|Year : 2021 | Volume
| Issue : 1 | Page : 31-36
Simple predictors of peripheral blood stem cell yield in healthy donors: A retrospective analysis in a tertiary care hospital
Thulasi Raman Ramalingam1, Lakshman Vaidhyanathan1, Anurekha Muthu1, Vikram Prabhakar1, Balasubramaniam Ramakrishnan2, Revathi Raj3, Jose M Easow4
1 Department of Hematology, Apollo Cancer Centre, Chennai, Tamil Nadu, India
2 Department of Biostatistics, Apollo Cancer Centre, Chennai, Tamil Nadu, India
3 Department of Pediatric Hematology and Blood and Marrow Transplantation, Apollo Cancer Centre, Chennai, Tamil Nadu, India
4 Medical Oncology, Hematology and Blood and Marrow Transplantation, Apollo Cancer Centre, Chennai, Tamil Nadu, India
|Date of Submission||15-Jun-2020|
|Date of Decision||15-Aug-2020|
|Date of Acceptance||11-Sep-2020|
|Date of Web Publication||15-Mar-2021|
Dr. Thulasi Raman Ramalingam
Ramalingam, Department of Hematology, Apollo Cancer Centre, Chennai - 600 035, Tamil Nadu
Source of Support: None, Conflict of Interest: None
BACKGROUND: Peripheral blood stem cells (PBSCs) are commonly used for hematopoietic stem cell transplant (HSCT) over other stem cell sources. The hematopoietic stem cells (HSCs) are mobilized from marrow by granulocyte colony-stimulating factor (G-CSF) and then harvested by apheresis technique. The HSC yield differs in donors that may be due to inadequate mobilization or difficulty in harvesting the mobilized stem cells.
MATERIALS AND METHODS: We retrospectively analyzed donor demographic and pre-apheresis hematological variables with circulating CD34+ cell (cir CD34) count and HSC yield in product in 100 normal donors. G-CSF was given for 5 consecutive days, and the stem cells were harvested on day 5. The cir CD34 count and pre-apheresis variables were recorded a day before harvest.
RESULTS: Of 100 donors, 77% were males and 23% were females. Male sex, younger age, and donor weight were significantly associated with better CD34 yield in the product. Among the pre-apheresis hematological variables, absolute neutrophil count, hematocrit, and absolute nucleated red blood cell count significantly correlated with post-GCF circulating CD34 and CD34 yield in the product. Donors with mean corpuscular volume <80 fL showed relatively poor CD34 cell harvest as compared to normal donors, though with adequate mobilization.
CONCLUSION: Selection of donors for PBSC apheresis is crucial for a good transplant outcome and recovery. Alternate strategies that improve the final CD34 yield should be employed for donors with high risk for poor CD34+ cell yield.
Keywords: Apheresis, granulocyte colony-stimulating factor, healthy donors, mobilization, peripheral blood stem cells
|How to cite this article:|
Ramalingam TR, Vaidhyanathan L, Muthu A, Prabhakar V, Ramakrishnan B, Raj R, Easow JM. Simple predictors of peripheral blood stem cell yield in healthy donors: A retrospective analysis in a tertiary care hospital. J Appl Hematol 2021;12:31-6
|How to cite this URL:|
Ramalingam TR, Vaidhyanathan L, Muthu A, Prabhakar V, Ramakrishnan B, Raj R, Easow JM. Simple predictors of peripheral blood stem cell yield in healthy donors: A retrospective analysis in a tertiary care hospital. J Appl Hematol [serial online] 2021 [cited 2021 Apr 20];12:31-6. Available from: https://www.jahjournal.org/text.asp?2021/12/1/31/311323
| Introduction|| |
Hematopoietic stem cell(HSC) transplantation is a curative option in multiple conditions, including aplastic anemia, red cell disorders, and primary immunodeficiencies. Mobilization and collection of the peripheral blood stem cells (PBSCs) are done routinely for autologous and allogeneic bone marrow transplantation. PBSCs are commonly used over bone marrow and umbilical cord stem cell. The PBSCs show better HSC yield, painless collection at an outpatient setting, faster engraftment, and low risk of graft failure and also have graft versus leukemia effect., However, they also carry higher risk of Graft versus host disease (GVHD) due to increased content of T-cells in the graft. The donor is also exposed to granulocyte colony-stimulating factor (G-CSF).
