Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield

BACKGROUND: In immunomagnetic selection of CD34+ cells from HPC transplants, not all factors that affect yield and purity of CD34+ cells are known. METHODS: Forty-three consecutive procedures of immunomagnetic selection of CD34+ cells from peripheral blood HPCs and bone marrow harvests (autologous harvests, n = 27; allogeneic harvests; n=16) were performed by use of a cell selection system (Isolex 300i, Baxter Immunotherapy). The composition of the starting component and the subsets of CD34+ cells were analyzed for correlation with the yield and purity of the final component. RESULTS: The mean purity of the final components was 84.3 percent (range, 27-99%), and the mean yield was 51.4 percent (range, 9.4-80. 4%). Partial regression analysis showed that, among the factors correlating with purity and/or yield, the RBC volume in the starting fraction had the highest predictive impact on the purity and yield of CD34+ cells, even after the exclusion of procedures using bone marrow harvests as an HPC source (beta coefficient, -0.704; p = 0. 001). CONCLUSION: The use of the Isolex 300i system allows efficient recovery of CD34+ cells in routine selection procedures. The volume of RBCs in the starting component should be minimized to ensure a high yield and purity of the final component.     

Isolation and flow cytometric analysis of T-cell-depleted CD34+ PBPCs

BACKGROUND: To extend allogeneic HPC transplantation to a greater range of patients, the use of partially matched related donors is under development. Because of the inherently higher degree of histoincompatibility in such transplants, there is increased risk of GVHD as well as of graft failure. Ex vivo depletion of donor-derived T-lymphocytes from PBPCs is one of the most effective methods of preventing GVHD. Thus far, individual centers have used custom-developed procedures to deplete the graft of T cells that are responsible for alloreactivity, often employing relatively impure, nonstandardized reagents such as soybean agglutinin and complement. In addition, with improved methods of T-cell depletion, it has been difficult to accurately assess the number of T cells remaining. Because different centers have used different protocols to assay T cells, it has been difficult to reproduce and validate the results between institutions, and this has limited direct comparison of data between centers. STUDY DESIGN AND METHODS: A standardized approach for T-cell depletion was developed by using a Good Manufacturing Practice-manufactured magnetic cell separator (Isolex 300i, Nexell Therapeutics) and commercially available OKT3 antibody. T-cell depletion was performed on PBPCs from six haploidentical donors. RESULTS: CD34+ cell recovery was 47 percent (range, 31-63%) with a median purity of 94 percent (range, 75-99%) and median T-cell log depletion of 4.72 (range, 3.90-5.83). Because this high degree of depletion makes it challenging to accurately quantitate the remaining T cells, two highly sensitive flow cytometric protocols were developed, each of which accurately detects T cells with a sensitivity of 2 per 10,000 (0.02%). The purified CD34+ cells administered to the patients (dose range, 6.13-13.50 x 10(6)/kg) provided rapid neutrophil and platelet engraftment. CONCLUSION: With the Isolex 300i and a MoAb directed against T cells, a high degree of T-cell depletion is obtained. Sensitive, accurate, and reproducible assays have now been developed for T-cell enumeration in these highly purified cell populations.     

