Alloimmunization to red blood cell antigens after universal leucodepletion. A regional multicentre retrospective study

Leucodepletion has been shown to reduce human leucocyte antigen immunization, but studies on the effect of leucodepletion on red cell alloimmunization reported discordant results. We conducted a retrospective multicentre study to determine whether prestorage filter leucodepletion alters the development of clinically significant red blood cell alloimmunization against the Rhesus, Kell, Duffy, Kidd and MSs blood group systems. Two periods were investigated, 2 years before and 2 years after universal leucodepletion. Comparisons were made between the transfused patient cohorts. To control for changes not related to leucoreduction, we compared antibody incidence with antibody prevalence in the two study periods. Newly detected antibodies (n = 4770) were found in 4115 patients from 19 participating hospitals. Of these, 857 antibodies in 659 patients were because of transfusions given in the study periods. The immunization risk was 0.13% for both periods. No differences were found regarding incidence of new antibodies, nor for patients regarding age, sex, previous antibodies, multiple antibodies, additional antibodies, number of transfusions, transfusions episodes and days from transfusion to date of immunization. In conclusion, compared with buffy-coat leucoreduction, universal prestorage filter leucodepletion did not alter the development of clinically significant red blood cell alloimmunization.     

Apoptosis in leucodepleted packed red blood cells

BACKGROUND AND OBJECTIVES: Packed red blood cells (pRBCs) contain apoptotic white cells. We studied apoptotic cells in pRBCs after filtration and at various time-points during storage. MATERIALS AND METHODS: To maintain the same subset of cells, seven pRBC units were pooled in a single bag and divided equally into seven aliquots. Two series of five experiments were performed: in the first we utilized the Biofil R01 Max filter, and in the second the Pall BPF4 filter was used. One aliquot was immediately leucodepleted while the others were stored at 4 degrees C and filtered on days 3, 7, 10, 14, 21 and 42 of storage. The postfiltration leucocyte counts and apoptotic evaluations were performed by using the Nageotte chamber and flow cytometry. RESULTS: The absolute number of residual leucocytes was always less than 0.5 x 106 in each experiment. Nageotte chamber counts showed a greater number of white blood cells than flow cytometry during the 42 days of storage. On day 0, the percentage of apoptotic cells in non-leucodepleted pRBCs was 1.1 +/- 0.4 and 1.2 +/- 0.4, while in filtered pRBCs it was high from day 0, at 53.5 +/- 16.3 and 52 +/- 18.5, respectively, with Biofil and Pall filters. On day 10 of storage, apoptotic cells reached a percentage of 42.5 +/- 15.8 and 41.6 +/- 18.6 in non-leucodepleted pRBCs, while in filtered units an average value of approximately 90% was found with both filters. CONCLUSIONS: The percentage of apoptotic cells was higher in leucodepleted than in non-leucodepleted pRBCs. After filtration, the degree of apoptosis was already high on day 0, and reached a mean of approximately 90% by day 10. The difference in residual WBC counts between the Nageotte chamber and flow cytometry could be related to the presence of a high percentage of apoptotic cells in filtered blood components, and to the method used to distinguish viable from apoptotic cells.     

Separation of whole blood into plasma and red cells by using a hollow-fibre filtration system

BACKGROUND AND OBJECTIVES: The aim of this study was to assess the separation of whole blood into red cells and plasma by using the Sangofer device, which is a gravity-fed, hollow-fibre system. The components would then be compared with those produced by the use of more elaborate technical equipment. MATERIALS AND METHODS: Ten whole-blood units were leucoreduced by using a WBF2 filter and immediately separated into red cells and plasma by using the Sangofer blood-separation device. Red cells were stored in additive solution and tested on days 1 and 42. The plasma was assayed for levels of various coagulation factors and for markers of both coagulation and complement activation. RESULTS: The red-cell parameters were similar to those obtained when routine centrifugation methods were used. The filter did not cause haemolysis. Levels of plasma factor VIII and factor XI were lower than those seen in routinely produced leucoreduced plasma units but there was no evidence of activation of the coagulation and complement systems. CONCLUSIONS: The Sangofer device is simple and straightforward to use and eliminates the need for both centrifugation and automated separation steps during the processing of whole blood into red cells and plasma components. Minor changes are required to make the procedure easier to incorporate into routine use.     

