Method comparison between hematopoietic progenitor cell and CD34+ cell counts in hematopoietic stem cell collection.

BACKGROUND: The reference method for hematopoietic stem cell enumeration is flow cytometric CD34+ cell analysis. We evaluated using the hematopoietic progenitor cell (HPC) count on the Sysmex hematology analyzer to safely replace some flow cytometric measurements performed in peripheral blood samples to guide apheresis timing. STUDY DESIGN AND METHODS: We compared HPC and CD34+ cell counts in 133 preharvest peripheral blood samples and 124 apheresis products. RESULTS: Pre-apheresis HPC counts ≥24 × 106/L in healthy donors and ≥36 × 106/L in lymphoma patients predicted adequate mobilization with 100% specificity and positive predictive value, saving 79% and 63% of flow cytometry analyses, respectively. Due to a positive bias (mean bias 50.26; 95% CI 36.24-64.29), a higher threshold was needed in multiple myeloma patients (HPC ≥ $$ \ge $$ 132 × 106/L), saving only 24% of flow cytometry analyses. CONCLUSION: When the HPC count is above the corresponding threshold, apheresis could be safely initiated without waiting for the flow cytometry result, thereby reducing time-to-decision. Lower HPC values, however, require confirmation by flow cytometry.

CD34+ cell yield among healthy donors: Large-scale model development and validation.

BACKGROUND: Successful engraftment in hematopoietic stem cell transplantation necessitates the collection of an adequate dose of CD34+ cells. Thus, the precise estimation of CD34+ cells harvested via apheresis is critical. Current CD34+ cell yield prediction models have limited reproducibility. This study aims to develop a more reliable and universally applicable model by utilizing a large dataset, enhancing yield predictions, optimizing the collection process, and improving clinical outcomes. MATERIALS AND METHODS: A secondary analysis was conducted using the Center for International Blood and Marrow Transplant Research database, involving data from over 17 000 healthy donors who underwent filgrastim-mobilized hematopoietic progenitor cell apheresis. Linear regression, gradient boosting regressor, and logistic regression classification models were employed to predict CD34+ cell yield. RESULTS: Key predictors identified include pre-apheresis CD34+ cell count, weight, age, sex, and blood volume processed. The linear regression model achieved a coefficient of determination (R2) value of 0.66 and a correlation coefficient (r) of 0.81. The gradient boosting regressor model demonstrated marginally improved results with an R2 value of 0.67 and an r value of 0.82. The logistic regression classification model achieved a predictive accuracy of 96% at the 200 × 106 CD34+ cell count threshold. At thresholds of 400, 600, 800, and 1000 × 106 CD34+ cell count, the accuracies were 88%, 83%, 83%, and 88%, respectively. The model demonstrated a high area under the receiver operator curve scores ranging from 0.90 to 0.93. CONCLUSION: This study introduces advanced predictive models for estimating CD34+ cell yield, with the logistic regression classification model demonstrating remarkable accuracy and practical utility.

High CD34-positive cell dose in matched unrelated donor allogeneic hematopoietic stem cell transplant is not associated with graft-versus-host disease or mortality.

BACKGROUND: CD34+ stem cells serve as the primary graft source for allogeneic transplants, with a minimum of 2-4 × 106 cells/kg needed for engraftment. There are conflicting data on outcomes at high stem cell doses, with studies limited by few patients receiving doses far above the minimum target. STUDY DESIGN AND METHODS: In this retrospective, single-center study of patients with hematologic malignancies who underwent matched unrelated donor transplants, we assessed outcomes for engraftment, survival, relapse, and graft-versus-host disease (GVHD) for the highest CD34+ dose quintile (>13 × 106 cells/kg, n = 36) compared to the remaining patients (n = 139). Similar analysis was performed correlating T cell dose and outcomes. RESULTS: There was no difference between the groups in neutrophil engraftment, with a trend toward faster platelet engraftment. There was no significant difference in mortality (adjusted risk ratio [aRR] = 1.02, 95% confidence interval [CI] = 0.85-1.22), relapse (aRR = 1.10, 95% CI = 0.85-1.42), or overall survival by Kaplan-Meier analysis (p = .44). High CD34+ dose was not associated with higher incidence of acute GVHD (aRR = 0.99 grades II-IV, aRR = 1.18 grades III-IV) or chronic GVHD (aRR = 0.87 overall, RR = 1.21 severe). There was limited correlation between CD34+ and T cell dose (R2 = .073), and there was no significant difference in survival, relapse, or GVHD in the highest T cell dose quintile (n = 33) compared to the remaining quintiles (n = 132). DISCUSSION: We found no difference in survival, relapse, or GVHD incidence or severity in patients receiving CD34+ doses above prior cutoffs reported in the literature. These data do not support the routine use of graft CD34+ dose reduction.