Preterm birth and the availability of cord blood for HPC transplantation

BACKGROUND: Cord blood from deliveries at term can be used for HPC transplantation. The objective of this study was to determine the amounts of cord blood nucleated cells (NCs) and HPCs that were collectable from preterm deliveries. STUDY DESIGN AND METHODS: Cord blood collected from preterm deliveries between 22 and 36 weeks of gestation was compared with regard to volume, NC count (/mL), CD34+ cell count (/mL), and the NC and CD34+ cell counts per cord blood sample and at different gestational ages. RESULTS: A correlation was found between gestational age and NC count (r = 0.52, p<0.001), and an inverse relation was found between gestational age and CD34+ cell count (r = - 0.68, p<0.001). The CD34+ cell count per cord blood sample was independent of gestational age (r = - 0.13, p = NS), and no significant difference between early (22-32 week) and late (33-36 week) preterm deliveries was found (p = 0.870). Comparison with published data from cord blood transplantations revealed that up to one-third of preterm samples contained at least as many NCs (or CD34+ cells) as the median cell dose transplanted (calculated for the median recipient weight) in the respective study. Furthermore, 77 percent of all preterm samples contained at least 1 x 10(7) NCs (and 42% at least 1 x 10(5) CD34+ cells) per kg for transplantation in a recipient of 20-kg body weight, which corresponds to the lower threshold of cells per kg in the graft recommended by Eurocord. CONCLUSION: Preterm delivery should not be a reason to exclude cord blood collection if allogeneic cord blood transplantation in a sibling is planned.     

High platelet contamination in progenitor cell concentrates results in significantly lower CD34+ yield after immunoselection

BACKGROUND: Selection of CD34+ cells by specific immunoselection leads to a significant loss of those cells. The factors influencing the yield and purity are not well identified. The results of CD34+ selection from peripheral blood progenitor cells (PBPCs) with high and low platelet contamination that are harvested with two different cell separators are reported. STUDY DESIGN AND METHODS: A progenitor cell concentrator (Ceprate SC, CellPro) was used to select CD34+ cells from 41 PBPC concentrates from 23 consecutive patients with relapsed non-Hodgkin's lymphoma (n = 3), breast cancer (n = 17), and multiple myeloma (n = 3). PBPC collection was performed by using two cell separators (CS3000 Plus, Fenwal: Group A, n = 11; and Spectra, COBE: Group B, n = 9). To reduce platelet contamination in the Spectra PBPC concentrates, an additional low-speed centrifugation was performed before CD34+ cell selection (Group C, n = 3). Leukapheresis components were stored overnight at 4 degrees C and combined with the next day's collection before the CD34+ selection procedure in 19 patients. RESULTS: A median of 1.5 leukapheresis procedures per patient were performed. Pooled PBPC concentrates showed no statistical difference in median numbers of white cells and CD34+ cells in Groups A and B: 3.2 (0.8-9.2) versus 4.4 (1.6-8. 3) x 10(10) white cells per kg and 15.0 (4.7-24.0) versus 12.0 (5. 6-34.0) x 10(6) CD34+ cells per kg. Platelet contamination was significantly higher in Group B: 0.67 (0.15-2.4) versus 2.3 (0.5-7. 1) x 10(11) (p = 0.0273). After the selection process, there was a significantly greater loss of CD34+ cells in Group B than in Group A: 39.1 versus 63.2 percent (p = 0.0070), with a median purity of 78. 0 percent versus 81.0 percent. An additional low-speed centrifugation before CD34+ cell selection seemed to reduce CD34+ cell loss in Group C with 16.9, 31.9, and 37.5 percent, respectively. CONCLUSION: CD34+ cell selection from PBPC concentrates resulted in an increased loss of CD34+ cells in concentrates with a higher platelet content. To improve CD34+ yield, PBPC concentrates with an initially low platelet contamination should be used, or additional low-speed centrifugation should be performed.     

Immunologic changes after transfusion of autologous or allogeneic buffy coat-poor versus white cell-reduced blood to patients undergoing arthroplasty. I. Proliferative T-cell responses and the balance of helper and suppressor T cells

