Manipulation,and cryopreservation of autologous peripheral blood stem cell products in Italy: A survey by GITMO,SIDEM and GIIMA societies

There is considerable heterogeneity in manipulation and cryopreservation of hematopoietic stem cells (HSC) for autologous HSC transplantation across Europe and Italy. To better address this point, three Italian Scientific Societies (GITMO- Gruppo Italiano per il Trapianto di Midollo Osseo; SIDEM- Società Italiana Emaferesi e Manipolazione Cellulare; and GIIMA- Gruppo Italiano Interdisciplinare Manipolazione e Aferesi per Terapie Cellulari), in collaboration with the Competent Authority “National Transplant Center” (CNT) sent to 85 Italian transplant centers (TC) a survey, which included 12 questions related to the most critical elements in graft processing. Fifty-nine centers (70 %) responded to the questionnaire. Overall, this survey demonstrates that the majority (>90 %) of responding TC used standardized procedures for HSC processing; however, an intercenter heterogeneity was clearly documented in several standard operating procedures adopted by different TC. These results seem to suggest that further standardization and efforts are needed to provide recommendations and guidelines on HSC manipulation, cryopreservation and functional assessment of cryopreserved material for autologous HSCT     

Impact of the CD4:CD8 ratio in bone marrow on stem cell mobilization and engraftment in autologous stem cell transplant patients

Bone marrow (BM) damage after previous chemotherapy, such as that involving alkylating agents, and radiation therapy alone cannot explain poor hematopoietic progenitor cell mobilization. We examined the T lymphocytes of BM in 67 autologous peripheral blood stem cell transplant (auto-PBSCT) patients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM) to establish whether the cellular phenotype predicts mobilization and engraftment between January 2000 and January 2020 at the Japanese Red Cross Society Wakayama Medical Center. The total number of mobilized CD34+ cells was <2 × 10⁶/kg in 30 patients (group A) and ≥2 × 10⁶/kg in 37 (group B). The median absolute number of CD3+CD4+ cells was lower in group A than in group B (P = .013), and the median absolute number of CD3+CD8+ cells was higher in group A than in group B (P = .016). A low CD4:CD8 ratio was observed in all patients in group A, whereas all patients in group B showed a normal CD4:CD8 ratio (P < .001). A strong correlation was found between the CD4:CD8 ratio and median total CD34+ cells yield (r = .723, P < .001). The present results showed that a lower CD4:CD8 ratio correlated with later neutrophil and platelet engraftment (r = .662, P = .007 and r = .571, P = .008, respectively). The present results indicate that the CD4:CD8 ratio in BM contributes to the prediction of mobilization and engraftment in auto-PBSCT patients.     

Effects of cell concentration,time of fresh storage,and cryopreservation on peripheral blood stem cells: PBSC fresh storage and cryopreservation

IntroductionPeripheral blood stem cells are widely used in autologous or allogeneic transplantation. The quality of the product directly impacts clinical outcomes, and the cell quality and/or functionality may be influenced by the storage conditions as time, temperature, total nucleated cells (TNC) concentration and cryopreservation requirement.ObjectiveTo verify the effects of time, cell concentration, and cryopreservation/thawing in the viability and functionality of stem cells for transplantation.MethodsWe evaluated TNC, CD45⁺ viable cells, CD34⁺ viable cells, and cell viability and functionality of 11 samples. Measurements were performed immediately and 24 h, 48 h, 72 h, and 96 h after sample collection at high and low TNC concentrations. The same parameters were also evaluated after cryopreservation and thawing of the samples.ResultDuration of storage and TNC concentration exhibited a negative effect on cell quality (CD45⁺ viable cells, CD34⁺ viable cells and functionality). Moreover, the association of these parameters increased the negative effect on graft quality. Cryopreservation and thawing also negatively affected the collected sample regarding viable CD34⁺ cells (recovery 66.2 %), viable CD45⁺ cells (recovery 56.8 %), and 7-AAD viability. No significant losses in viable CD45⁺/CD34⁺ cells and functionality were observed in the first 24 h in both TNC conditions.ConclusionThese results emphasize the importance to consider carefully the storage conditions until transplantation, measuring TNC/μL until 24 h after collection (diluting the product when TNC > 300 × 10³/μL) and infusing fresh graft as soon as possible.     