Extended storage of whole blood before the preparation of blood components: in vitro effects on red blood cells in Erythro-Sol environment

Background and Objectives  Routine procedures for extended storage of whole blood (WB) before the preparation of blood components are of interest primarily for logistical reasons. We stored red cell units in either Erythro-Sol 2 (E-Sol 2, test units, 150 ml added) or in saline-adenine-glucose–mannitol (SAG-M) (reference units, 100 ml added) that were prepared after storage of WB at room temperature for 8, 12, 16 or 19 h after blood collection.
Study Design and Methods  Red blood cells were stored for 42 days. We measured pH, glucose, lactate, haemolysis, red blood cell adenosine triphosphate and 2,3-diphosphoglycerate on days 1, 7, 14, 21, 28, 35 and 42.
Results  Haematocrits were significantly lower in E-Sol 2 than in SAG-M due to the higher volume of E-Sol 2 added compared to SAG-M. Significantly reduced levels were found in E-Sol 2 of extracellular pH (throughout storage after 8-h hold and initially after 12-, 16- or 19-h hold), of lactate (initially after 8-h hold and throughout storage after 12-, 16- or 19-h hold), and of haemolysis from day 35 in the 8-h and on day 42 in the 12-h hold group. Sigificantly increased levels of adenosine triphosphate were seen in E-Sol 2 after 8-h hold (from day 14) and after 12-h hold (at days 21, 35 and 42) compared to SAG-M. Significantly higher concentrations of 2,3-diphosphoglycerate were noticed primarily after 8-h hold of WB.
Conclusion  The use of E-Sol 2 as a replacement for SAG-M does not significantly improve in vitro data after extended storage of WB at room temperature before preparation of blood components. However, after 8-h hold in vitro characteristics similar to or better than in fresh blood will be maintained for several weeks in E-Sol 2, a situation that makes E-Sol 2 superior to SAG-M when storage of WB is limited to 8 h. Some improvement was noted after 12-h hold as well.     

A new density gradient system for the separation of human red blood cells

A new density system for the separation of human red blood cells by density-gradient centrifugation is described. The gradient medium is made with colloidal silica particles coated with polyvinylpyrrolidone suspended in aqueous solution of meglamine diatrizoate. By this method, more than 10 red-cell fractions can be separated. These show different ages (by creatine content and ⁵⁹Fe in vivo labelling) and different characteristics (ie, potassium content). A 40-fold enrichment in reticulocytes can be obtained in the top layers, with a great improvement of specific activity in labelling procedures of newly synthesized globin chains. The method is simple, rapid, inexpensive, reliable, nontoxic for erythrocytes, and is suitable for globin synthesis and other studies of erythrocyte metabolism.     

Temporal sequence of major biochemical events during Blood Bank storage of packed red blood cells

Background.

We used sensitive spectroscopic techniques to measure changes in Band 3 oligomeric state during storage of packed red blood cells (RBC); these changes were compared to metabolic changes, RBC morphology, cholesterol and membrane protein loss, phospholipid reorganisation of the RBC membrane, and peroxidation of membrane lipid. The aim of the study was to temporally sequence major biochemical events occurring during cold storage, in order to determine which changes may underlie the structural defects in stored RBC.

Materials and methods.

Fifteen RBC units were collected from normal volunteers and stored under standard blood bank conditions; both metabolic changes and lipid parameters were measured by multiple novel assays including a new mass spectrometric measurement of isoprostane (lipid peroxidation) and flow cytometric assessment of CD47 expression. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy, and RBC morphology by microscopy of glutaraldehyde-fixed RBC.

Results.

Extracellular pH decreased and extracellular potassium increased rapidly during cold storage. Band 3 on the RBC membrane aggregated into large oligomers early in the storage period and coincident with changes in RBC morphology. Membrane lipid changes, including loss of unesterified cholesterol, lipid peroxidation and expression of CD47, also changed early during the storage period. In contrast loss of acetylcholinesterase activity and haemolysis of RBC occurred late during storage.

Discussion.