Modified lentiviral globin gene therapy for pediatric ß0/ß0 transfusion-dependent ß-thalassemia: A single-center, single-arm pilot trial.

ß0/ß0 thalassemia is the most severe type of transfusion-dependent ß-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a ß-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in ß0/ß0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5 months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22 months. The treatment extended the lifespan of red blood cells by over 42 days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all ß-thalassemia.

Effects of CD34+ cell dose on engraftment and long-term outcomes after allogeneic bone marrow transplantation.

BACKGROUND: The number of CD34+ cells in the graft is generally associated with time to engraftment and survival in transplantation using cord blood or allogeneic peripheral blood stem cells. However, the significance of abundant CD34+ in bone marrow transplantation (BMT) remained unclear. METHODS: We retrospectively reviewed 207 consecutive adult patients who underwent their first BMT at Jichi Medical University between January 2009 and June 2021. RESULTS: The median nucleated cell count (NCC) and CD34+ cell dose were 2.17 × 108/kg (range .56-8.52) and 1.75 × 106/kg (.21-5.84), respectively. Compared with 104 patients in the low CD34+ group (below the median), 103 patients in the high CD34+ group (above the median) showed faster engraftment at day +28 in terms of neutrophil (84.6% vs. 94.2%; p =  .001), reticulocyte (51.5% vs. 79.6%; p < .001), and platelet (39.4% vs. 72.8%; p < .001). There were no significant differences in overall survival, relapse, nonrelapse mortality, acute or chronic graft-versus-host disease, or infectious complications between the two groups in univariate and multivariate analyses. Low or high NCC had no significant effect on overall survival, nonrelapse mortality, cumulative incidence of relapse and graft-versus-host disease, either. While a positive correlation was observed between NCC and the CD34+ cell dose, a high CD34+ cell dose was associated with rapid hematopoietic recovery, even in patients with NCC below the median. CONCLUSION: Measurement of CD34+ cell dose in addition to NCC was useful for predicting hematopoietic recovery, but seemed to have little influence on the long-term outcome in BMT.

G-CSF + plerixafor versus G-CSF alone mobilized hematopoietic stem cells in patients with multiple myeloma and lymphoma: a systematic review and meta-analysis.

AIM: The combination of granulocyte-colony stimulating factor (G-CSF) and plerixafor is one of the approaches for hematopoietic stem cell mobilization in patients with multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), and Hodgkin's lymphoma (HL). This systematic review and meta-analysis aimed to determine the ability of G-CSF + plerixafor to mobilize peripheral blood (PB) CD34+ cells and examine its safety profile. METHODS: We performed a database search using the terms 'granulocyte colony stimulating factor', 'G-CSF', 'AMD3100', and 'plerixafor', published up to May 1, 2023. The methodology is described in further detail in the PROSPERO database (CRD42023425760). RESULTS: Twenty-three studies were included in this systematic review and meta-analysis. G-CSF + plerixafor resulted in more patients achieving the predetermined apheresis yield of CD34+ cells than G-CSF alone (OR, 5.33; 95%, 4.34-6.55). It was further discovered that G-CSF + plerixafor could mobilize more CD34+ cells into PB, which was beneficial for the next transplantation in both randomized controlled (MD, 18.30; 95%, 8.74-27.85) and single-arm (MD, 20.67; 95%, 14.34-27.00) trials. Furthermore, G-CSF + plerixafor did not cause more treatment emergent adverse events than G-CSF alone (OR, 1.25; 95%, 0.87-1.80). CONCLUSIONS: This study suggests that the combination of G-CSF and plerixafor, resulted in more patients with MM, NHL, and HL, achieving the predetermined apheresis yield of CD34+ cells, which is related to the more effective mobilization of CD34+ cells into PB.