Tempo of hematologic recovery correlates with peripheral blood CD34+ cell level in patients undergoing stem cell mobilization

This study was undertaken to evaluate the relationship between the time to recovery of peripheral blood counts and CD34+ cells in the peripheral blood (PB) and apheresis collections of patients undergoing intensive chemotherapy followed by rhG-CSF. Twenty-three patients with a median age of 42 years (range 17–64) with malignancies underwent peripheral blood stem cell (PBSC) collection after cyclophosphamide (CY) 4 g/m² and etoposide (600 mg/m²) followed by rhG-CSF (10 μg/kg/day). The WBC, platelet counts, CD34+ cell counts per ml of PB, and CD34+ cells in apheresis products were followed in all patients. The relationship of the time to recovery of WBC >1,000/μl, >3,000/μl, >10,000/μl and platelets >20,000/μl and 50,000/μl was compared to the average daily CD34+ cells/ml in each patient using the Spearman Correlation test. The tempo of recovery of WBC and platelets were highly correlated with the average CD34+ cell count in blood. In order to derive some useful guidelines for the timing of apheresis, the patients were divided into two groups, early recover (ER) and late recover (LR) based on the median time (day 10) to reach WBC count greater than 1,000/μl. ER patients had an average daily PB CD34+ cell count of 9.04 × 10⁴/ml (range 0.44–17.5) and a median yield of CD34+ cells of 10.43 × 10⁶/kg (range 0.60–25.95) compared to LR patients, who had 1.87 × 10⁴/ml (range 0.32–5.44) in the PB (P = .001) and a yield 3.20 × 10⁶/kg CD34+ cells (range 0.037–9.39) (P = .001). Patients recovering their WBC to 1,000/ml within 10 days of completing this regimen may undergo PBSC collection and achieve minimum-target cell doses of >2.5 × 10⁶ CD34+ cells/kg—100% of the time. J. Clin. Apheresis 13:1–6, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of Amicus and COBE Spectra for allogenic peripheral blood stem cell harvest: Study from tertiary care centre in India

Introduction

Most common source of stem cell graft for both autologous and allogenic haematopoietic transplants are peripheral blood haematopoietic progenitor stem cells. Adequate collection of the CD34+ cells and safety of the allogenic donor during the leukapheresis are of prime importance to an apheresis physician. Our retrospective analysis is a comparison between of two platforms namely, COBE Spectra and Amicus, for CD34+ mononuclear cell collection.

Evaluation of the Sysmex XN automated hematopoietic progenitor cell enumeration for timing of peripheral blood stem cell harvest

BackgroundEnumeration of stem cells is essential in the management of peripheral blood stem cell (PBSC) harvest. An alternative to the gold standard flow cytometric CD34+ stem cell count is the fully automated hematopoietic stem cell (HPC) count on the Sysmex XN hematology analyzer.Materials and methodsEighty-nine patients and healthy stem cell donors who underwent PBSC harvest were included in the study. Stem cells were enumerated in pre-harvest peripheral blood and the apheresis yield by both flow cytometric CD34+ stem cell enumeration and by the Sysmex XN HPC count.ResultsThe Sysmex HPC concentration overestimated the CD34+ stem cell concentration by a ratio of 1.3 in average. The agreement between the two methods was poor at concentration <40 stem cells/μL (Bias: 45 %, 95 % limits of agreement: -71 - 160 %). CD34+ stem cell concentration and HPC concentration correlated well in pre-harvest peripheral blood (R=0.73, slope=0.96). We established a positive cut off >43.5 HPC/μL, where PBSC harvest can be initiated. And a negative cut off <16.5 HPC/μL, where harvest should be postponed or other mobilizing regimens or bone marrow harvest should be considered. 33 % of measurements were in between the negative and positive cut-off and would require a supplementary CD34+ cell count.ConclusionAlthough Sysmex HPC count correlates well with CD34+ cell count in peripheral blood, the agreement between the two methods is poor, especially at low concentrations, namely in the clinical decision range. Sysmex HPC count as a surrogate for CD34+ cell count should, therefore, be used with caution.     