BACKGROUND: Donor white cells (WBCs) contained in red cell (RBC) transfusions are thought to provoke down-regulation of T-cell-mediated immunity. This study investigated this topic in otherwise healthy patients receiving buffy coat-depleted or WBC-filtered RBCs and undergoing standardized perioperative management. STUDY DESIGN AND METHODS: Patients undergoing elective orthopedic surgery (primary hip and knee replacement surgery) were enrolled in a prospective study. Perioperative changes in T-cell proliferation (stimulation with phytohemagglutinin and mixed lymphocyte culture) and T-cell balance (T-lymphocytes, helper T cells, and suppressor T cells) were compared after random assignment to allogeneic buffy coat-depleted (Group 2, n = 8) or WBC-reduced RBC (Group 3, n = 11) transfusion regimens. Recipients of autologous buffy coat-depleted RBC transfusions (n = 15) served as controls (Group 1). RESULTS: Compared to that in autologous transfusion recipients, alloantigen-induced T-cell proliferation was significantly reduced in recipients of allogeneic WBC-reduced RBCs (Day 3, p = 0.0274). After the transfusion of allogeneic buffy coat-depleted RBCs, a weak trend toward decreased T-cell proliferation was observed (p = 0.0933) and the numbers of CD4+ T cells were also significantly lower (Day 7, p = 0.0389). On Day 10, alloantigen-induced T-cell proliferation remained significantly below baseline after transfusion of WBC-reduced RBCs (p = 0.05), the numbers of CD3+ cells decreased in allogeneic RBC recipients (Group 2, p = 0.078; Group 3, p = 0.05), and those of CD8+ cells decreased significantly after the transfusion of allogeneic buffy coat-depleted RBCs (p = 0.0234) concomitant with an increased CD4:CD8 ratio (p = 0.0391). CONCLUSION: Results of the present study confirm the hypothesis of impaired T-cell-mediated immunity after allogeneic transfusion.     

The predictive value of white cell or CD34+ cell count in the peripheral blood for timing apheresis and maximizing yield

BACKGROUND: The collection of peripheral blood stem and progenitor cells (PBPCs) for transplantation can be time-consuming and expensive. Thus, the utility of counting CD34+ cells and white cells (WBCs) in the peripheral blood was evaluated as a predictor of CD34+ cell yield in the apheresis component. STUDY DESIGN AND METHODS: The WBC and CD34+ cell counts in the peripheral blood and the apheresis components from 216 collections were assessed. Sixty-three patients underwent mobilization with chemotherapy plus filgrastim, and 17 patients and 14 allogeneic PBPC donors did so with filgrastim alone. The relationship between the number of WBC and CD34+ cells in the peripheral blood and in the apheresis component was analyzed by using rank correlation and linear regression analysis. RESULTS: The correlation coefficient for CD34+ cells per liter of peripheral blood with CD34+ cell yield (x 10(6)/kg) was 0.87 (n = 216 collections). This correlation existed for many patient and collection variables. However, patients with acute myeloid leukemia had fewer CD34+ cells in the apheresis component at any level of peripheral blood CD34+ cell count. Components collected from patients with CD34+ cell counts below 10 x 10(6) per L in the peripheral blood contained a median of 0.75 x 10(6) CD34+ cells per kg. When the WBC count in the blood was below 5.0 x 10(9) per L, the median number of CD34+ cells in the peripheral blood was 5.6 x 10(6) per L (range, 1.0-15.5 x 10(6)/L). A very poor correlation was found between the WBC count in the blood and the CD34+ cell yield (p = 0.12, n = 158 collections). CONCLUSION: The number of CD34+ cells, but not WBCs, in the peripheral blood can be used as a predictor for timing of apheresis and estimating PBPC yield. This is a robust relationship not affected by a variety of patient and collection factors except the diagnosis of acute myeloid leukemia. Patients who undergo mobilization with chemotherapy and filgrastim also should undergo monitoring of peripheral blood CD34+ cell counts, beginning when the WBC count in the blood exceeds 1.0 to 5.0 x 10(9) per L.     