Impact of plerixafor use in the mobilization of blood grafts for autologous hematopoietic cell transplantation.

Plerixafor (PLER), a reversible antagonist of the CXC chemokine receptor type 4, has been in clinical use for mobilization of blood grafts for autologous hematopoietic cell transplantation (AHCT) for about 15 years. Initially PLER was investigated in placebo-controlled trials with the granulocyte colony-stimulating factor (G-CSF) filgrastim. It has also been used in combination with chemotherapy plus G-CSF in patients who had failed a previous mobilization attempt or appeared to mobilize poorly with current mobilization (preemptive use). This review summarizes what is known regarding addition of PLER to standard mobilization regimens. PLER increases mobilization of CD34+ cells, decreases the number of apheresis sessions needed to achieve collection targets and increases the proportion of patients who can proceed to AHCT. It appears also to increase the amount of various lymphocyte subsets in the grafts collected. In general, hematologic recovery after AHCT has been comparable to patients mobilized without PLER, although slower platelet recovery has been observed in some studies of patients who mobilize poorly. In phase III studies, long-term outcome has been comparable to patients mobilized without PLER. This also appears to be the case in patients receiving plerixafor for poor or suboptimal mobilization of CD34+ cells. In practice, PLER is safe and has not been shown to increase tumor cell mobilization.

An evidence-based and risk-adapted GSF versus GSF plus plerixafor mobilization strategy to obtain a sufficient CD34+ cell yield in the harvest for autologous stem cell transplants.

BACKGROUND: Plerixafor is a bicyclam molecule with the ability to reversibly bind to receptor CXCR-4 thus leading to an increased release of stem cells (SC) into the circulation. This study aims to evaluate the efficacy of G-CSF plus plerixafor versus G-CSF alone mobilizing regimens on the basis of CD34+ cell yield and engraftment kinetics following hematopoietic SC transplants. METHODS: The study incorporated 173 patients with plasma cell neoplasms (PCN), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), undergoing mobilization and following autologous SC-transplant. For patients with mobilization failure and those predicted to be at risk of harvesting inadequate CD34+ yields (poor-responders), plerixafor was administered. Data was collected and compared in relation to the harvesting protocols used, cell quantification, cell-engraftment potential and overall clinical outcome. RESULTS: A total of 101 patients received plerixafor (58.4 %) and the median CD34+increase was 312 %. Chemotherapy-mobilized PCN-patients required less plerixafor administration (p = 0.01), no difference was observed in lymphoma groups (p = 0.46). The median CD34+cell yield was 7.8 × 106/kg bm. Patients requiring plerixafor achieved lower, but still comparable cell yields. Total cell dose infused was in correlation with engraftment kinetics. Patients requiring plerixafor had delayed platelet engraftment (p = 0.029). CONCLUSIONS: Adequately selected plerixafor administration reduces "mobilization-related-failure" rate and assure a high-level cell dose for SC transplants, with superior "therapeutic-potential" and safety profile. The mobilization strategy that incorporates "just-in-time" plerixafor administration, also leads to a reduction of hospitalization days and healthcare resource utilization. For definitive conclusions, further controlled/larger clinical trials concerning correlation of CD34+ cell count/yield, with hematopoietic reconstitution are required.

Loss of CD34+ Cells and Effect of the Number of Viable Cryopreserved CD34+ Cells in the Infused Blood Grafts on Hematologic Recovery, Progression-Free Survival and Overall Survival in NHL Patients After Autologous Stem Cell Transplantation.

