Bone Marrow Transplantation, (1998) 22, 125–130 1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt Peripheral blood CD34+ cell count reliably predicts autograft yield P Chapple1,2, HM Prince1, M Quinn1, I Bertoncello3, S Juneja1,2, M Wolf1, H Januszewicz1, M Brettell1, J Gardyn1, C Seymour1 and D Venter2 1Blood and Marrow Transplant Service and 2Department of Pathology, Division of Haematology and Medical Oncology and 3Stem Cell Biology Laboratory, Division of Research, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia Summary: routinely at many centers with threshold values of 2– 5 × 106 CD34+ cells/kg predicting rapid and durable A reliable measure to predict peripheral blood progeni- engraftment following PBPCT.4 tor cell (PBPC) autograft CD34+ cell content is required However, obtaining PBPCs by leukapheresis is expens- to optimize the timing of PBPC collection. We prospec- ive and there is morbidity, albeit small, associated with the tively examined the peripheral blood (PB) CD34+ cell procedure. Consequently, a reliable assay to predict the count in 59 consecutive patients with various malig- CD34+ cell yield is required to optimize the timing of col- nancies and analyzed the correlation between the PB lections. To determine the optimal time of PBPC collection, CD34+ cell count and various parameters in the PBPC we prospectively examined a number of parameters in the autograft. Two hundred and thirty-five collections were peripheral blood (PB) of patients on the day of planned performed with a median of 4.0 collections per patient leukapheresis, and correlated these with various parameters (range, 2–10). The median PB CD34+ cell count at the in the autograft product. time of collection was 39 × 106/l (range, 0.0–285.6). The PBPC autograft parameters measured were the CD34+ cell, colony-forming unit granulocyte–macrophage Methods (CFU-GM) and mononuclear cell (MNC) content. There was a strong linear correlation between PB CD34+ cells/l Immediately prior to 235 consecutive PBPC collection pro- and autograft CD34+ cells/kg (r = 0.8477). The corre- cedures, the PB white blood cell (WBC), MNC, platelet lation with CFU-GM/kg (r = 0.5512) was weaker. There and CD34+ cell counts were determined and correlated with was no correlation between autograft CD34+ cells/kg various autograft parameters namely, CD34+ cell, CFU-GM and PB WBC (r = 0.0684), PB MNC (r = 0.1518) or PB and MNC content. These autograft parameters were platelet count (r = 0.2010). At our institution we aim to expressed as a function of patient body weight. CD34+ obtain a minimum of 0.5 × 106 CD34+ cells/kg with each cells/kg in the autograft was also compared to the other day of collection. We demonstrate that such a collection autograft parameters. can be reliably obtained if the PB CD34+ cell count exceeds 5.0 × 106/l. Patient characteristics Keywords: CD34; transplantation; apheresis All patients undergoing PBPC harvesting at our institute between June 1996 and June 1997 were included and their disease characteristics are summarized in Table 1. This pro- Rapid and durable hematopoietic recovery is required fol- vided 235 complete data sets for analysis. lowing intensive therapy and autologous peripheral blood progenitor cell transplantation (PBPCT). Although, no definitive assay for the pluripotent hematopoietic stem cell Apheresis procedure 1 exists, a number of surrogate laboratory measures are used The mobilization strategy employed various chemothera- to predict hematopoietic recovery following PBPC trans- peutic agents supplemented with rhGCSF (10 ␮g/kg), or plantation.2 These include the autograft mononuclear cell count (MNC), clonogenic assays in semi-solid media, usu- = ally the colony-forming unit granulocyte–macrophage Table 1 Patient characteristics (n 59) (CFU-GM), and more recently the number of hematopoietic cells marked by the progenitor cell antigen CD34.3 Indeed, Diagnosis Number Number of days + of apheresis as the CD34 cell content in the autograft correlates well mean (range) with the rate of hematopoietic recovery, it is now assessed All patients 59 4.0 (2–10) Non-Hodgkin’s lymphoma 23 3.6 (2–8) Correspondence: Dr HM Prince, Blood and Marrow Transplant Service, Hodgkin’s disease 3 3 (3–3) Division of Haematology and Medical Oncology, Peter MacCallum Breast cancer 18 3.6 (2–9) Cancer Institute, Locked Bag 1, A’Beckett St, Melbourne 3000, Victoria, Multiple myeloma 8 5.1 (2–10) Australia Others 7 5.3 (2–9) Received 21 July 1997; accepted 8 March 1998 Blood CD34 and autograft yield P Chapple et al 126 utilized rhGCSF (10 ␮g/kg) alone (Table 2). Following cytometry using phycoerythrin-conjugated class III anti- chemotherapy and cytokine mobilization, PBPC collection bodies to the CD34 antigen (clone HPCA-2) and peridium was initiated when the patient’s PB WBC returned to chlorophyl protein (PerCP) conjugated anti-CD45 (all anti- Ͼ1.5 × 109/l and/or the PB absolute neutrophil count bodies from Becton