Dextran sedimentation in a semi-closed system for the clinical banking of umbilical cord blood

BACKGROUND: The results of current processing procedures for reducing volume and recovering HPCs from umbilical cord blood (UCB) before cryopreservation vary. STUDY DESIGN AND METHODS: Dextran was added to bags containing UCB, followed by sedimentation for 30 minutes. The processed UCB was then frozen. RBCs, nucleated cells, MNCs, CD34+ cells, CFUs and long-term culture-initiating cells (LTC-ICs), viability, and sterility were evaluated. Fractionations in ficoll-hypaque and hydroxyethyl starch (HES) were also run in parallel for comparison. RESULTS: The nucleated cell (NC) recovery and RBC depletion were 86.1 percent and 94.3 percent, respectively (n = 50). Sedimentation with dextran also enabled the recovery of 80.7 percent MNCs and 82.6 percent CD34+ cells (n = 30). Postsedimentation samples displayed no impairment of CFU growth (n = 42, 108.7% CFU-C, 104.6% CFU-GEMM, 107% CFU-GM, and 95.7% BFU-E). Long-term cultures on five paired samples before and after sedimentation generated similar numbers of CFU-C each week (p = 0.88). Limiting dilution analysis of 12 paired pre/postsedimentation samples showed comparable median proportions of LTC-ICs (1/6494 vs. 1/5236; p = 0.18). The cell viability of 24 samples of thawed UCB after sedimentation was 90.3 percent (77.5-96%) and the recovery of CFU-C, CFU-GEMM, CFU-GM, and BFU-E of 11 postsedimentation samples was 93.4 percent, 84.9 percent, 92.3 percent, and 83.4 percent, respectively. NC recovery was significantly higher after treatment with dextran than with ficoll-hypaque (n = 30; 88.5% vs. 29.1%; p<0.005) and HES treatment (n = 21; 88.5% vs. 76.4%; p = 0.004). However, MNCs, CD34+ cells, CFUs, LTC-ICs, and RBCs were comparable. Two cycles of dextran sedimentation recovered 93.9 percent of NCs with cell viability of 98.6 percent (96.5-100%), whereas 11.7 percent of RBCs were retained (n = 20). The final yield volume was 33.5 (28-41) mL. CONCLUSION: In a semi-closed system, dextran sedimentation enabled volume reduction of UCB without significant quantitative and qualitative losses of HPCs.     

Ex vivo evaluation of PBMNCs collected with a new cell separator

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.     

Uncontrolled-rate freezing and storage at -80 degrees C, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations

BACKGROUND: Although controlled-rate freezing and storage in liquid nitrogen are the standard procedure for peripheral blood progenitor cell (PBPC) cryopreservation, uncontrolled-rate freezing and storage at -80 degrees C have been reported. STUDY DESIGN AND METHODS: The prospective evaluation of 109 autologous PBPC transplantations after uncontrolled-rate freezing and storage at -80 degrees C of apheresis products is reported. The cryoprotectant solution contained final concentrations of 1-percent human serum albumin, 2.5-percent hydroxyethyl starch, and 3.5-percent DMSO. RESULTS: With in vitro assays, the median recoveries of nucleated cells (NCs), CD34+ cells, CFU-GM, and BFU-E were 60.8 percent (range, 11.2-107.1%), 79.6 percent (6.3-158.1%), 35.6 percent (0.3-149.5%), and 32.6 percent (1.7-151.1%), respectively. The median length of storage was 7 weeks (range, 1-98). The median cell dose, per kg of body weight, given to patients after the preparative regimen was 6.34 x 10(8) NCs (range, 0.02-38.3), 3.77 x 10(6) CD34+ cells (0.23-58.5), and 66.04 x 10(4) CFU-GM (1.38-405.7). The median time to reach 0.5 x 10(9) granulocytes per L, 20 x 10(9) platelets per L, and 50 x 10(9) reticulocytes per L was 11 (range, 0-37), 11 (0-129), and 17 (0-200) days, respectively. Hematopoietic reconstitution did not differ in patients undergoing myeloablative or nonmyeloablative conditioning regimens before transplantation. CONCLUSION: This simple and less expensive cryopreservation procedure can produce successful engraftment, comparable to that obtained with the standard storage procedure.     

