流式细胞术计数CD34阳性细胞的标准化与质量控制

用流式细胞术计数脐带血,外周血及其采集物中的ＣＤ３４＾＋细胞数已被普遍采用,然而,不同实验之间的ＣＤ３４＾＋细胞计数差异非常大,本就影响结果的主要因素,目前国际上采用的标准化方案和不同方案之间的比较进行综述,我们实验室应用ＰｒｏＣＯＵＮＴ试剂盒检测了４５例脐带血,１２份正常骨髓和４例外周血采集物中的ＣＤ３４＾＋细胞的数量,结果表明ＰｒｏＣＯＵＮＴ为标准化程度较高的方法,有利于减少不同实验之间ＣＤ３４＾＋细胞计数的差异。     

Semi-automated flow cytometric analysis of CD34-expressing hematopoietic cells in peripheral blood progenitor cell apheresis products

BACKGROUND: The measurement of CD34+ cells is the most important step in the quality control of peripheral blood progenitor cell apheresis products. For this purpose, flow cytometry is applied. Recently, a new test kit has been introduced for the enumeration of CD34-expressing cells, in combination with software support for semi-automation of data acquisition and analysis. STUDY DESIGN AND METHODS: This study evaluated the ProCOUNT kit. Ninety samples obtained from peripheral blood progenitor cell apheresis products from 39 patients with hemato-oncologic diseases were analyzed. For data acquisition and analysis, ProCOUNT software was used. Data comparison was performed with parallel measurements according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) guidelines and the German reference protocol for analysis of CD34-expressing cells. RESULTS: Correlation of the German and ISHAGE techniques was excellent (r2 = 0.99). The initial correlation coefficient of ProCOUNT analysis with the German protocol was r2 = 0.89. In 21 (23.3%) of 90 ProCOUNT analyses, a warning message was encountered from the ProCOUNT software. Following manual reevaluation of these data with CellQUEST software, a correlation of r2 = 0.96 with the German protocol and r2 = 0.97 with the ISHAGE analyses was obtained. ANOVA testing revealed significant differences between ProCOUNT and ISHAGE techniques (p<0.05) and between ProCOUNT and the German protocol (p<0.05). No statistically significant difference between ISHAGE and German protocol was observed (p = 0.19). CONCLUSION: The ProCOUNT kit and software for semi-automated data acquisition and analysis represents a further step toward standardization of CD34 cell quantitation in peripheral blood progenitor cell apheresis products. However, the occurrence of software warnings is high, and analysis or data reevaluation by experienced staff is still mandatory. Therefore, currently there is no definite advantage of the kit and software over the existing guidelines for CD34+ analysis in peripheral blood progenitor cell grafts.     

Toward standardization of CD34+ cell enumeration: an international study

BACKGROUND: An international multicenter study, involving six sites in North America and six sites in Europe, was undertaken to assess the performance of standardized methods for the enumeration of CD34+ cells in peripheral blood over the dynamic range from 200 cells per microL to zero. Two commercially available techniques were studied, a flow cytometry method and a microvolume fluorimetry method. STUDY DESIGN AND METHODS: Coded samples were centrally prepared and sent to test sites by overnight mail. Samples included internal replicates, linear dilutions, and specimens at the lower limit of detection. In addition, commercially available reagent positive control cells were sent to a subset of laboratories. RESULTS: Over the sample range studied, the intersite precision among different laboratories was good with coefficients of variation ranging from 14 percent to 24 percent for microvolume fluorimetry and from 20 percent to 31 percent for flow cytometry. Intrasite precision ranged from 7 percent to 21 percent. Test linearity was excellent with sites demonstrating a mean r2 = 0.992 for microvolume fluorimetry and r2 = 0.984 for flow cytometry. The lower limit of detection was 5 CD34+ cells per microL for both commercial assays. Over the range of 5 to 50 CD34+ cells per microL, the microvolume fluorimetry assay reported slightly higher values than the flow cytometry assay. Preliminary analysis of reagent positive control cells showed very good precision and accuracy. CONCLUSIONS: Standardization of CD34+ cells enumeration is improving and commercially available assays provide accurate and precise methods. More investigation of reagent positive control cells is warranted.     