Dickinson Immunocytometry Systems, (ANC) reached 1.0 × 109/l following the nadir. A minimum San Jose, CA, USA), using an in-house whole blood lysis platelet count of 30 × 109/l was also required. For patients methodology which has been verified against the Inter- mobilized with cytokine alone, apheresis was commenced national Society of Hematotherapy and Graft Engineering 5 days following initiation of G-CSF. By employing these (ISHAGE) guidelines.5,6 Briefly, 1 × 106 cells, either in standard trigger points for initiating apheresis, patients were whole blood or the apheresis collection product, were added harvested between 5–16 days following mobilization. Dur- to an aliquot of anti-CD34 PE and anti-CD45 PerCP which ing the study, the PB CD34+ cell count was not used as a had previously been titrated to provide a saturating anti- criterion for initiating apheresis. body concentration, and incubated for 20 min at room tem- Apheresis was performed using a Haemonetics V50, perature. This was followed by addition of red cell lysing CS3000plus (Baxter Healthcare Corp, Deerfield, IL, USA) solution (FACS lysing solution, Becton Dickinson), fol- or Spectra (Cobe, Denver, CO, USA) cell separators. Blood lowed by brief vortex mixing and incubation for 10 min at was collected via a central venous access device or from room temperature. The cells were washed once by adding peripheral veins with 1.5–2.5 times the patient’s estimated imidazole buffered saline with 2% bovine serum and 0.1% blood volume processed. Na azide and centrifuging at 600 g. Without delay they were analyzed on a Becton Dickinson FACSCalibur flow cytometer by acquiring florescence and forward light scatter Cell counts data on 120 000 ungated events. Subsequent data analysis Following appropriate dilution with Isoton III (Coulter, was performed on the listmode data files using Becton Hialeah, FL USA), total WBC counts for PB and PBPC Dickinson Cellquest software v3.0 (Becton Dickinson). In samples were determined on a Coulter STKS analyzer all list mode data analysis ‘true’ CD34+ events were defined (Coulter). A 100 cell manual differential count was used to as having the dual characteristics of bright CD34 determine the ANC. The MNC count was calculated by expression and low-to-intermediate CD45 expression subtracting the ANC from the total WBC count. (Figure 1). Using the autograft WBC count, %CD34+ cells and the patient’s actual body weight, CD34+ cells/kg body weight was determined. Colony assays CFU-GM assays were performed by culture in Iscove’s Statistical methods modified Dulbecco’s medium (Gibco Laboratories, New York, USA) containing 3% agar, 15% fetal calf serum A correlation matrix was used to determine all possible per- (FCS) supplemented with rhIL-6 (6.66 ng/ml), rhG-CSF mutations of the available parameters and statistical analy- (3.33 ng/ml), rhGM-CSF (1.33 ng/ml) and rhSCF sis of correlation between the various measured and calcu- (1.0 ng/ml). Cultures were plated in duplicate at 1 × 105 lated parameters was performed using linear regression cells ml and incubated at 37°C for 14 days in a humidified analysis. This included three separate sets of correlations; environment with 5% CO2. Colonies containing more than a correlation of the PB parameters to one another, corre- 50 cells were scored. The CFU-GM content of the autograft lation of the PB parameters to the PBPC parameters and was then calculated from the colonies scored per ml and correlation of the PBPC parameters with the overall PBPC the total cells/kg. yield (CD34+ cells/kg). Statistics were performed on StatXact 3 for windows (CYTEL Software, Cambridge, MA, USA) and S-PLUS version 3.3 for Windows (StatSci; CD34 analysis MathSoft, Seattle, WA, USA). The PBPCs and their accompanying PB samples were ana- lyzed for the percentage of CD34+ cells by bivariate flow Results Table 2 Mobilization strategies employed Two hundred and thirty-five consecutive apheresis pro- Mobilization regimen Number of cedures were performed. The median and mean number of patients procedures per patient was 4.0 (range: 2–10) (Table 1). At the time of initiating apheresis, the median peripheral blood 2 + a Cyclophosphamide 1.5 g/m rhGCSF 29 + × 9 Cyclophosphamide 4.0 g/m2 + rhGCSFa 5 WBC, MNC and CD34 cell counts were 27.1 10 /l 9 Ifosfamide 5 g/m2 + doxorubicin 50 mg/m2 + 2 (range, 1.5–139.6), 15.2 × 10 /l (range, 0.1–56.9) and rhGCSFa 39 × 106/l (range, 0–285.6), respectively. The median %PB Ifosfamide 5 g/m2 + paclitaxel 175 mg/m2 + rhGCSFa 4 CD34+ cells was 0.21 (range, 0–2.65). In the 235 PBPC 2 + 2 Cyclophosphamide 1.5 g/m doxorubicin 50 mg/m 5 products, there was a median WBC count of 310.6 × 109/l + rhGCSFa × 8 Other chemotherapy and rhGCSFa 7 (range, 21.0–668.0), MNC/kg of 2.27 10 (range, 0.02– rhGCSF alone 7 20.55), CFU-GM/kg of 7.84 × 104 (range, 0.08–95.67), %CD34+ cells of 0.81 (range, 0–10.67) and CD34+ cells/kg arhGCSF (10 ␮g/kg) commenced 24 h after completion of chemotherapy.