Immune reconstitution after autologous selected peripheral blood progenitor cell transplantation: comparison of two CD34+ cell-selection systems

BACKGROUND: Selection of CD34+ PBPCs has been applied as a method of reducing graft contamination from neoplastic cells. This procedure seems to delay lymphocyte recovery, while myeloid engraftment is no different from that with unselected PBPC transplants. STUDY DESIGN AND METHODS: Lymphocyte recovery was studied in two groups of patients who underwent autologous CD34+ PBPC transplant with two different technologies (Ceprate SC, Cellpro [n = 17]; CliniMACS, Miltenyi Biotech [n = 13]). The median number of CD34+ cells transfused was 3.88 x 10(6) per kg and 3.32 x 10(6) per kg, respectively. Residual CD3 cells x 10(6) per kg were 4.97 and 0.58, respectively (p = 0.041). Residual CD19 cells x 10(6) per kg were 1.33 and 0.73, respectively (NS). RESULTS: No differences were found between the two groups in total lymphocyte recovery to >0.5 x 10(9) per L, which achieved a stable count by Day 30. During the study period, the CD4+ cell count remained below 0.2 x 10(9) per L, and the B-cell subset showed a trend toward normalization. CD3/HLA-DR+ and CD16/56 increased markedly in both groups by Day 30. An increase in CMV (13%) and adenovirus (17.4%) infection was found in both groups. CONCLUSION: Both CD34+ cell selection technologies used here determined an excellent CD34+ cell purity and an optimal depletion of T cells. The high rate of viral complications is probably due to the inability of residual T cells left from the CD34+ cell selection to generate, immediately after transplant, an adequate number of virus-specific lymphocytes.     

Collection of peripheral blood progenitor cells with an automated leukapheresis system

BACKGROUND: Apheresis devices designed for the collection of mature blood elements are being used for the collection of peripheral blood progenitor cells (PBPCs).The collection of PBPCs differs from that of other cells in the rarity of the target cell and in the fact that donors may undergo several days of collection. A consequence of this process may be a depletion of blood cells such as platelets from the blood. The disposable set and operating software for an apheresis device (Spectra, COBE BCT) was modified by the manufacturer to automate the collection of PBPCs and reduce the collection of unwanted blood cells. STUDY DESIGN AND METHODS: A study was initiated to compare the collection of PBPCs with the new device, the AutoPBSC (version [V]6.0 with AutoPBSC tubing set), and that with the MNC (mononuclear cell) procedure (V4.7 with white cell tubing set), for patients and healthy donors. RESULTS: Patients whose blood was processed by either theV6.0 orV4.7 procedure achieved the target dose of 5 x 10(6) CD34+ cells per kg of patient weight in similar numbers of procedures, even though the calculated collection efficiency for CD34+ cells using the automated V6.0 procedure was significantly less than that with the V4.7 procedure for both allogeneic donors and patients donating PBPCs. The collection efficiency for platelets was lower with the V6.0 procedure, and components collected in this manner contained fewer platelets. Apheresis by the V6.0 procedure required 30 to 60 more minutes per procedure than apheresis by the V4.7 procedure. Review of engraftment kinetics after transplantation did not reveal any effect of the collection procedure on recipients of either allogeneic or autologous transplants. CONCLUSION: The collection efficiencies of the V6.0 procedure for both CD34+ cells and mature blood cells are lower than those of the V4.7 procedure.The lower collection efficiency for platelets results in a smaller drop in peripheral blood platelet count after the procedure.The automated features of the V6.0 procedure may simplify PBPC collection, but this procedure requires a longer apheresis.     