PATIENTS: This post-hoc study aimed to find out factors affecting graft viable CD34+ cell loss during processing and cryopreservation in 129 non-Hodgkin lymphoma (NHL) patients receiving autologous stem cell transplantation (auto-SCT) and the impact of a low (< 2.0 × 106/kg, group A) and a decent number (≥ 2 × 106/kg, group B) of viable CD34+ cells infused on the hematologic recovery, progression-free survival (PFS) and overall survival (OS) after auto-SCT. RESULTS: The median loss of viable CD34+ cells during cryopreservation was higher in group A (47% vs. 19%, p < .001). A higher yield of CD34+ cells at the first apheresis in group B (p = .002) was linked with greater loss of viable graft CD34+ cells after cryopreservation. Filgrastim (FIL) use for mobilization seemed to associate with higher viable CD34+ cell loss compared to pegfilgrastim (PEG) or lipegfilgrastim (LIPEG) in both groups (in group A FIL 66 vs. PEG 35%, p = .006; in group B FIL 37 vs. PEG 15 vs. LIPEG 13%, p < .001). Hematologic recovery after auto-SCT was faster in group B. Neither viable CD34+ cell loss during storage nor viable CD34+ cell number < 2.0 × 106/kg infused affected on PFS or OS. CONCLUSIONS: G-CSF type used in mobilization and mobilization capacity were found to correlate with viable CD34+ cell loss during processing and storage. Most importantly, low infused viable CD34+ cell count did not seem to impact on PFS or OS.

Selection of Cord Blood Unit by CD34+ Cell and GM-CFU Numbers and Allele-Level HLA Matching in Single Cord Blood Transplantation.

In Japan, only single-unit cord blood transplantations (CBTs) are typically performed, and their number has increased over the last 23 years, with ongoing improvement in results. In most cases, CBTs with multiple HLA mismatches are used, owing to a low HLA barrier, and lower engraftment rate is a problem that must be overcome. Here, as part of an effort to improve guidelines for the selection and processing of CB units for transplantation, we sought to assess the present status of CBT in Japan and to elucidate factors contributing to the favorable outcomes, focusing in particular on selection by cell components of CB unit and HLA allele matching. We conducted a nationwide study analyzing 13,443 patients who underwent first CBT between in Japan between December 1997 and December 2019 using multivariate regression analysis. Both patient- and transplantation-related variables, such as age and Hematopoietic Cell Transplantation Comorbidity Index, as well as selected CB unit characteristics, were included in the analysis. The interaction analysis elucidated that CB unit selection favoring higher counts of CD34+ cells and granulocyte macrophage colony-forming units (GM-CFU)/kg, but not of total nucleated cells, contributed to improved engraftment after transplantation. Moreover, a higher CD34+ cell dose was associated with improved overall survival (OS). Distinctive HLA allele matching was observed. A 0 or 1 HLA allele mismatch between patient and donor had favorable engraftment and carried significantly lower risks of acute GVHD and chronic GVHD but had a significantly higher leukemia relapse rate, compared with a 3-HLA allele mismatch. HLA-DRB1 mismatches were associated with reduced risk of leukemia relapse. Notably, the number of HLA allele mismatches had no incremental effect on engraftment, acute and chronic GVHD, or relapse incidence. As a result, 5-year overall survival did not differ significantly among patients receiving CB units with 0 to 7 HLA allele mismatches. The main points of CB unit selection are as follows. First, selection according to a higher number of CD34+ cells/kg and then of CFU-GM/kg is recommended to obtain favorable engraftment. A unit with .5 × 105 CD34+ cells/kg is minimally acceptable. For units with a CD34+ cell dose of .5 to 1.0 × 105 cells/kg, applying the parameter of ≥20 to 50 × 103 GM-CFU/kg (66.5% of transplanted CB units in this cohort) is associated with a neutrophil engraftment rate of approximately 90%. A unit with ≥1.0 × 105 CD34+ cells/kg can achieve a ≥90% mean neutrophil engraftment rate. Subsequently, HLA allele matching of HLA-A, -B, -C, and -DRB1 at the 2-field level should be searched for units with 0 or 1 HLA allele mismatch in the host-versus-graft direction for favorable engraftment. Units with 2 to 6 HLA allele mismatches are acceptable in patients age ≥15 years and units with 2 to 4 HLA allele mismatches are acceptable in patients age ≤14 years. Units with HLA-DRB1 and/or -B allele mismatch(es) might not be preferable owing to an increased GVHD risk. Our analysis demonstrates that single-unit CBT with the selection of adequate CD34+/kg and GM-CFU/kg and HLA allele matching showed favorable outcomes in both pediatric and adult patients.

