Summary:GVHD is a life-threatening complication of allogeneic BMT. It can be either acute (aGVHD) or chronic (cGVHD), In our BMT Unit, we have observed a high frequency with the latter form behaving like an autoimmune disease. of skin rash associated with fever and other clinical finClassically, GVHD is thought to be the response of donor dings during engraftment of autologous BM and/or lymphocytes to the foreign histocompatibility antigens of PBSC. Thirty patients with breast cancer and 12 the recipients. 1 Recent studies in humans, however, indicate patients with Hodgkin's or non-Hodgkin's lymphoma, that aGVHD-like syndrome can occur after BMT perfortreated with the same regimen, were analyzed retromed between identical twins (syngeneic) or after ABMT. 2-7 spectively or prospectively to characterize the clinicalIn fact, an incidence of 8% of this aGVHD is reported in syndrome, its frequency, and its clinical course, as well autologous or syngeneic BMT recipients. 8 These early as to define the factors affecting its incidence. In reports were met with skepticism because they challenged patients developing skin rash, the median and range for the universal concept that histocompatibility differences time to onset of skin rash and for time to increase in between donor and host are absolute requirements for the WBC after reinfusion of stem cells were identical (8 induction of GVHD, as once postulated by Billingham. 1 days, range 5-13) and did not differ significantly (P = More recent reports suggest that GVHD may include a dis-0.533). Twenty-three patients (55%) had skin rash, 18 regulation of self-nonself discrimination or a failure of both patients had fever. Other, less frequent manifestations central and peripheral mechanisms that govern self-tolerinclude platelet transfusion refractoriness (PTR), ance. 9 Several studies have shown that the induction of diarrhea, diffuse alveolar hemorrhage, and autoimmune syngeneic/autologous GVHD requires (1) the inhibition of thrombocytopenia or hemolytic anemia. A higher prothymic-dependent clonal deletion of autoreactive T cells by portion of breast cancer patients developed the syncyclosporine, and (2) the elimination of the peripheral regudrome in comparison to lymphoma patients (67% vs latory mechanism by the preparative regimen before 25%, P = 0.051). Acute GVHD grade I-II was estab-BMT. [10][11][12][13] The absence of this peripheral regulatory system lished histologically in six patients with the syndrome.creates a permissive environment for the activation and Comparison of the incidence of the syndrome by differexpansion of the autoreactive effector T lymphocytes. ent variables using Fisher's exact test revealed signifiSimilar mechanisms may be responsible for the cance for disease category (P = 0.02) and number of cutaneous eruptions seen frequently in immunocomproprevious treatment regimens (P = 0.002) as predictive mised patients undergoing intensive chemotherapy in the factors for developing the autoaggression syndrome. In treatment of malignancies. 14,15 In a ...

Conventional flow cytometric methods for CD34+ cell counting may be affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple flow cytometric no-wash procedure that avoids these shortcomings because it provides absolute CD34+ cell counts and assesses cell viability. Samples were incubated with phycoerythrin (PE)-labeled anti-CD34 (Becton Dickinson Immunocytometry Systems [BD], San Jose, CA) and peridinin chlorophyll protein (PerCP)-labeled anti-CD45 (BD) in bead-containing TRUCOUNT tubes (BD). After red cell lysis with a fixative-free reagent, the impermeant nucleic acid dye YO-PRO-1 (Molecular Probes, Eugene, OR) was added and samples were analyzed on a single-laser FACSCalibur (BD). A comparison with the ProCOUNT progenitor cell assay (BD) in 57 samples revealed excellent correlation of results (r = 0.98, intercept -0.2 cells/microl, slope 1.01). Precision studies conveyed coefficients of variation of 6.4 and 8.9% at concentrations of 35 and 16 CD34+ cells/microl, respectively. In untreated and leukocyte-enriched cord blood 4.5+/-3.8% of CD34+ cells were stained by YO-PRO-1, representing apoptotic or necrotic cells. In post-thawing cryopreserved samples this number increased to 10.4+/-5.5%. Isotype controls showed very low blank values of viable cells (0.1+/-0.4 cells/microl, maximum 2.4) and seemed unnecessary. We found no washing-related alteration of results in 35 samples, indicating that the method may also be performed with cell washing. Replacing YO-PRO-1 with TO-PRO-3 facilitated four-color analysis of subpopulations of viable CD34+ cells on a FACSCalibur equipped with a second (diode) laser. We found the proposed method to be a rapid, efficient, and flexible procedure that improved validity of CD34+ cell counts.

The cost of harvesting, processing, and freezing multiple peripheral blood stem cell (PBSC) products could easily exceed that of bone marrow harvest. To reduce costs while maintaining product viability, we examined the effect of overnight storage on PBSC products. Sixteen consecutive leukapheresis samples from 12 patients were examined prospectively. Each initial leukapheresis product was stored overnight on ice (median temperature 15 degrees C) after adding an equal amount of M199 culture medium containing heparin. After overnight storage, the product was combined with the next day PBSC harvest if required and processed/frozen per protocols. Parameters measured before and after storage include cell count and differential, viability, bacterial cultures, and colony-forming unit (CFU) assays. The results show that the median cell concentration during storage was 7.12 x 10(7)/ml and the median length of storage was 20 h. After storage, the median viability and nucleated cell recovery were 100% and 99.5%, respectively. In addition, 98% recovery of CFU-GM was achieved. No clotting or bacterial contamination was detected. All 12 patients studied engrafted promptly. In addition, 124 similarly treated patients were retrospectively analyzed. Of these, 48% required > or = 2 large-volume leukaphereses to achieve the target cell dose. As a result of overnight storage, 150 final products, instead of 224, were processed and cryopreserved. This difference is equivalent to 33% cost savings. Again, all patients were transplanted and engrafted successfully. In conclusion, overnight storage and pooling of two consecutive PBSC products are safe, reduce cost, and allow for optimum laboratory staffing.