A controlled comparison of two different clinical grade devices for CD34+ cell selection of autologous blood stem cell grafts

Six patients who were to undergo autologous PBSC transplantation with positively selected CD34+ cells were included in this study to compare the efficiency of two devices for clinical grade stem cell selection, the Isolex 300i (Baxter, Munich, Germany) and CEPRATE SC (CellPro, Bothell, WA). PBSC were mobilized by chemotherapy and G-CSF and were collected by leukapheresis on a CS3000 cell separator on 2 consecutive days. The two apheresis products were pooled for CD34 selection. The pooled apheresis products from each patient were divided into two equal portions to be separated on each of the two devices. Cell selection was performed according to the manufacturers' instructions. Enumeration of CD34+ cells was performed by flow cytometry using the HPCA-2 MAb. Purity and yield were significantly better with Isolex than with CEPRATE. Median purity was 93.0% (range 80%-98%) for Isolex and 61.5% (range 27%-72%) for CEPRATE (p = 0.03); median yields for Isolex and for CEPRATE were 48.0% (range 18%-73%) and 23.0% (range 17%-29%), respectively (p = 0.03). The number of CD34+ cells/kg body weight was also significantly higher with Isolex (median 3.8x10(6), range 1.7-5.2) compared with CEPRATE (median 2.35x10(6), range 0.7-4.3) (p = 0.03). Thus, the Isolex 300i device gave products of higher purity and recovered a higher proportion of the CD34+ cells in the harvest before separation. The yield was still poor with both devices, however, and further optimization of the technique for clinical grade stem cell selection is warranted.     

In vitro collection and posttransfusion engraftment characteristics of MNCs obtained by using a new separator for autologous PBPC transplantation

BACKGROUND: A clinical study was performed to evaluate the peripheral blood progenitor cell (PBPC) collection, transfusion, and engraftment characteristics associated with use of a blood cell separator (Amicus, Baxter Healthcare). STUDY DESIGN AND METHODS: Oncology patients (n = 31) scheduled for an autologous PBPC transplant following myeloablative therapy were studied. PBPCs were mobilized by a variety of chemotherapeutic regimens and the use of G-CSF. As no prior studies evaluated whether PBPCs collected on the Amicus separator would be viable after transfusion, to ensure patient safety, PBPCs were first collected on another cell separator (CS-3000 Plus, Baxter) and stored as backup. The day after the CS-3000 Plus collections were completed, PBPC collections intended for transfusion were performed using the Amicus instrument. For each transplant, >2.5 x 10(6) CD34+ PBPCs per kg of body weight were transfused. RESULTS: Clinical data collected on the donors immediately before and after PBPC collection with the Amicus device were comparable to donor data similarly obtained for the CS-3000 Plus collections. While the number of CD34+ cells and the RBC volume in the collected products were equivalent for the two devices, the platelet content of the Amicus collections was significantly lower than that of the CS-3000 Plus collections (4.35 x 10(10) platelets/bag vs. 6.61 x 10(10) platelets/bag, p<0.05). Collection efficiencies for CD34+ cells were 64 +/- 23 percent for the Amicus device and 43 +/- 14 percent for the CS-3000 Plus device (p<0.05). The mean time to engraftment for cells collected via the Amicus device was 8.7 +/- 0.7 days for >500 PMNs per microL and 9.7 +/- 1.5 days to attain a platelet count of >20,000 per microL-equivalent to data in the literature. No CS-3000 Plus backup cells were transfused and no serious adverse events attributable to the Amicus device were encountered. CONCLUSIONS: The mean Amicus CD34+ cell collection efficiency was better (p<0.05) than that of the CS-3000 Plus collection. Short-term engraftment was durable. The PBPCs collected with the Amicus separator are safe and effective for use for autologous transplant patients requiring PBPC rescue from high-dose myeloablative chemotherapy.     

Inverse relationship between patient peripheral blood CD34+ cell counts and collection efficiency for CD34+ cells in two automated leukapheresis systems

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of > or =2.5 x 10(6) CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per microL needed to collect a single-apheresis autograft. However, at > or =40 circulating CD34+ cells per microL, both cell separators achieved the target of > or =2.5 x 10(6) CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at <40 cells per microL to 51.2 percent at > or =40 cells per microL on the AS104 (p = 0.017) and from 50.1 percent to 41.3 percent on the Spectra (p = 0.033). CONCLUSION: Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.     