Our results demonstrate that changes in the macromolecular organisation of membrane proteins on the RBC occur early in storage and suggest that lipid peroxidation and/or oxidative damage to the membrane are responsible for irreversible morphological changes and loss of function during red cell storage.     

A multicentre evaluation of a new filtration protocol for leucocyte depletion of high-haematocrit red blood cells collected by an automated blood collection system

BACKGROUND AND OBJECTIVES: A multicentre trial was set up to evaluate the performance of a new leucodepletion protocol. MATERIALS AND METHODS: Filtration at high haematocrit was started during collection of red blood cell (RBC) products by apheresis with Trima. SAG-M was added after filtration through the filter. Haematocrits and haemoglobin of the filtered RBCs were measured. Residual leucocytes were determined by Nageotte counting. RESULTS: One-hundred and forty seven procedures were carried out. The haematocrit and haemoglobin contents were 57.3 +/- 3.0% and 55.1 +/- 4.3 g/unit, respectively. All products showed low residual leucocyte levels (< or = 0.75 x 106/unit; 99.31% < 1 x 106). CONCLUSION: Immediate, on-line, high-haematocrit filtration of red cells collected on Trima resulted in leucoreduced RBCs, which met the AABB and Council of Europe criteria.     

Preferential localisation of red cell vacuoles to pathologically shaped human red blood cells

The percentage of pitted erythrocytes and Howell-Jolly bodies in peripheral blood samples of 51 individuals following posttraumatic splenectomy and 20 patients splenectomized because of various haematological diseases differed significantly from each other (p less than 0.001) and from that of healthy controls (p less than 0.001). The percentage of pitted erythrocytes was significantly higher in pathologically shaped red blood cells (RBCs) (acanthocytes, schizocytes, elliptocytes) than in normal discoid shaped RBCs (p less than 0.001). As the number of pits per RBC showed great individual variations, a scoring system for the evaluation of pitted RBCs is proposed.     

Some characteristics of human red blood cells separated according to their size: a comparison with density-fractionated red blood cells

During their in vivo ageing, red blood cells (RBC) increase in density and become smaller. Age-defined RBC subpopulations are usually collected by centrifugation. A fractionation according to RBC volume has been proposed as an improved alternative to such age separation. Because a few data reported in the literature indicate some discrepancies between the two methods, blood samples were separated either by centrifugation or by counterflow centrifugation, and some characteristics of the RBC thus fractionated were studied. The enzyme activities decrease either when the density rises or when the volume (MCV) decreases. However, the comparison of other RBC characteristics strongly suggests that these two procedures do not lead to the collection of the same RBC subpopulations: for instance, the hemoglobin content increases when the MCV rises, whereas it remains constant whatever the RBC density is. With radiolabelled cells, it is shown 1) that the most dense RBC are recovered in all the size-separated RBC subpopulations, even though they tend to concentrate in the fractions with the largest MCV, and 2) that the smallest RBC are almost fairly distributed in all the RBC subpopulations, whatever their density, whereas the largest RBC are mainly, but not exclusively, present in the high-density fractions. Thus, fractionation according to size does not match separation according to density. Taken together with results from in vivo experiments carried out in mice and with the fact that reticulocytes are present in all the size-separated fractions, these data suggest that counterflow centrifugation may be a very questionable procedure to achieve a RBC fractionation according to age and therefore that RBC volume might not be a reliable criterion of RBC age.     

The long and winding road to pathogen reduction of platelets,red blood cells and whole blood

Pathogen reduction technologies (PRTs) have been developed to further reduce the current very low risks of acquiring transfusion-transmitted infections and promptly respond to emerging infectious threats. An entire portfolio of PRTs suitable for all blood components is not available, but the field is steadily progressing. While PRTs for plasma have been used for many years, PRTs for platelets, red blood cells (RBC) and whole blood (WB) were developed more slowly, due to difficulties in preserving cell functions during storage. Two commercial platelet PRTs use ultra violet (UV) A and UVB light in the presence of amotosalen or riboflavin to inactivate pathogens’ nucleic acids, while a third experimental PRT uses UVC light only. Two PRTs for WB and RBC have been tested in experimental clinical trials with storage limited to 21 or 35 days, due to unacceptably high RBC storage lesion beyond these time limits. This review summarizes pre-clinical investigations and selected outcomes from clinical trials using the above PRTs. Further studies are warranted to decrease cell storage lesions after PRT treatment and to test PRTs in different medical and surgical conditions. Affordability remains a major administrative obstacle to PRT use, particularly so in geographical regions with higher risks of transfusion-transmissible infections.     

Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells

Background

Routines for the storage of whole blood (WB) overnight for the preparation of blood components on the following day are of increasing interest primarily for logistic reasons. The present study focuses on in vitro effects during storage for 6 weeks on red blood cells (RBC) prepared in different blood containers after being held overnight.

Study design and methods

Five different blood collection systems were used with either inline leucocyte reduction red cell filters for the preparation of RBC, buffy coat (BC) and plasma or WB filters for the preparation of RBC and plasma. A new container with an integrated WB filter removing leucocytes but not platelets was also included for the preparation of leucocyte-reduced RBC, BC and plasma units. Standard CPD solution (63 or 70 mL) and SAG-M solution (100 or 110 mL) were used for the collection of either 450 or 500 mL blood. All WB units were stored at room temperature, either overnight for 18–24 hours (test groups, n=104) or for up to 8 hours (reference groups, n=20). In addition, five test units were stored overnight under refrigeration.

Results

In test groups (overnight storage at room temperature) we found significantly lower levels of extracellular potassium, 2,3-DPG and pH (up to day 28). During storage, higher levels of ATP (Terumo, CaridianBCT until day 35, Fresenius until day 14, Fenwal throughout storage) were seen in test groups than in reference groups. When WB was stored overnight at 2–6°C before WB filtration, the levels of ATP and haemolysis were higher than in the corresponding reference.

Conclusion

Significant differences in in vitro parameters were observed between RBC prepared within 8 hours and 18–24 hours after blood collection. The results were consistent irrespective of the blood container used. New alkaline solutions may decrease the differences.     

Pilot study of oxygen transport rate of banked red blood cells

Background and Objectives  Dynamic oximetry provides a new way to assess the effect of blood storage on the oxygen transport rate (OTR).
Materials and Methods  In dynamic oximetry, the rate at which oxyhemoglobin becomes deoxyhemoglobin is measured optically, thereby, indirectly measuring the rate at which oxygen leaves the red blood cell (RBC) making it available for transfer to tissues. Extending the physiologic diffusion time in an in vitro apparatus, consisting of a diffusion system and gas exchanger capable of controlling the surface area and the time of exposure for oxygenation and deoxygenation, makes OTR measurement feasible. Eight normal blood donor units, collected in adenine, dextrose, sorbitol, sodium chloride and mannitol , were stored for 8 weeks under standard conditions and serially sampled for OTR.
Results  We report that the OTR at the time of blood bank donation appears to be singular for each donor, that the interdonor differences are maintained over time, and that the individual OTR increased 1·72-fold (95% CI 1·51, 1·95) over 8 weeks, adjusting for sex, age and plasma cholesterol level.
Conclusion  Oxygen transport rate increases during storage; blood units with similar haemoglobin content may have significant differences in OTR. Studies examining blood parameters at the time of donation and blood storage on patient outcomes should consider measuring OTR, as it may contribute to differences in observed efficacy of tissue oxygenation.     

Spurious counts and spurious results on haematology analysers: a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes

Haematology analysers provide quick and accurate results in most situations. However, spurious results, related either to platelets (part I of this report) or to other parameters from the cell blood count (CBC) may be observed in several instances. Spuriously low white blood cell (WBC) counts may be observed because of agglutination in the presence of ethylenediamine tetra-acetic acid (EDTA). Cryoglobulins, lipids, insufficiently lysed red blood cells (RBC), erythroblasts and platelet aggregates are common situations increasing WBC counts. In most of these instances flagging and/or an abnormal WBC differential scattergram will alert the operator. Several situations lead to abnormal haemoglobin measurement or to abnormal RBC count, including lipids, agglutinins, cryoglobulins and elevated WBC counts. Mean (red) cell volume (MCV) may be also subject to spurious determination, because of agglutinins, excess of glucose or salts and technological considerations. In turn, abnormality related to one measured parameter will lead to abnormal calculated RBC indices: mean cell haemoglobin content (MCHC) is certainly the most important RBC indices to consider, as it is as important as flags generated by the haematology analysers (HA) in alerting the user to a spurious result. In many circumstances, several of the measured parameters from CBC may be altered, and the discovery of a spurious change on one parameter frequently means that the validity of other parameters should be considered. Sensitive flags now allow the identification of several spurious counts, but only the most sophisticated HA have optimal flagging and more simple HA, especially those without a WBC differential scattergram, do not possess the same sensitivity for detecting anomalous results. Reticulocytes are integrated now into the CBC in many HA, and several situations may lead to abnormal counts.     