Repetitive administration of cultured human CD34+ cells improve adenine-induced kidney injury in mice.

BACKGROUND: There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease (CKD). AIM: To examine the efficacy of cultured human CD34+ cells with enhanced proliferating potential in kidney injury in mice. METHODS: Human umbilical cord blood (UCB)-derived CD34+ cells were incubated for one week in vasculogenic conditioning medium. Vasculogenic culture significantly increased the number of CD34+ cells and their ability to form endothelial progenitor cell colony-forming units. Adenine-induced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice, and cultured human UCB-CD34+ cells were administered at a dose of 1 × 106/mouse on days 7, 14, and 21 after the start of adenine diet. RESULTS: Repetitive administration of cultured UCB-CD34+ cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group. Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group (P < 0.01). Microvasculature integrity was significantly preserved (P < 0.01) and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group (P < 0.001). CONCLUSION: Early intervention using human cultured CD34+ cells significantly improved the progression of tubulointerstitial kidney injury. Repetitive administration of cultured human UCB-CD34+ cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Effect of CD34+ Cell Dose on the Outcomes of Allogeneic Stem Cell Transplantation with Post-Transplantation Cyclophosphamide.

The impact of infused CD34+ cell dose on outcomes after allogeneic hematopoietic stem cell transplantation (alloHSCT) using standard graft-versus-host disease (GVHD) prophylaxis remains controversial. Information on this subject is scarce for alloHSCT using high-dose post-transplantation cyclophosphamide (PTCy). We aimed to assess the effect of CD34+ cell dose in peripheral blood stem cell (PBSC) grafts on the outcome of alloHSCT using PTCy-based GVHD prophylaxis. To do so, we conducted a single-center retrospective analysis of 221 consecutive adult patients who underwent PTCy alloHSCT from HLA-matched sibling donors (MSDs; n = 22), HLA-matched unrelated donors (MUDs; n = 83), mismatched unrelated donors (MMUDs; n = 73), and haploidentical donors (n = 43). Based on the binary partitioning method, 5 × 106/kg was used as the optimal cutoff for CD34+ cell dose. According to our institutional protocol, the maximum CD34+ cell dose was capped at 8 × 106/kg. The study cohort was divided into 2 groups based on CD34+ cell dose: high dose (>5 to 8 × 106/kg) and low dose (≤5 × 106/kg). Patients receiving high-dose CD34+-containing grafts had significantly shorter median times to neutrophil engraftment and platelet engraftment compared to those who received low-dose CD34+ (19 days versus 21 days [P = .002] and 16 days versus 22 days [P = .04], respectively). There were no differences between the high-dose and low-dose groups in the cumulative incidence of day +100 acute GVHD (grade II-IV: 25% versus 23% [P = .7]; grade III-IV: 5% versus 4% [P = .4], respectively) or 2-year chronic GVHD (moderate/severe GVHD: 9% versus 6%; P = .5). There was no impact of CD34+ cell dose on survival outcomes with the use of MSDs, MUDs, or MMUDs. Recipients of haploidentical alloHSCT using low-dose CD34+ cells had significantly worse overall survival (hazard ratio [HR], 6.01; P = .004) and relapse-free survival (HR, 4.57; P = .004). In recipients of PBSC PTCy alloHSCT, infused CD34+ cell doses >5 to 8 × 106/kg were associated with faster neutrophil and platelet engraftment, independent of donor type. Our study suggests an impact of CD34+ cell dose on survival outcomes only with haploidentical donors, for whom the administration of a CD34+ cell dose ≤5 × 106/kg significantly decreased survival outcomes.