Allogeneic blood progenitor cell collection in normal donors after mobilization with filgrastim: the M.D. Anderson Cancer Center experience

BACKGROUND: Information on the safety and efficacy of allogeneic peripheral blood progenitor cell (PBPC) collection in filgrastim-mobilized normal donors is still limited. STUDY DESIGN AND METHODS: The PBPC donor database from a 42-month period (12/94-5/98) was reviewed for apheresis and clinical data related to PBPC donation. Normal PBPC donors received filgrastim (6 microg/kg subcutaneously every 12 hours) for 3 to 4 days and subsequently underwent daily leukapheresis. The target collection was > or =4 x 10(6)CD34+ cells per kg of recipient's body weight. RESULTS: A total of 350 donors were found to be evaluable. Their median age was 41 years (range, 4-79). Their median preapheresis white cell count was 42.8 x 10(9) per L (range, 18.3-91.6). Of these donors, 17 (5%) had inadequate peripheral venous access. Leukapheresis could not be completed because of apheresis-related adverse events in 2 donors (0.5%). Of the 324 donors evaluable for apheresis yield data, 221 (68%) reached the collection target with one leukapheresis. The median CD34+ cell dose collected (first leukapheresis) was 462 x 10(6) (range, 29-1463).The main adverse events related to filgrastim administration in donors evaluable for toxicity (n = 341) were bone pain (84%), headache (54%), fatigue (31%), and nausea (13%). These events were rated as moderate to severe (grade 2-3) by 171 (50%) of the donors. In 2 donors (0.5%), they prompted the discontinuation of filgrastim administration. CONCLUSION: PBPC apheresis for allogeneic transplantation is safe and well tolerated. It allows the collection of an "acceptable" PBPC dose in most normal donors with one leukapheresis, with minimal need for invasive procedures.     

Comparison of intermittent- and continuous-flow cell separators for the collection of autologous peripheral blood progenitor cells in patients with hematologic malignancies

BACKGROUND: The transplantation of autologous peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy is a valuable therapy for patients with hematologic and solid malignancies. Several methods are used for harvesting PBPCs. The efficiency of intermittent- and continuous-flow blood cell separators in collecting progenitor cells from the blood of patients undergoing myeloablative treatment for cancer was compared. STUDY DESIGN AND METHODS: PBPC components (n = 133) were obtained from 72 patients by leukapheresis with continuous-flow machines (Spectra, COBE; CS 3000 Plus, Baxter) and with an intermittent-flow machine (MCS 3P, Haemonetics). The data were analyzed retrospectively. Blood samples obtained from the patients before leukapheresis and samples of the leukapheresis components themselves were analyzed for their content of RBCs, WBCs, platelets, and CD34+ cells. RESULTS: The Spectra processed more than twice the blood volume in the shortest time (15 L in 178 min), whereas the Baxter CS 3000 Plus (10 L in 185 min) and the MCS 3P (4.8 L in 239 min) processed significantly smaller volumes in a longer time. The mean ACD consumption was 403 mL with the MCS 3P, 900 mL with the CS 3000 Plus, and 1000 mL with the Spectra. The product volumes were 50 mL (CS 3000 Plus), 69 mL (MCS 3P), and 166 mL (Spectra). In all groups, differences in the preapheresis hemograms were not significant, but the Spectra group had fewer CD34+ cells than the other groups. Despite this, the differences in the number of CD34+ cells in the leukapheresis components of all groups were without statistical significance. In the Spectra group, the collection of MNCs of 104 percent and CD34+ cells of 154 percent was significantly more efficient than that in the MCS 3P group (42.2% and 56%, respectively) or the CS 3000 Plus group (50.8% and 47.15%) as related to the patients' blood volume. CONCLUSION: PBPC collection can be performed successfully with continuous-flow and intermittent-flow blood cell separators. The Spectra had the best recovery of CD34+ cells within the shortest time. Leukapheresis with the MCS 3P is indicated if only a single venous access is available.     