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.     

Quality assurance of progenitor cell content of apheresis products: a comparison of clonogenic assays and CD34+ enumeration. The Canadian Apheresis Group and Canadian Bone Marrow

The most common methods used for evaluation of the haematopoietic stem cell content of peripheral blood apheresis products are the colony forming cell assay and the enumeration of CD34+ cells by flow cytometry. The Canadian Apheresis Group and the Canadian Bone Marrow Transplant Group established a multicentre study to compare the reproducibility of colony forming cell assays and CD34+ enumeration by flow cytometry in six transplant centres routinely performing haematopoietic stem cell apheresis. Over a 5-month period in 1996, 31 fresh apheresis samples were shipped by overnight courier for testing at six centres to perform CD34+ enumeration by flow cytometry and clonogenic assays. The mean coefficient of variation and range for the following assays were: cell count 36% (2.6-148%), CFU-GM 82% (46-123%), CD34+ absolute/kg 60% (14-174%) and CD34+ per cent 42% (12-84%). The wide variation in cell count in this pilot study highlights the difficulties related to provision of samples for quality assessment programmes. Results showed poor interinstitutional reproducibility even among selected samples with similar cell counts for both CFC and CD34+ assays demonstrating the need for development and implementation of an interinstitutional quality assurance programme for haematopoietic stem cell assessment. Provision of a reliable source of testing material will be a necessary next step.     

不同标记法及去红细胞法对检测微量脐血CD34+造血干细胞含量的影响

目的 采用ISHAGE(international society of hematotherapy and graft engineering)法和常用的CD34^ 造血干细胞流式检测方法对同一脐血样品进行比较实验，确定更适用于微量脐血CD34 ^ 造血干细胞含量检测的方法。方法 分别采用ISHAGE造血干细胞计数协会推荐方法、低渗NH4C1去红细胞CD45／CD34双色标记法(NH4C1双标法)、OptilyseC溶血剂去红细胞CD34单色标记法(OptilyseC单标法)、低渗NH4C1去红细胞CD34单色标记法(NH4C1单标法)、羟乙基淀粉沉淀去红细胞CD45／CD34双色标记法(HES双标法)检测脐血造血干细胞含量，并比较不同方法测得数据的差异。结果 8份脐血标本用ISHAGE方法测得CD34^ 造血干细胞含量的中位数为0．278％，NH4C1双标法测得结果为0．297％，与ISHAGE方法相比差异无显性。用HES双标、OptilyseC单标法和NH4C1单标法测得的结果分别为5．715％，0．391％和0．741％，与ISHAGE方法相比差异有显性。结论 ISHAGE方法是一种公认的准确性高，重复性好的检测造血干细胞方法。OptilyseC单标法、NH4C1单标法和HES双标法测定结果比实际值偏高。     

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots

BACKGROUND: The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability.
STUDY DESIGN AND METHODS: The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration.
RESULTS: Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 ± 0.22 percent, the results after thawing were 0.98 ± 0.65 and 0.57 ± 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition.
CONCLUSIONS: The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.     

Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood

BACKGROUND: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation. STUDY DESIGN AND METHODS: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files. RESULTS: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per microL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per microL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files. CONCLUSION: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.     

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.     

Kinetics of PBPC mobilization by cyclophosphamide, as compared with that by epirubicin/paclitaxel followed by G-CSF support: implications for optimal timing of PBPC harvest

