The increased use of Peripheral Blood Stem Cells (PBSC) to reconstitute hematopoiesis in autotransplant and, more recently, allotransplant settings has not been associated with a consensus means to quality control the PBSC product. Since the small population of cells that bear the CD34 antigen are thought to be responsible for multilineage engraftment, graft assessment by flow cytometric quantitation of CD34+ cells should provide a rapid, reliable, and reproducible assay. Unfortunately, although a number of flow cytometric assays for CD34 enumeration have been described, the lack of a standardized method has led to the generation of widely divergent data. Furthermore, none of these assays has been validated as to interlaboratory reproducibility and suitability for widespread clinical application. In early 1995, the International Society of Hematotherapy and Graft Engineering (ISHAGE) established a Stem Cell Enumeration Committee, the mandate of which was to validate a simple, rapid, and sensitive flow cytometric method to quantitate CD34+ cells in peripheral blood and apheresis products. We also sought to establish its utility on a variety of flow cytometers in clinical laboratories and its reproducibility between transplant centers. Here, we describe the four-parameter flow methodology adopted by ISHAGE for validation in a multicenter study in North America.

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34 cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34 count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r 0.99), with no statistically significant difference or bias between methods (P G 0.05). Linearity of the absolute counting method generated an R 2 value of 1.00 over the range of 0-250/l. Precision of the absolute counting method measured at three concentrations of CD34-stabilised KG1a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34 cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34 cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibil-ity of CD34 determinations due to the reduction in sample handling and calculation of results. Cytometry (Comm. Clin. Cytometry) 34:61-70, 1998.

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Although storage duration influences the quality of red blood cell product, there is currently insufficient evidence to advocate shorter storage periods for red blood cell products.

Summary:Reduced CD34 + cell viability due to cryopreservation has unknown effects on subsequent hematopoietic engraftment in autologous transplantation. Thirty-six consecutive autologous peripheral stem cell collections were analyzed for absolute viable CD34 + cell numbers at the time of stem cell collection and prior to reinfusion. Viable CD34 + cells were enumerated using single platform flow cytometry and the molecular exclusion

The clinical consequences of prolonged storage of red cells have not been established. In this pilot study, we evaluated whether it would be feasible to provide a continuous supply of red cells stored <8 days. In addition, we examined the potential benefits attributed to "fresh" as compared to standard red cells in 66 critically ill and cardiac surgical patients. Nine patients were issued red cells but were not transfused. From the 57 remaining patients, the number of units transfused averaged 5.5 +/- 8.43 red cell units in the experimental group compared to 3.3 +/- 3.27 red cell units in the standard group (P = 0.25). The median storage time was 4 days in the experimental group compared to 19 days in the standard group (difference of 15 days; interquartile range of 12-16 days; P < 0.001). Overall, 73% of patients received red cells with storage times that corresponded to the treatment allocation more than 90% of the time. The group receiving red cells <8 days old tended to be older on average (68 +/- 8.54 yr versus 63 +/- 15.30 yr; P = 0.13) and have more comorbid illnesses (85% versus 65%; P = 0.09). In total, 27% of patients in the experimental group died or had a life-threatening complication as compared to 13% in the standard group (P = 0.31). There were no differences in prolonged respiratory, cardiovascular, or renal support after randomization (P > 0.05). A large clinical trial comparing red cell storage times is feasible and warranted given the limited available evidence.

Animal models of transfusion are employed in many research areas yet little is known about the storage-related changes occurring in the blood used in these studies. This study assessed storage-related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA-1 at 4 degrees C over a 4-week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 degrees C. RBC function was assessed by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2.2 +/- 0.2 micromol g(-1) Hb; human, 2.5 +/- 0.3 micromol g(-1) Hb). Baseline DPG concentrations were similar in rat and human RBCs (16.2 +/- 2.3 micromol g(-1) Hb and 13.7 +/- 2.4 micromol g(-1) Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post-transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (- 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA-1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species-specific differences in the structure and metabolism of these cells.

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