Transfusion of buffy coat-depleted blood components and risk of postoperative infection in orthopedic patients

BACKGROUND: Allogeneic blood transfusions have been reported to increase susceptibility to postoperative infection, but the findings were inconclusive. This study was designed to investigate the effect of buffy coat-depleted allogeneic and autologous transfusion on postoperative infection in patients undergoing orthopedic surgery. STUDY DESIGN AND METHODS: Patients (n = 385) undergoing elective orthopedic surgery (primary and revision joint replacement, spinal, or pelvic surgery) were included in a prospective observational study of the incidence of postoperative infection between April and December 1996. Infection rates in patients who received allogeneic buffy coat-depleted blood transfusions were compared with those in patients who received no transfusion or only autologous (buffy coat-depleted) blood. RESULTS: Patients without exposure to allogeneic blood (no blood or only autologous blood) had an infection rate of 3.9 percent, as compared to a rate of 12.2 percent for those with exposure to allogeneic blood (allogeneic blood, autologous plus allogeneic blood) (odds ratio 3.442; 95% CI, 1.349-10.40; p = 0.006). Of the 385 study patients, 309 underwent primary hip or knee replacement surgery. In this homogeneous subgroup, the postoperative infection rate was 4.6 percent after no transfusion or autologous transfusion and 11.9 percent after allogeneic transfusion (odds ratio 2.827; 95% CI 1.059-8.799; p = 0.036). Multivariate regression analysis confirmed buffy coat-depleted allogeneic blood transfusion as an independent variable associated with high risk for postoperative infection. CONCLUSION: Buffy coat-depleted allogeneic blood transfusion increases the incidence of postoperative infection in patients undergoing uncontaminated orthopedic surgery.     

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.     

Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device.

The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.     

A cell-kinetic model of CD34+ cell mobilization and harvest: development of a predictive algorithm for CD34+ cell yield in PBPC collections

BACKGROUND: Mobilization and homing of PBPCs are still poorly understood. Thus, a sufficient algorithm for the prediction of PBPC yield in apheresis procedures does not yet exist. STUDY DESIGN AND METHODS: The decline of CD34+ cells in the peripheral blood during apheresis and their simultaneous increase in the collection bag were determined in a prospective study of 18 consecutive apheresis procedures. A cell-kinetic, four-compartment model describing these changes was developed. Retrospective data from 136 apheresis procedures served to further improve this model. A predictive algorithm for the yield was developed that considered the sex, weight, and height of the patient, the number of CD34+ cells in peripheral blood before apheresis, the inlet flow, and the duration of the apheresis. The accuracy of this algorithm was evaluated by comparison of the predicted and the observed yields of CD34+ cells in 105 prospective autologous and 148 retrospective allogeneic apheresis procedures. RESULTS: The correlation between predicted and observed yields was good for the autologous and allogeneic groups with a correlation coefficient (r) of 0.8979 and 0.8311 (p<0.0001), respectively. The regression is described by the equations log (measured value [m]) = 1.0118 + 0.8595 x log (predicted value [p]) for the autologous and log (m) = 2.226 + 0.7559 x log (p) for the allogeneic group. The respective equations for the zero-point regression are log (m) = 1.014 x log (p) and log (m) = 1.026 x log (p). The probability that the measured value was 90 percent or more of the predicted value was 83.8 percent for the autologous and 90.5 percent for the allogeneic apheresis procedures. CONCLUSION: The predictive accuracy of the algorithm and the slope of the zero-point regression curve were higher for allogeneic than autologous PBPC collections. The predictive algorithm may be a useful tool in PBPC harvest, enabling the adaptation of the size of the apheresis to the needs of each patient.