BACKGROUND: Limited information is available on the mobilization kinetics of autologous PBPCs after induction with various chemotherapy regimens. With PBPC mobilization in patients with breast cancer used as a model for chemotherapy-induced PBPC recruitment, the kinetics of progenitor cells mobilized either with cyclophosphamide (CY) or epirubicin/paclitaxel (EPI-TAX) followed by the administration of G-CSF was compared. STUDY DESIGN AND METHODS: The study included a total of 86 patients with breast cancer (stage II-IV) receiving either CY (n = 39) or EPI-TAX (n = 47), both followed by G-CSF support. The progenitor cell content in peripheral blood and apheresis components was monitored by flow cytometric enumeration of CD34+ cells. PBPC collection was started when the threshold of >20 x 10(6) CD34+ cells per L of peripheral blood was reached. RESULTS: The PBPC collection was begun a median of 9 days after the administration of EPI-TAX followed by G-CSF support, as compared to a median of 13 days after mobilization with CY plus G-CSF. After treatment with CY, the total numbers of PBPCs peaked on Day 1 of apheresis, and they rapidly declined thereafter. In contrast, treatment with EPI-TAX followed by G-CSF administration led to a steady mobilization of CD34+ cells during leukapheresis. The difference in the mobilization patterns with CY and EPI-TAX resulted in a greater yield of CD34+ cells per L of processed blood volume. Compared to EPI-TAX, mobilization with CY required the overall processing of 30 percent less whole-blood volume to reach the target yield of > or = 10 x 10(6) CD34+ cells per kg of body weight. After a median of three apheresis procedures, however, both CY+G-CSF and EPI-TAX+G-CSF were equally effective in obtaining this target yield. CONCLUSION: These results imply that specific PBPC mobilization as part of a given chemotherapy regimen should be taken into consideration before the planning of a PBPC harvest.     

Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment

BACKGROUND: Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7‐aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions. STUDY DESIGN AND METHODS: To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony‐forming units (CFU) to determine if graft potency can be predicted using a “functional flow cytometry” approach. RESULTS: Significant numbers of CD34+ AnnV+ events were found within the 7AAD‐gated population. Nonapoptotic cell dose (CD34+ AnnV?) correlated well with CFUs in both a small‐scale (n = 10) and a large‐scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration. CONCLUSION: Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.     

Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.     

Validation of the Nordic flow cytometry standard for CD34+ cell enumeration in blood and autografts: report from the third workshop. Nordic Stem Cell Laboratory Group

Following two workshops on standardization of enumeration of CD34+ cells in blood and leukapheresis products, the Nordic Stem Cell Laboratory Group (NSCL-G) evaluated the Milan/Mulhouse/Nordic standard in clinical practice during the third workshop (WS-III). This report documents an acceptable interlaboratory variation in the most clinically active laboratories, with a coefficient of variation (CV) below 0.19 in 7 of 8 analyses performed. The introduction of a pan-CD45 antibody in the analysis did not improve the CV. Comparison of two different CD34 class II antibodies on a total of 99 samples and procedures with and without washing on a total of 96 samples revealed a significant correlation (r2 >0.99) for all analyses. Finally, subset analysis of uncommitted and lineage-specific progenitors revealed major gating difficulties, indicating that further improvements are necessary. In an analysis of more than 600 patients undergoing mobilization and harvest of blood progenitors, with about 500 patients autografted, we found a significant correlation between blood levels of CD34+ cells and recovery of CD34+ cells from each harvest as well as between CD34+ cell number reinfused and time to neutrophil and platelet recovery. This report documents for the first time that the very simple Milan/Mulhouse method (termed The Nordic Standard) can be used by a group of laboratories to obtain important clinical information. Consequently, we consider this method as the conventional method in quality assessment of autografts, which should provide a benchmark for development of second-generation improvements.     

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.     

Instrument- and protocol-dependent variation in the enumeration of CD34+ cells by flow cytometry

BACKGROUND: The information regarding the minimum number of CD34+ cells that are necessary to reconstitute hematopoiesis in patients undergoing peripheral blood progenitor cell transplantation is quite controversial. Some of the differences in these figures might be due to the selection of antibodies, staining protocols, and acquisition strategies for the flow cytometric enumeration of these cells. STUDY DESIGN AND METHODS: Twenty-seven human umbilical cord blood samples and 33 leukapheresis products were consecutively collected for this study. Cells were stained following two different protocols, both using monoclonal antibodies to CD45 and CD34, and analyzed by the same operator in two different flow cytometers to enumerate the percentage of CD34+ mononuclear cells. RESULTS: Relevant differences in the proportion of cells were encountered, and the correlation between the results yielded by both instruments and protocols, although statistically valid, was suboptimal. CONCLUSIONS: Both interinstrument and interprotocol variation can provide additional explanation for the redundantly reported discrepancies concerning the numbers of CD34 cells that suffice to secure hemopoietic grafting. These results point to the need for new and different standardization approaches in this clinically relevant field.     