A new video image analysis system to study red blood cell dynamics and oxygenation in capillary networks

OBJECTIVE: The authors present a Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) to study red blood cell (RBC) dynamics and oxygenation in capillary networks. The system enables analysis of capillaries to study geometry and morphology and provides values for capillary parameters such as diameter and segment length. It also serves as an analysis tool for capillary RBC flow characteristics, including RBC velocity, lineal density, and supply rate. Furthermore, the system provides a means of determining the oxygen saturation of hemoglobin contained within RBCs, by analysis of synchronized videotapes containing images at two wavelengths, enabling the quantification of the oxygen content of individual RBCs. METHODS: Video recordings of RBC flow at two wavelengths, 420 nm (isosbestic) and 436 nm (oxygen sensitive), are made using a dual camera video microscopy system. The 420-nm recording is used to generate images based on the variance of light intensity fluctuations that help to identify capillaries in a given field of view that are in sharp focus and exhibit flow of individual RBCs separated by plasma gaps. A region of interest enclosing the desired capillary is defined and a fixed number of successive video frames at the two wavelengths are captured. Next a difference image is created, which delineates the RBC column, whose width is used to estimate the internal diameter of the capillary. The 420-nm images are also used to identify the location and centroid of each RBC within the capillary. A space-time image is generated to compute the average RBC velocity. Lineal density is calculated as the number of RBCs per unit length of a capillary segment. The mean optical density (OD) of each RBC is calculated at both wavelengths, and the average SO(2) for each cell is determined from OD(436)/OD(420). RESULTS AND CONCLUSIONS: MASCOT is a robust and flexible system that requires simple hardware, including a SGI workstation fitted with an audio-visual module, a VCR, and an oscilloscope. Since the new system provides information on an individual cell basis from entire capillary segments, the authors believe that results obtained using MASCOT will be more accurate than those obtained from previous systems. Due to its flexibility and ease of extension to other applications, MASCOT has the potential to be applied widely as an analysis tool for capillary oxygen transport measurements.     

Humoral immune response to autologous blood transfusion in hip surgery: whole blood versus packed red cells and plasma.

BACKGROUND AND OBJECTIVES: The immune response to the transfused autologous buffy coat content in whole blood has, to date, not been studied in detail. SUBJECTS AND METHODS: Patients undergoing hip arthroplasty were studied according to whether they received autologous whole blood (WB) (n = 30), autologous fresh-frozen plasma and buffy coat-poor red cells (RC) (n = 40), or no transfusion (NT) (n = 27). Plasma levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and complement SC5b-9 were analysed by enzyme-linked immunosorbent assay (ELISA) 7 days after surgery. RESULTS: There were no significant between-group differences regarding the time course of TNF-alpha, IL-6 and complement SC5b-9 plasma level changes, the infection rate, or the length of hospital stay. CONCLUSION: In comparison to the impact of surgery on cytokine and complement levels, the transfusion of autologous buffy coat is not relevant.     

显微操作法在获取孕妇外周血中胎儿有核红细胞的应用

目的：探讨显微操作法对获取孕妇外周血中胎儿有核红细胞（MRBCs）的可行性和准确性。方法：将妊娠6-38周41例孕妇的外周血进行不连续密度梯度离心，将分离后的细胞进行制片，在光学显微镜下识别MRBCs，然后用微操作仪获取NRBCs，并用聚合酶链反应（PCR）在基因水平进行验证。结果：41例孕妇有22例在外周血中检出NRBCs，检出率为53.7％，PCR结果显示22例中有10例呈现Y特异带，总符合率为95.4％。结论：显微操作法是获取孕妇外周血中NRBCs准确可靠的方法。