Effects of CD34+ cell dose on haematopoietic recovery in acute lymphoblastic leukaemia patients with positive pretransplant measurable residual disease.

INTRODUCTION: A higher CD34+ cell dose in allografts is associated with faster haematopoietic recovery after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Leukaemia stem cells impair normal bone marrow (BM) niches and induce BM failure during leukemogenesis. However, whether measurable residual disease (MRD), known as the persistence of low-level leukaemic cells, could influence the effects of CD34+ cell dose on haematopoietic recovery after transplantation in acute lymphoblastic leukaemia (ALL) patients is unknown. METHODS: A total of 975 ALL patients were enrolled and classified into pre-HSCT MRD-positive and MRD-negative subgroups. Cox proportional hazard regression models were built for time-to-event outcomes. Multivariate analysis was performed to determine independent influencing factors from the univariate analysis. RESULTS: An appropriate CD34+ cell dose was positively associated with faster haematopoietic recovery in the total ALL population. More importantly, in pre-HSCT MRD-positive ALL patients, a higher CD34+ cell dose (≥2.76 × 106 /kg) was related to faster neutrophil (HR 1.330, 95% CI 1.045-1.692, p = 0.021) and platelet engraftment (HR 1.808, 95% CI 1.412-2.316, p < 0.001) in multivariate analysis. CD34+ cell dose was a crucial factor associated with either engraftment or transplant outcomes, although we did not demonstrate direct correlations of CD34+ cell dose with relapse, TRM, LFS or OS after allo-HSCT. CONCLUSION: Our results indicated that no additional CD34+ stem and progenitor cell harvests were needed to ensure successful haematopoietic recovery in pre-HSCT MRD-positive patients compared to pre-HSCT MRD-negative patients.

Advancements in Cell-Based Therapies for HIV Cure.

The treatment of human immunodeficiency virus (HIV-1) has evolved since the establishment of combination antiretroviral therapy (ART) in the 1990s, providing HIV-infected individuals with approaches that suppress viral replication, prevent acquired immunodeficiency syndrome (AIDS) throughout their lifetime with continuous therapy, and halt HIV transmission. However, despite the success of these regimens, the global HIV epidemic persists, prompting a comprehensive exploration of potential strategies for an HIV cure. Here, we offer a consolidated overview of cell-based therapies for HIV-1, focusing on CAR-T cell approaches, gene editing, and immune modulation. Persistent challenges, including CAR-T cell susceptibility to HIV infection, stability, and viral reservoir control, underscore the need for continued research. This review synthesizes current knowledge, highlighting the potential of cellular therapies to address persistent challenges in the pursuit of an HIV cure.

Analytical performance verification protocols and specifications of CD34 +cell enumeration by flow cytometry / 中华检验医学杂志

Objective:To investigate the analytical performance verification protocols and performance specifications of CD34+cell enumeration by flow cytometry for clinical laboratories.Methods:According to international guidelines and National Health Standard of China, we designed the performance verification protocols of CD34 +cell enumeration (including percent count and absolute count) by flow cytometry. Four quality assessment materials, three leukapheresis products and three samples of peripheral blood were selected to verify the precision, linearity, carryover, trueness and accuracy of FACSCanto Ⅱ measurement system, and the assessment criterion was set according to the detection technologies of clinical laboratories. Results:The CVs of intra-run precision of percent count and absolute count were 2.5% to 8.9% and 3.0% to 9.0%; the CVs of inter-run precision were 2.8% to 10.5% and 3.8% to 9.9%, respectively. The slopes of linearity regression equation of low range (3.6/μl to 123.6/μl) and high range (113.2/μl to 1196.3/μl) were 0.993 2 and 0.965 2, and R2 were 0.999 6 and 0.993 9, and the biases were -8.67% to 0.22%. The carryover of percent and absolute count were 0.07% and 0.00%. When percent count≤0.2% or absolute count≤20/μl, the absolute biases of trueness were in the range of ±0.006% or ±0.5/μl, and the absolute biases of accuracy were in the range of ±0.02% or ±0.9/μl; when percent count>0.2% or absolute count>20/μl, the relative biases of trueness were in the range of ±5.65%, and the relative biases of accuracy were in the range of ±8.19%. The verification results met the assessment criterion set in this study. Conclusions:The performance verification protocols and assessment criterion formulated in this study not only conform to the recommendations of domestic and foreign guidelines, but also conform to state of the detection technologies of native clinical laboratories, which can be taken as a reference of performance verification for clinical laboratories.