The HSCs reside in the marrow and are exceedingly rare in the peripheral blood, accounting to 0.01–0.005% of nucleated cells. Hence, the HSCs from the marrow are mobilized into the peripheral blood using growth factors and harvested by apheresis technique. Adequate mobilization, day of harvest, collection efficiency, and other factors influencing the apheresis are crucial for good HSC yield in the final product. The HSC yield differs in donors that may be due to inadequate mobilization or difficulty in harvesting the mobilized stem cells. A stem cell dose of 4.0–5.0 × 106/kg of recipient is desired for a successful engraftment. Up to 20% of healthy donors are proved to be poor mobilizers. As the numbers of unrelated volunteers for stem cell donation are improving, identifying a good donor is crucial. Donor characteristics and pre-apheresis variables after G-CSF are proposed to predict the PBSC yield in healthy donors. No well-established predictive factors exist in identifying a donor with poor stem cell yield. Currently, circulating pre-aphaeresis CD34+ cell count (circulating pre aphaeresis CD34 + cell count) is considered as a reliable indicator for adequate mobilization and harvest. Some authors have studied the donor demographic data in association with stem cell yield.
We retrospectively studied the various pre-aphaeresis hematological variables in healthy donors. We also correlated these variables to the donor cir CD34 and HSC yield in the product so that it can be used as a predictor of adequate mobilization and harvest. Various authors have commented on total white blood cell (WBC) count and platelet count as the predictors for good stem cell yield. We also analyzed the factors that impede the apheresis harvest of HSC after adequate mobilization. Low mean corpuscular volume (MCV) is also proposed to affect the collection of HSC during aphaeresis. Hence, we studied the cir CD34 and apheresis product CD34+ cells in microcytic and normal donors. Factors that predict or indicate a better quality and greater quantity of HSC graft can improve patients' transplant outcome and recovery.
| Materials and Methods|| |
Study design and setting
Healthy voluntary donors who underwent PBSC mobilization and harvested at Apollo Cancer Hospitals from April 2018 to March 2019 were recruited for this observational study. The study was approved by our institutional ethics committee.
The donors were selected for stem cell collection after obtaining written consent. A thorough physical examination, evaluation of complete medical history, laboratory tests (complete blood counts, liver and renal function tests, screening hemoglobin (Hb) electrophoresis, Vitamin B12, Vitamin D and folate levels), electrocardiogram, and chest X-ray were done. Infectious disease workup was also done for the safety of the recipient. Healthy donors who fulfill and satisfy the above assessment were selected for PBSC collection and enrolled in the study. Those who did not give consent were excluded from the study. G-CSF (filgrastim) was given subcutaneously at 10 μg per kg recipient weight for 5 consecutive days. Stem cells were collected on day 5. A preprocedure CD34 count was performed a day before harvest.
Peripheral blood stem cell collection
All the PBSC collection procedures were performed using Spectra Optia apheresis system, Version 11.0 (Terumo Blood and cell technologies) aided by Continuous Mononuclear Cell Collection (CMNC) software protocol. This system uses continuous-flow centrifugation and optical detection technology for establishing the mononuclear cell (MNC) interface, which is continuously monitored. The Spectra Optia Cell Separator is loaded with the prescribed CMNC collection kit and primed with saline and ACD-A solution as per the touch screen guidelines. The donor data consisting of height, weight, sex, and hematocrit (HCT) were entered. A common protocol was to process 2.5–3 times the donor blood volume or about 10–12 l of blood over 150–180 min. However, using the collection efficiency factor and preprocedure CD34 count, the total blood volume processed can be optimized to get the desired target yield. The target yield was aimed to provide at least a stem cell dose for two transplants. A second apheresis, if required, was done if the target yield was not achieved in the first collection. The procedure was performed with continuous ECG monitoring and calcium supplements as and when indicated. The establishment of MNC interface and collection of MNC into the bag were monitored at frequent intervals and by adjusting the collection preference if required.