移植物中CD34+细胞及T细胞亚型对HLA相合同胞异基因外周血造血干细胞移植预后的影响

目的 探讨移植物中CD34+细胞及T细胞剂量对HLA相合同胞异基因外周血造血干细胞移植(allo-PBSCT)预后的影响.方法 流式细胞术检测移植物中CD34+细胞,CD3+、CD3+CD4+及CD3+CD8+T细胞含量,按患者体重计算出移植物中单个核细胞(MNC),CD34+细胞,CD3+、CD3+CD4+及CD3+CD8+T细胞数量,根据中位数分别将患者分为高剂量组和低剂量组,比较高剂量和低剂量组患者移植后造血重建、移植物抗宿主病(GVHD)、移植相关死亡(TRM)、复发、总体生存(OS)率以及无病生存(DFS)率的发生情况.结果 CD34+细胞高剂量组(34例)移植后中性粒细胞和血小板的恢复速度显著加快(P值均<0.05).CD3+CD4+、CD3+CD8+T细胞高剂量组和相应低剂量组相比,Ⅱ-Ⅳ度aGVHD发生率有增高趋势(P值分别为0.089和0.098).CD3+CD4+及CD3+CD8+T细胞高剂量组和相应低剂量组相比,TRM显著增高(P值均<0.05);多因素分析显示,CD3+CD4+和CD3+CD8+T细胞输注剂量是患者TRM的影响因素(RR分别为13.12和25.90,P值均<0.05).各高剂量组和相应低剂量组比较复发率差异无统计学意义(P值均>0.05).CD3+ CD4+及CD3+CD8+T细胞高剂量组分别和相应低剂量组相比,OS显著降低(P值均<0.05);多因素分析显示,CD3+CD4+和CD3+CD8+T细胞输注剂量是患者OS的影响因素(RR分别为3.71和3.01,P值均<0.05);CD3+CD4+T细胞高剂量组和低剂量组相比,DFS显著降低(P值均<0.05);多因素分析显示,CD3+CD4+T细胞输注剂量(RR=6.91,P=0.011)是患者DFS的影响因素.结论 高剂量CD34+细胞移植可加快移植后造血重建;移植物中高含量的CD3+CD4+及CD3+CD8+T细胞会增加患者TRM,降低OS或DFS.     

芜湖市非亲缘外周血造血干细胞捐献志愿者招募情况调查

目的:探讨芜湖市非亲缘外周血造血干细胞(PBSC)捐献志愿者的招募情况。方法:选择2007-2020年,安徽省芜湖市中心血站招募的3 810例PBSC捐献志愿者中,人类白细胞抗原(HLA)低分辨配型全相合,并且同意参加高分辨配型的75例志愿者为研究对象,并且纳入研究组。采用整群随机抽样方法,选择本站2007-2020年...     

Outcomes of stem cell mobilization and engraftment in patients with multiple myeloma according to CD56 expression status

BackgroundThe molecular mechanism underlying the mobilization and engraftment of CD34+ cells is poorly understood. The most relevant factors in the regulation of stem cell release and engraftment include chemokines, adhesion molecules, and chemokine receptors. Previously, it was suggested that the absence of CD56 expression could be used as a predictive factor for mobilization failure at the time of diagnosis. Here, we investigated the effect of CD56 expression status on both mobilization and engraftment processes. Additionally, other factors affecting mobilization and engraftment efficacy were investigated.MethodsData from 79 multiple myeloma patients undergoing autologous stem cell transplantation between 2015 and 2020 were analyzed for peripheral stem cell mobilization and posttransplant neutrophil and platelet engraftment according to CD56 expression on myeloma cells.ResultsNo difference in either the median number of CD34+ cells collected or time to engraftment was found between the CD56+ and CD56- groups. The age of the patients (p = 0.025) and peak number of circulating CD34+ cells in peripheral blood (p = 0.005) were important predictors for a higher number of collected CD34+ cells. The average time to recovery of leukocytes and platelets after transplantation was markedly correlated with the number of transplanted stem cells and peak number of circulating CD34+ cells in peripheral blood, respectively (p = 0.049 and p = 0.003).ConclusionsOur results indicated no effect of CD56 expression status on the mobilization and engraftment of PBSCs. Our results also support the notion that the peak number of circulating CD34+ cells in peripheral blood is clinically important for rapid platelet engraftment following HPC transplantation.     