Subclones of bone marrow CD34+ cells in acute myeloid leukemia at diagnosis confer responses of patients to induction chemotherapy.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a prognosis that varies with genetic heterogeneity of hematopoietic stem/progenitor cells (HSPCs). Induction chemotherapy with cytarabine and anthracycline has been the standard care for newly diagnosed AML, but about 30% of patients have no response to this regimen. The resistance mechanisms require deeper understanding. METHODS: In our study, using single-cell RNA sequencing, we analyzed the heterogeneity of bone marrow CD34+ cells from newly diagnosed patients with AML who were then divided into sensitive and resistant groups according to their responses to induction chemotherapy with cytarabine and anthracycline. We verified our findings by TCGA database, GEO datasets, and multiparameter flow cytometry. RESULTS: We established a landscape for AML CD34+ cells and identified HSPC types based on the lineage signature genes. Interestingly, we found a cell population with CRIP1high LGALS1high S100Ashigh showing features of granulocyte-monocyte progenitors was associated with poor prognosis of AML. And two cell populations marked by CD34+ CD52+ or CD34+ CD74+ DAP12+ were related to good response to induction therapy, showing characteristics of hematopoietic stem cells. CONCLUSION: Our study indicates the subclones of CD34+ cells confers for outcomes of AML and provides biomarkers to predict the response of patients with AML to induction chemotherapy.

A comparison of four leukapheresis methods to harvest an optimal dose of CD34+ cells: A single center experience.

BACKGROUND: Chemokine receptor CXCR4 antagonist plerixafor (Px) as well as high volume (HV) leukapheresis have been shown to reduce hematopoietic stem progenitor cell (HSPC) mobilization failure rates. However, no direct comparisons of such methods currently exists. METHODS AND MATERIALS: We compared the HSPC collection yield based on basal peripheral blood CD34+ cell numbers in patients diagnosed with multiple myeloma or non-Hodgkin's lymphoma undergoing autologous stem cell transplantation in a retrospective chart review. The leukapheresis methods used included HV versus regular volume (RV) with or without Px. There were 116 patients in the study group while the historical control group had 34 patients. RESULTS AND CONCLUSIONS: Control group underwent RV leukapheresis without Px. Addition of Px or HV in the study group failed to display significant improvement in CD34+ cell collection yield; however, when basal CD34+ cell numbers were taken into account, both Px + RV and HV without Px increased CD34+ cell collection yield. The collection failure rates of HV without Px group were comparable to Px + RV when the basal CD34+ cell numbers were over 20/µl. Of interest, multivariate linear regression analysis did not detect any significant difference between HV versus Px + RV or other leukapheresis methods in CD34 yields or collection failure rates from a single collection after controlling for other factors (sex, age, or underlying disease). In multivariate analysis, pre apheresis CD34+ cell number was significantly and positively associated with the CD34+ cell yields from a single apheresis. In our studies, the majority of patients can be rescued without Px by HV alone as a potential cost saving approach. In summary, trend in our studies reflects that both Px and HV are capable of reducing the mobilization failure rates except the poorest mobilizers, which will need to be validated in larger studies.

Successful Mobilization of Autologous Hematopoietic Peripheral Blood Stem Cells after Salvage Chemotherapy in Patients with Low CD34 Blood Cell Counts.

