in RBCs drawn from pathological β-thalassemia blood samples.sup.17,22. In this Hb disorder, an imbalance in the biosynthesis of globin chains lead to an excess of α-chains. Precipitation of α-chains, and oxidative damage in erythroid precursors and RBCs, resulting in inefficient erythropoiesis, have all been observed. In the most severe forms of β-thalassemia, the α-Hb pool values are higher than 1,000 ppm (very high in comparison to the α-Hb pool observed in RBC units) and this correlates well with the clinical severity of the disease. Here, the detected α-Hb pool value is negligible compared to the amount of functional Hb in RBC units and would have had almost no impact on the quality of the stored RBC units. Furthermore, most blood units are transfused to patients between D8 and D18, and the α-Hb pool remains practically stable within this timeframe (FIG. 2A), thus supporting the view that the values of α-Hb would not affect the choice of RBC units to be transfused.

[0116] The wide dispersion of values from different RBC units observed throughout the storage period, but also increasing towards the end (interquartile range 33 at D3-D8 vs 48.5 at D38-D42), may reflect the well-known variability between different blood bags obtained from the same donor.sup.14. The units were verified for the lack of the sickle-cell trait but were not genotyped for globin genes. In a previous study, out of 50 healthy volunteers genotyped for globin genes, 20% had an abnormal α-globin genotype and α-Hb pool values lower than those observed in normal α-globin subjects.sup.26. This could be due to the presence of an α-thalassemia mutation.sup.17, 22. Thus, it would be of particular interest to know more about the α-globin genotype of those two RBC units with α-Hb values lower than the average of the other units tested (FIG. 2A; see at D3-D8).

[0117] It is important to remember that the use of cryopreserved blood units with rare phenotypes can be required for transfusion in certain circumstances, particularly for sickle-cell anaemia (SCA) patients in painful acute crisis or those experiencing severe haemolytic episodes.sup.20. The results we obtained before and after freezing some blood units clearly showed that the α-Hb pool increased only slightly (FIG. 2B). Thus, such a freezing/thawing procedure required to transfuse previously cryopreserved rare blood units does not significantly modify the soluble α-Hb pool. Finally, as a control, we verified the impact of storage time on Hb functionality in stored RBCs (FIG. 1) with spectra evaluated for soluble Hb in cytosols of RBCs from blood units. The characteristics of soluble Hb were correctly preserved during a lengthy period of refrigerated storage, as shown for other RBC parameters in previous rheological studies by our team.sup.14, 20.

Conclusions

[0118] This study evaluated the Hb spectra and the presence of a soluble α-Hb pool in the RBC units throughout the 42-day storage period, to determine the impact of the storage time on quality of Hb.

[0119] Considering the conservation of RBC units, we can conclude that a higher storage temperature leads to an increase of α-Hb pool value. In conclusion, this study shows in RBC units no modifications of Hb absorbance spectra depending on the storage time indicating that the quality of Hb during the storage period is maintained.

[0120] We also show for the first time the presence of a soluble α-Hb pool in RBC units with a great variability from one RBC unit to another as well as the significant increase of this pool after a storage period of 42 days.

[0121] In conclusion, inventors show here, for the first time, the presence of a soluble α-Hb pool in RBC units, with a high variability between RBC units and a significant increase in this pool after storage for 42 days, although the final quantity of the α-Hb pool remained relatively small.

[0122] The authors demonstrate here that the increase of soluble α-Hb pool in RBCs units observed at intermediate time D13-D17 was not significantly higher than that obtained at beginning storage, important result since the most RBC units are used for transfusion between day 8 and day 18. Thus, α-Hb pool evaluation can be used in the future as a new supplementary quantitative parameter for the follow-up of the quality of RBC units for transfusion. This may improve the selection of particular blood units for the transfusion of specific populations of recipients, such as sickle-cell disease patients, who are highly dependent to the quality of the blood products they receive during transfusion.

REFERENCES

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