Hematopoietic progenitor cell count, but not immature platelet fraction value, predicts successful harvest of autologous peripheral blood stem cells

Mobilized stem cells in the peripheral blood (PB) must be efficiently harvested at the appropriate time before autologous PB stem cell (PBSC) transplantation. Enumeration of CD34+ cells in the PB before apheresis predicts the number of PBSCs that can be collected, but the cytometric techniques used are complex and expensive. Therefore, it is necessary to identify an alternative to the CD34+ cell count in PBSC harvest-time monitoring. Fully automated flow cytometry using blood cell counters now allows reliable quantification of immature myeloid cells in the PB, referred to as hematopoietic progenitor cells (HPC), and reticulated platelets, expressed as the immature platelet fraction (IPF). Immature or reticulated platelets are thought to correlate with thrombopoietic activity of the marrow. Following a chemotherapy nadir, the recovery of white blood cell and platelet counts has been used to determine the right time for apheresis. Therefore, we examined whether the HPC count and IPF value could be used to predict PBSC mobilization in 20 patients with hematological malignancies. The HPC count was found to be correlated with the CD34+ cell count (r = 0.84, P < 0.01), whereas the IPF value was not (r = 0.37, P = 0.44). Therefore, the HPC count, but not the IPF value, is a possible predictor of the timing of autologous stem cell transplantation.     

原核表达重组人白介素-3及其对脐带血造血干细胞体外增殖的影响

目的研究原核表达重组人白介素-3(rhIL-3)及其对脐带血造血干细胞体外增殖的影响。方法构建pET-43.1-rhIL-3表达载体,克隆至BL21菌株,并进行中试发酵生产,所得包涵体经透析复性后,采用阳离子交换柱纯化,纯化产物送N-端测序。Ficoll密度梯度离心结合免疫磁珠法富集人脐带血中CD34+细胞,以不同浓度rhIL-3及干细胞因子(SCF)、Fms样酪氨酸激酶受体-3配体(FL)和促血小板生成素(TPO)因子组合培养1周,测定细胞总数,计算CD34+细胞增殖倍数。结果在SCF和FL存在下,rhIL-3可明显促进造血干祖细胞增殖,但较无rhIL-3时CD34+细胞占细胞总数的比例有所下降。与SCF、FL共同作用时的最佳浓度在15 ng/mL左右,与SCF、FL、TPO共同作用时的最佳浓度在50 ng/mL左右。结论高效表达及纯化的rhIL-3与SCF、FL和TPO细胞因子组合对脐带血造血干细胞具有良好的增殖作用。     

Donor lymphocyte apheresis for adoptive immunotherapy compared with blood stem cell apheresis

Donor lymphocyte transfusion has gained considerable interest as adoptive cellular immunotherapy for prevention or treatment of relapse after allogeneic stem cell transplantation. This study was designed to compare the yield of CD3⁺, CD3⁺4⁺, CD3⁺8⁺, CD19⁺, CD3^?56⁺16⁺, and CD34⁺ cells contained in apheresis products from 61 consecutive non‐cytokine treated, human leukocyte antigen (HLA)‐matched donors for lymphocyte collection with the corresponding apheresis‐derived cell yield from 112 consecutive, HLA‐matched donors for blood stem cell collection who received recombinant human granulocyte colony stimulating factor (rhG‐CSF, filgrastim) 6 μg/kg every 12 hours until cell collection was completed. Apheresis was started on day 4 or 5 of rhG‐CSF treatment. The yield of lymphoid subsets was significantly different in the two sample groups, rhG‐CSF treated product yields exceeding untreated product yields by a median of 2.1‐fold (range: 1.3–2.6). However, the CD34⁺ cell yield in rhG‐CSF‐treated apheresis products exceeded untreated products by 26‐fold. A single untreated apheresis procedure was usually sufficient to collect a target dose of 1 × 10⁸/kg CD3⁺ cells. Untreated apheresis products contained a median of 0.2 × 10⁶/kg CD34⁺ cells. A potential engraftment dose of ≥0.5 × 10⁶ CD34⁺ cells per kg of recipient body weight was contained in 16% of 57 untreated apheresis products. One single apheresis performed in a normal, untreated donor provides a sufficient amount of CD3⁺ cells for adoptive immunotherapy. Compared with that of an rhG‐CSF stimulated apheresis product, the CD34⁺ cell count is usually, but not always, below the engraftment dose range. RhG‐CSF treatment has little effect on the yield of lymphoid subsets collected by apheresis but is highly selective of the release of CD34⁺ cells. This report provides baseline data for studies that will show whether other cytokines such as granulocyte macrophage colony stimulating factor (GM‐CSF) and/or Flt‐3 Ligand can immunomodulate allotransfusates in vivo to improve the graft‐vs.‐leukemia (GVL) effect after allogeneic stem cell transplantation, while lowering the incidence and severity of graft‐vs.‐host disease (GVHD). J. Clin. Apheresis. 16:82–87, 2001. © 2001 Wiley‐Liss, Inc.     