A major barrier for proceeding to autologous stem cell transplantation (ASCT) is an inability to mobilize and collect an adequate number of peripheral blood (PB) stem cells (PBSC) for the transplant graft. Plerixafor added to granulocyte colony stimulating factor (G-CSF) alone, without prior chemotherapy, significantly improves the mobilization of autologous PBSC in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). However, the efficacy of plerixafor and the best timing to give the drug to poorly mobilizing patients with very low PB CD34+ cell counts after salvage chemotherapy and G-CSF are not well defined. We hypothesized that PBSC mobilization and collection might be improved in heavily treated patients who mobilized very poorly after salvage chemotherapy and G-CSF by alternating the days of plerixafor administration and leukapheresis. A day of rest between plerixafor doses, while continuing G-CSF, could allow time for some replenishment of the marrow stem/progenitor cell pool before the next mobilization. A retrospective review of collection results in poorly mobilizing patients at our center was undertaken. Three cohorts were identified: those who got every-other-day plerixafor and leukapheresis, those who got sequential plerixafor and leukapheresis and those who got risk adapted plerixafor. Overall, 69% of patients with NHL and MM with PB CD34+ cell counts <5/µL after salvage chemotherapy and G-CSF were ultimately able to collect adequate CD34+ cells to support ASCT using daily plerixafor and leukapheresis. On the alternating plerixafor and leukapheresis schedule, all 17 patients achieved the cumulative CD34+ cell product goals required for ASCT. This positive observation after salvage chemotherapy and G-CSF led to the incorporation at our center of an alternate-day schedule of plerixafor and leukapheresis into our real-time risk adapted strategy for poor mobilizers. © 2023 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Isolation and Culture of Non-adherent Cells for Cell Reprogramming.

Coronary heart disease (CHD) is a leading cause of death globally, while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration, and CD34+ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However, the isolation and culture of non-adherent CD34+ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34+ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment, isolation, and expansion. The culture was subsequently observed for their viability, adherence, and confluence. Day 0 observation of the culture showed a healthy CD34+ cell with a round cell shape, without any adherent cells present yet. Day 4 of observation showed that CD34+ cells within the blood plasma medium became adherent, indicated by their transformations into spindle or oval morphologies. Meanwhile, CD34+ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34+ cells in blood plasma medium, and which had 75% of confluence. In conclusion, the CD34+ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium, but not in cultures using fibronectin and vitronectin.

Association of CD 34 positive cell dose with engraftment kinetics in autologous peripheral blood stem cell transplant patients of multiple myeloma.

Background: Treatment with high-dose chemotherapy and stem cell transplantation has prolonged survival in patients of multiple myeloma (MM). A dose-response relationship between number of CD34+ cells infused and leukocyte and platelet recovery, exists. Patients receiving dose of <2.0 × 106 CD34+ cells/kg have delayed engraftment. The level of optimal cutoff for accelerated engraftment is yet to be validated. Hence, this study was undertaken to study the association of CD 34+ cell dose with engraftment kinetics in patients of MM who underwent autolgous peripheral blood stem cell transplant (PBSCT). Methods: We retrospectively analyzed 19 patients of MM who underwent PBSCT at our center between December 2016 to December 2018. Complete blood counts were carried out daily after transplantation to record neutrophil and platelet engraftment. Results: Based on the CD34+ cell dose given : <5 × 106/kg (category 1), 5-10 × 106/kg (category 2), >5 × 106/kg (category 3), the mean (SD) neutrophil engraftment time was 11.3 (0.5) days, 10.6 (0.9) days, and 10.2 (1.3) days respectively. Platelet engraftment time was 12.4 (2.60) days, 10.6 (1.14) days, and 11.2 (1.64) days for category 1, 2, and 3 patients, respectively. Correlation co-efficient between CD 34+cell dose and days for neutrophil and platelet engraftment was found to be -0.24 and -0.20, respectively. Time for neutrophil engraftment was found to be significantly associated with CD34+ cell dose category. Conclusion: CD 34+ cell dose appears as the strongest predictor of leukocyte and platelet engraftment. CD 34+ cell dose of >5.0 × 106 cells/kg leads to an accelerated neutrophil and platelet engraftment in patients of MM.