CD34+ cell enumeration
The cir CD34 and the product PBSC samples were obtained from a single-stage, no wash-no lyse ISHAGE protocol, run on Stem One Software in FC500 flow cytometer (FCM). The Stem Kit Reagents are commercially available and manufactured by Beckman Coulter. The sample is diluted with PBS to obtain a total nucleated cell concentration of <30 × 109 cells/L. 20 μl of anti-CD45 FITC and anti-CD34 PE is taken in a 12-mm × 75-mm polystyrene tube. To this, 20 μl of 7-AAD viability dye and 100 μl of well mixed sample are added. After incubation for 20 min at room temperature, 2 ml of ammonium chloride red blood cell (RBC) lysis buffer is added to the samples and incubated for 10 min. Just before acquiring on the FCM, 100 μl of well-mixed suspended stem-count fluorosphere beads is added and gently mixed. A minimum of 75,000 CD45+ events and a minimum of 100 CD34+ cells are acquired. The data are analyzed by creating regions and logical gates using software. The number of CD34+ cells is determined from the viable CD34+ cells.
Pre-apheresis blood counts and indices
The complete hematological indices including total WBC count, total RBC count, Hb, HCT, platelet count, MCV, mean platelet volume (MPV), red cell distribution width (RDW), absolute neutrophil count (ANC), and absolute nucleated red cell count (nRBCs) of the donor peripheral blood before apheresis were obtained with a DxH 800 Beckman Coulter automated cell counter. This is an automated hematology analyzer which uses the VCS technology and FCM analysis for WBC differentials and nRBCs.
Our objective of the study is to find the reliable predictors of successful PSBC collection in healthy donors. Sample size is derived with a significant correlation coefficient of 0.331 reported by van Oostrum et al. With this idea using the following formula, N = ([Zα + Zβ]/C)2 + 3, where N = number of subjects required, Zα = the standard normal variate for α = 1.96, Zβ = the standard normal variate for β = 0.84, C = 0.5 × ln ([1 + r]/[1 − r]) = 0.30945, r = expected correlation coefficient. The minimum sample required for the study is 85 cases, and hence, we recruited 100 cases for the study.
All nonnormally distributed continuous variables are represented as median (interquartile range). Categorical variables are represented as percentage. Comparisons of categorical variables are done by either Chi-square test or Fisher's exact test. Comparisons of normally distributed continuous variables are done by unpaired t-test. Comparisons of nonnormally distributed continuous variables are done by Mann–Whitney U-test. Linear regression models are developed based on stepwise regression method to avoid multicollinearity. Using stepwise regression model, best models are developed by avoiding multicollinearity and controlled variation inflation factor (<10). Data entry is done in MS Excel Spread sheet. Data validation and analysis are carried out using SPSS statistics, version 25.0, Armonk: New York; IBM corp, 2017. A P < 0.05 is considered as statistically significant.
| Results|| |
A total of 100 normal donors were enrolled into this study. There was predominance of male donors (77%) compared to females (23%), with a mean age of 31.6 and 25.6, respectively. To study the donor factors that may determine the quantity of stem cell collection, we analyzed the relationship between the cir CD34 and CD34 yield in product with donor demographic variables (age, sex, and weight) and pre-apharesis hematological indices such as WBC count, RBC count, Hb, HCT, platelet count, MCV, MPV, ANC, and nRBCs. The pre-apheresis hematological indices and cir CD34 were recorded a day before harvest. The CD34 count of first apheresis product was only taken.