A new model for predicting the timing of leukapheresis on the basis of CD34+ cell and hematopoietic progenitor cell levels

We developed a model (depending on peripheral CD34(+) cell count and hematopoietic progenitor cell count) to determine the optimal timing of 3-day leukapheresis in patients pretreated with chemotherapy and G-CSF. Marrow potentials were identified on the basis of three patterns of leukapheretic yield. Pattern 1 predicted good marrow potential. The positive predictive value of a first-day leukapheretic yield of >1 x 10(6) CD34(+) cells/kg (mean 3-day yield = 8.18 x 10(6) CD34(+) cells/kg, n = 11) was 100%. Pattern 2 predicted poor marrow potential. The negative predictive value of a 3-day leukapheretic yield of >1 x 10(6) CD34(+) cells/kg (3-day yield = 0.26 x 10(6) CD34(+) cells/kg, n = 1) was 100%. Pattern 3 met neither of the above criteria (mean 3-day yield = 1.37 x 10(6) CD34(+) cells/kg, n = 19). The marrow potential was borderline and patients could be further divided into two subgroups according to peripheral CD34(+) cell counts when WBC reached >10,000/microl. The mean yield differed significantly between pattern 1 and 3 (P < 0.001). For patients with good marrow potential, leukapheresis should begin as soon as the WBC count is >5,000/microl. Patients with borderline marrow potential may benefit from delaying leukapheresis until the WBC level is >10,000/microl and leukapheresis extended more than 3 days.     

Impact of apheresis automation on procedure quality and predictability of CD34+ cell yield

Success of peripheral blood stem cell (PBSC) collections depends on patient biological parameters and stable apheresis device performance. We investigated product quality and factors influencing main apheresis procedure outcomes including CD34⁺ collection efficiency (CE), product volume or platelet CE. We also assessed different CD34⁺ cell yield prediction algorithms. Autologous PBSC collections by Spectra Optia from myeloma and lymphoma patients were analyzed. Complete blood count (CBC) from patient preprocedure and from collected products were assessed. (1) Product yield was calculated, (2) Product CBC was correlated with patient preprocedure variables, and (3) Predictions of CD34⁺ yields based on (a) product CD34⁺ cell concentration in samples after two or four chamber flushes or (b) traditional CE2 benchmark, were compared. 62 procedures in 41 patients were analyzed. 84% of all procedures were run without operator intervention. Median CD34⁺ CE2 was 56.9% (48.8%‐65.2%) and quite stable irrespective of patient conditions, with minor influence from patient white blood cell (WBC) precounts (r_s = –.47; P < .001). Platelet loss correlated with WBC precount (r_s = .46; P < .001), product volume (r_s = .71; P < .0001) and number of chambers collected (r_s = .72; P < .0001). CD34⁺ cell yield was better predicted based on (a) product CD34⁺ cell concentration from samples after 2 and 4 chamber flushes, respectively (r_s = .969; P < .0001 and r_s = .9648; P < .0001) than based on (b) CE2 formula (r_s = .8262, P < .0001). Spectra Optia provides good quality PBSC products with stable and predictable yield regardless of starting conditions. CD34⁺ sampling of product after few chamber flushes could be used to predict CD34⁺ yield.     

不同细胞因子组合对脐血造血干细胞体外扩增效果分析

目的观察不同细胞因子组合对脐血干细胞体外扩增效果的影响。方法用含15%胎牛血清的改良细胞培养液,按加入的不同细胞因子组合分成7个实验组(A—G)进行细胞培养。分别于培养0d、3d、7d、14d取培养液,台盼蓝染色计数单个核细胞的总数、流式细胞仪分析CD34+细胞含量。结果与对照组相比,含细胞因子的7个实验组单个核细胞的数量均显著增加,除F组在培养d14时扩增达到峰值(15.60±10.90)×109/L外,其它各实验组在扩增d7时,均达到了扩增峰值并以E组为最高(17.11±6.12)×109/L,培养延续到d14时,各组均有不同程度的下降。所有实验组CD34+细胞的百分比也显著提高,A、D、E、F、G组均在d7达到了峰值,B、C组则在第14d达到峰值。经计算,扩增7d时CD34+细胞单位体积含量范围在(34.7±10.4)×107/L(A组)—(168.6±43.5)×107/L(这种表述似乎不太合适),对应的扩增达10—50倍。结论不同细胞因子组合可以提高脐血单个核细胞和CD34+细胞的扩增效率,SCF+IL-3,-6,-2,-4的组合在d7时达到了最佳的扩增。