Isolation of CD34+ progenitor cells from peripheral blood by use of an automated immunomagnetic selection system: factors affecting the results

BACKGROUND: The isolation of CD34+ cells from mobilized peripheral blood is being increasingly used in the setting of allogeneic or autologous hematopoietic cell transplantation. Investigation of variables that may influence the effectiveness of CD34+ cell selection is of interest. STUDY DESIGN AND METHODS: Fifty-one CD34+ cell selections from peripheral blood progenitor cells (PBPCs) (39 allogeneic and 12 autologous) were performed using a magnetic cell separator (Isolex 300i, Baxter), including version 2.0 software. The results obtained were analyzed for different processing variables. The feasibility of transplanting these isolated CD34+ cells was also analyzed. RESULTS: The isolated CD34+ cell fraction had a median purity of 88.9 percent (range, 47.8-98.3). The median recovery of CD34+ cells was 45.1 percent (13.8-76.2), and the median colony-forming unit- granulocyte-macrophage (CFU-GM) content was 17. 2 percent (0.8-58.6). Logarithms of T- and B-cell depletion had median values of 3.7 and 2.8, respectively. The version 2.0 software of the Isolex 300i gave a higher CD34+ cell recovery in the enriched cell fraction (median 57.8%) than did version 1.11 (39.4%) or 1.12 (44.4%) (p = 0.01). The use of recombinant human deoxyribonuclease I during cell processing yielded more CD34+ cells (53% vs. 41%, p = 0. 01) and higher purity (92.8% vs. 87%, p = 0.03). There was a correlation between the percentage of CD34+ cells labeled with the monoclonal antibody 8G12 clone and the percentage of CD34+ cells labeled with the monoclonal antibody used during the processing technique (9C5 clone) in the initial, enriched, and depleted CD34+ cell fractions (R(2) = 0.95; 0.92; 0.78, p< 0.005, respectively). Median times for recovering >0.5 x 10(9) per L of granulocytes and >20 x 10(9) per L of platelets were 13 and 16 days in the allograft patients and 13 and 14 days in the autograft patients. CONCLUSION: CD34+ cells can be highly and effectively isolated from allogeneic and autologous grafts by use of this automated technique, with a high grade of T- and B-cell depletion. These purified CD34+ cell components can engraft normally.     

Preapheresis peripheral blood CD34+ mononuclear cell counts as predictors of progenitor cell yield

BACKGROUND: Peripheral blood progenitor cells, harvested by apheresis after mobilization, provide rapid hematologic recovery after high-dose chemotherapy. However, because harvesting these cells is expensive and time-consuming, there has been much interest in optimizing collection protocols. An investigation was made to determine whether, in this clinical setting, peripheral blood progenitor cell yields may be predicted from preapheresis progenitor cell counts, allowing the length of each procedure to be "fine tuned" to achieve specific target goals. STUDY DESIGN AND METHODS: Preapheresis peripheral blood CD34+ cell and total colony-forming cell counts were assessed before 78 peripheral blood progenitor cell collections from 13 consecutive patients were performed. Preapheresis counts were correlated with actual progenitor cell yields. Factors affecting this correlation were analyzed. RESULTS: With the use of linear regression analysis preapheresis progenitor cell counts were found to correlate significantly but weakly with actual yields per kg of body weight per liter of blood processed (CD34+ cells: r = 0.43; colony-forming cells: r = 0.56). Further analysis revealed two possible causes: 1) circulating progenitor cell concentrations fluctuate widely during harvest, which implies that preapheresis counts are not representative of actual concentrations during apheresis, and 2) the efficiency with which apheresis machines extract mononuclear cells varies greatly between procedures. CONCLUSION: Preapheresis CD34+ and colony-forming cell counts correlated poorly with subsequent yields in this clinical setting, which suggests that it is not practical to use such counts to predict with certainty the length of apheresis needed to achieve a target yield.     