The data of hematological variables in males and females are shown in [Table 1]. All donors tolerated G-CSF administration and apheresis procedure with no major side effects. Males showed significantly more cir CD34 and CD34 yield in final product (mean: 121.8 × 106/L and 6.22 × 106/kg) compared to females (mean: 70.8 × 106/L and 5.47 × 106/kg) though volume of product was almost matching. Hence, graft taken from male donors is likely to have more PBSCs. Of 100 donors, only six donors showed final CD34+ yield of <3 × 106/kg, while all others (94%) showed a CD34 harvest of more than 3 × 106/kg. Among the hematological variables, RBC and its associated indices were low in females. Pre-apheresis platelet count was found to be low in males than females and was statistically significant.
|Table 1: Comparison of donor characteristics and variables in males and females|
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Stepwise regression analysis of cir CD34 cells and CD34+ cell yield in product with all demographic and pre-apheresis variables was done [Table 2] and [Table 3]. Significant correlation (P < 0.05) of cir CD34 with age, weight, ANC, and nRBCs of donor was observed. The other parameters did not correlate with cir CD34. The absolute CD34+ cell count and yield in the final product showed significant correlation with age, weight, HCT, ANC, and nRBCs.
|Table 2: Regression model (stepwise) of donor characteristics and variables with cir CD34|
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|Table 3: Regression model (stepwise) of donor characteristics and variables with CD34+ yield in product|
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On comparing the variables between the donors with MCV <80 fL and MCV >80 fL [Table 4], the volume of product obtained in microcytic donor (MCV <80 fL) is less compared to donors with normal MCV. Hb and HCT were low in microcytic donors, and they showed high RBC count and RDW. The cir CD34 was low in microcytic donors, but it was not statistically significant. On the contrary, the total CD34 + cells in product and yield was significantly low in microcytic donors as compared to normal donors. Hence, there may be a difficulty in harvesting the stem cells in microcytic donors even after adequate mobilization.
|Table 4: Comparison of donor characteristics and other variables with mean corpuscular volume <80 and =80 fL|
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| Discussion|| |
The cir CD34 enumeration is considered as a reliable indicator for adequate mobilization and harvest of PBSC. However, CD34 enumeration by FCM is a sophisticated test, demanding infrastructure, and technical expertise and may not be available at resource-limited setting. Hence, an alternate simple predictor for stem cell mobilization and harvest is desirable. Hence, we studied the donor demographic details and pre-apheresis hematological indices with circulating CD34 and final PBSC yield in product. Younger donors showed higher CD34+ cells in the apheresis product compared to older donors. Similarly, a direct correlation was noted with weight and CD34 yield. In our present study, males showed a better mobilization and harvest of CD34+ cells compared to females. This is in concordance with previous studies.,,,
ANC is a good predictive factor for final stem cell harvest as it independently correlated with CD34+ cell yield in the product. It also showed correlation with cir CD34. Some authors found significant correlation of WBC count with CD34+ yield., However, we found correlation only with ANC but not with total WBC count which is concordant with Wang et al. It is known that G-CSF activated neutrophils leading to release of proteases which cleave the intimation between stem cell and bone marrow micro environment. It is also proposed that interleukin (IL)-8 is mediated by neutrophils and release of MMP-9 that induce rapid mobilization of HSC., The above mechanism in the stem cell mobilization explains the correlation of pre-apheresis neutrophil numbers and final CD34+ cell yield after G-CSF stimulation. Pruijt et al. found a correlation between the number of circulating neutrophils and the number of circulating hematopoietic progenitor cells (HPCs). They also reported with experiment models that IL-8-induced mobilization of HPCs was reduced significantly during induced neutropenia which later increased proportionally during the neutrophilic phase. Hence, neutrophils and its number have strong association with HPCs and can be a predictor for mobilization and harvest of CD34+ cells.