Minimal number of circulating CD34+ cells to ensure successful leukapheresis and engraftment in autologous peripheral blood progenitor cell transplantation

BACKGROUND: The number of peripheral blood (PB) CD34+ cells has been widely used to monitor the timing of leukapheresis for autologous transplantation. However, no cutoff value for CD34+ cells in PB has been defined as a guideline for the identification of patients in whom the harvest would be effective and those in whom there was a high probability of failure. STUDY DESIGN AND METHODS: The present study investigated the best threshold of CD34+ cells in PB for successful harvesting and engraftment, using 263 PB samples with their corresponding leukapheresis components. In addition, that measure has been compared to other commonly used criteria such as the white cell count, the number of mononuclear cells, and the number of colony- forming units-granulocyte macrophage in PB. RESULTS : Time to engraftment of both granulocytes and platelets was significantly influenced by the number of CD34+ cells transfused, but all patients receiving > or = 0.75 × 10(6) CD34+ cells per kg achieved engraftment within a reasonable number of days (> 0.5 × 10(9)/L granulocytes by Day 11 and > 20 × 10(9)/L platelets by Day 13). A clear correlation between the number of CD34+ cells per microL in PB and of CD34+ cells per kg collected was found at each apheresis (r = 0.9, p < 0.0001). Moreover, the number of CD34+ cells per microL measured in PB the day the first leukapheresis was initiated displayed an excellent correlation with the total amount of CD34+ cells per kg finally collected (r = 0.81, p < 0.0001). On the basis of the regression curve obtained and the clinical engraftment results, it was found that the presence of > 5 CD34+ cells per microL in PB ensured a good yield from the harvest in 95 percent of patients and would avoid an unsuccessful harvest in 81 percent of cases. CONCLUSION: A dose of only 0.75 × 10(6) CD34+ cells per kg guarantees hematopoietic recovery within a reasonable number of days. To initiate a leukapheresis from which enough progenitor cells may confidently be obtained, a minimum of 5 CD34+ cells per microL in PB is required.     

A sensitive quantitative single-platform flow cytometry protocol to measure human platelets in mouse peripheral blood

BACKGROUND: The NOD/SCID mouse is a widely used model for human cord blood (CB) transplantation. Engraftment is generally estimated with semiquantitative methods, measuring the percentage of human cells among mouse cells. To compare protocols aiming to improve hematopoietic recovery, quantitative methods to enumerate human cells would be preferred. This study describes a single-platform protocol to count human platelets (hPLTs) after transfusion and CB transplantation in the peripheral blood (PB) of the mouse. METHODS: With an anti-human CD41 antibody against hPLTs and counting beads, the sensitivity to detect hPLTs in mouse blood by flow cytometry was validated. PLT recovery after hPLT transfusions and PLT kinetics after transplantation with CB CD34+ cells was followed in time in NOD/SCID mice. RESULTS: hPLTs could be reliably detected to a level as low as 1 PLT per microL with this single-platform protocol, what appeared to be at least 10 times more sensitive than detection with the dual-platform protocol. To verify the applicability for mouse studies, hPLTs were measured serially in transfusion and transplantation studies in NOD/SCID mice. The results showed that earlier detection of PLT recovery was feasible with the single-platform protocol. CONCLUSION: A single-platform flow cytometry method can repeatedly measure low numbers of circulating hPLTs in the PB of the same mouse. This method may be helpful in search of new protocols aiming at accelerating PLT recovery after CB transplantation, but also in a number of clinical settings, such as monitoring PLT reconstitution after hematopoietic stem cell transplantation.