The other pre-apheresis variable which significantly correlated with cir CD34 and stem cell yield in product is the nRBCs. Quantification of nRBCs by automated hematology analyzers using the principle of FCM is precise, accurate, and reproducible. Hence, pre-apheresis nRBC count can certainly act as a predictor for final HSC yield. The CD34+ cell count directly correlated with nRBCs. G-CSF is postulated to enhance production of late normoblasts than the early forms. It also causes mobilization of few late normoblasts and newly synthesized reticulocytes into circulation. Hence, nRBCs in circulation can be an indicator for responsiveness of marrow to G-CSF. No literature was found on circulating nRBCs in normal donor after G-CSF and its significance.
Donors with high HCT showed better CD34 cell harvest in apheresis product. This is concordant with Pornprasertsud et al., who found direct correlation between HCT and CD34 + harvest and reported as a strong predictor for PBSC collection yield. The donor HCT is used by the apheresis machine to set the interface for collection of MNCs. However, the exact reason for the above association is unclear. In this study, female patient showed higher pre-apheresis platelet count than males as in normal physiological sate. This is in concordance to the study done by Wang et al. Although platelet count showed positive correlation with CD34+ cell yield, it was not statistically significant. Similar result was found by Lysák et al. in their study of 111 healthy donors. The baseline platelet count has been proposed as an accurate indicator of PBSC mobilization and collection in one study.
The interference of microcytic RBCs with apheresis harvest of CD34 cells was assessed by comparing the variables between two groups (MCV <80 fL and MCV >80 fL). The final volume of apheresis product was significantly lower in patient with MCV <80 fL compared to MCV >80 fL. RBC, Hb, HCT, and RDW showed significant variation between both the groups, as the above variables are collinearly related to MCV. Although the cir CD34 did not show significant correlation between the two groups, the final harvest showed lower yield in patients with MCV <80 fL compared to donor having normocytic RBCs. Wang et al. opined that donor with low MCV was associated with poor apheresis outcomes. They also observed that the mobilization of CD34+ cells in these donors was adequate. We also found a similar result in our study as the pre-apheresis circulating CD34 showed no significant variation in two MCV groups. However, the final apheresis CD34+ yield varied significantly between the two groups. Hence, donors with microcytic RBCs have adequate stem cell mobilization but low collection efficiency during apheresis procedure. Wang et al. speculated that the poor harvest is possibly due to the interference of microcytic RBCs with buffy coat stratification during leukapheresis. Constantinou et al. also found poor CD34+ cell harvest in some thalassemic patients, despite adequate mobilization. However, these findings must be confirmed in a larger cohort of healthy donors. The constitutions of more females in the microcytic donors (40.9%) compared to normal donors (8.9%) is a limiting factor in the present study.
| Conclusion|| |
Male sex, younger age, and donor weight were significantly associated with better CD34 yield in the product. Among the pre-apheresis hematological variables, ANC and absolute nRBC count significantly correlated with post-GCF circulating CD34 and CD34 yield in the product. Microcytic donors showed relatively poor CD34 cell harvest compared to normal donors, though with adequate mobilization. Hence, these factors should be considered when selecting donors for apheresis and alternate strategies that improve the final CD34 yield should be employed.
We are very much grateful for the altruistic donors enrolled in this study. We thank Dr. Dhaarani Jayaraman, Assistant Professor, Department of Pediatrics–Hemato Oncology Division, SRIHER, for reviewing the article. We also extent our thanks to Mr. Selvaraj for performing CD34 count FCM assay and Mrs. Mirunalini and Mr. Devarajan for assisting us in data collection.
Informed and written consent was obtained from each PBSC donor enrolled in this study. Ethical guidelines were adhered as per the Declaration of Helsinki. This study has been approved by the institutional ethical committee (IEC) on January 2019, 23/19.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]