Comparison of peripheral blood automated hematopoietic progenitor cell count and flow cytometric CD34+ cell count.

BACKGROUND: Stem cell apheresis in the context of autologous stem cell transplantation requires an accurate cluster of differentiantion 34 (CD34+) count determined by flow cytometry as the current gold standard. Since flow cytometry is a personnel and time-intensive diagnostic tool, automated stem cell enumeration may provide a promising alternative. Hence, this study aimed to compare automated hematopoietic progenitor enumeration carried out on a Sysmex XN-20 module compared with conventional flow cytometric measurements. METHODS: One hundred forty-three blood samples from 41 patients were included in this study. Correlation between the two methods was calculated over all samples, depending on leukocyte count and diagnosis. RESULTS: Overall, we found a high degree of correlation (r = 0.884). Furthermore, correlation was not impaired by elevated leukocyte counts (>10 000/µL, r = 0.860 vs <10 000/µL, r = 0.849; >20 000/µL, r = 0.843 vs <20 000/µL, r = 0.875). However, correlation was significantly impaired in patients with multiple myeloma (multiple myeloma r = 0.840 vs nonmyeloma r = 0.934). SUMMARY: Stem cell measurement carried out on the Sysmex XN-20 module provides a significant correlation with flow cytometry and might be implemented in clinical practice. In clinical decision-making, there was discrepancy of under 15% of cases. In multiple myeloma patients, XN-20 should be used with caution.

Circulating CD34+ cells of primary myelofibrosis patients contribute to myeloid-dominant hematopoiesis and bone marrow fibrosis in immunodeficient mice.

INTRODUCTION: Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how these aberrant myeloid cells and fibroblasts are produced remains unclear. AIM AND METHODS: In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination. RESULTS: Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms. CONCLUSION: A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.

Associations Between Inflammation, Cardiovascular Regenerative Capacity, and Cardiovascular Events: A Cohort Study.

Objective: Circulating progenitor cells possess immune modulatory properties and might mitigate inflammation that is characteristic of patients with coronary artery disease. We hypothesized that patients with fewer circulating progenitor cells (CPCs) will have higher inflammatory markers and worse outcomes. Approach and Results: Patients with stable coronary artery disease were enrolled in a prospective study enumerating CPCs as CD (cluster of differentiation)-34-expressing mononuclear cells (CD34+) and inflammation as levels of IL (interleukin)-6 and high-sensitivity CRP (C-reactive protein) levels. Patients were followed for 5 years for the end points of death and myocardial infarction with repeat inflammatory biomarkers measured after a median of 2 years. In the entire cohort of 392 patients, IL-6 and high-sensitivity CRP levels remained unchanged (0.3+/-2.4 pg/mL and 0.1+/-1.0 mg/L; P=0.45) after 2 years. CPC counts (log-transformed) were inversely correlated with the change in IL-6 levels (r, -0.17; P<0.001). Using linear regression, IL-6 and high-sensitivity CRP levels declined by -0.59 (95% CI, -0.90 to -0.20) pg/mL and -0.13 (-0.28 to 0.01) mg/L per 1 log higher CPC counts after adjustment for the demographic and clinical variables, as well as medications. Using Cox models adjusted for these risk factors, a rise in 1 pg/mL of IL-6 was associated with a 11% (95% CI, 9-13) greater risk of death/myocardial infarction. We found that the change in IL6 level partly (by 40%) mediated the higher risk of adverse events among those with low CPC counts. Conclusions: Reduced cardiovascular regenerative capacity is independently associated with progressive inflammation in patients with coronary artery disease that in turn is associated with poor outcomes.

Evaluation of circulating CD34+ stem cells in peripheral venous blood in patients with varying degrees of periodontal disease.

INTRODUCTION: Periodontal disease presents a challenge for modern medicine, and research on the use of stem cells as a treatment is currently underway. MATERIAL AND METHODS: The study included 45 patients who were given a thorough physical examination. Additionally, an evaluation of their medical history of the disease, degree of progression of periodontal disease, and the level of CRP in the blood was carried out. Patients were divided into 4 groups: 4 patients were in the first group with no periodontal disease, 8 patients in the second group with a moderate level, 20 patients in the third group with an advanced level, and 13 patients in the fourth group were toothless. For each group, the use of stem cells as a treatment of antibody-labeled CD34+ stem cells, lymphocytes, and leukocytes was conducted. RESULTS: A statistically significant positive correlation was observed in CD34+ stem cells in proportion to lymphocytes in the moderate (0.80), in the advanced (0.75), and in the toothless groups (0.70). The ratio of CD34+ stem cells to leukocytes was statistically significant in the toothless group (0.92) and in the advanced group (0.91). A statistically significant increase was noted in the level of CRP in the previously mentioned groups of patients, and the highest concentration of CD34+ stem cells in the advanced group. CONCLUSIONS: The highest concentration of CD34+ cells was observed in the group of patients with advanced periodontal disease.

Strongyloides stercoralis and HTLV-1 coinfection in CD34+ cord blood stem cell humanized mice: Alteration of cytokine responses and enhancement of larval growth.

Viral and parasitic coinfections are known to lead to both enhanced disease progression and altered disease states. HTLV-1 and Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. Independently, HTLV-1 induces a Th1 response and S. stercoralis infection induces a Th2 response. However, coinfection with the two pathogens has been associated with the development of S. stercoralis hyperinfection and an alteration of the Th1/Th2 balance. In this study, a model of HTLV-1 and S. stercoralis coinfection in CD34+ umbilical cord blood hematopoietic stem cell engrafted humanized mice was established. An increased level of mortality was observed in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected group. The mortality was not correlated with proviral loads or total viral RNA. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. HTLV-1 infected and coinfection groups showed a significant, yet non-clonal expansion of the CD4+CD25+ T-cell population. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group and no autoinfective larvae were found. However, infective larvae recovered from the coinfection group showed an enhancement in growth, as was seen in mice with S. stercoralis hyperinfection caused by treatment with steroids. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections in vivo in the context of human immune cells.

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

Associations between handgrip strength and hypertension in relation to circulating CD34-positive cell levels among Japanese older men: a cross-sectional study.

BACKGROUND: A positive association between handgrip strength and blood pressure has been reported. Since these factors are linked to the condition of the endothelium, the activity of endothelial repair might influence the association between handgrip strength and hypertension. METHODS: A cross-sectional study was conducted with 257 Japanese men aged 60-69 years who underwent an annual health checkup. As individuals with high level of circulating CD34-positive cells might show active endothelial repair, which plays an important role in vascular homeostasis, participants were stratified by circulating CD34-positive cell levels, using the median value of this population (0.96 cells/µL) as the cutoff. RESULTS: Independent of known cardiovascular risk factors, for participants with a high CD34-positive cell, handgrip strength is significantly positively associated with hypertension (odds ratio and 95% confidence interval of hypertension for 1 standard deviation increment of handgrip strength were 1.85 (1.19, 2.88) but not for participants with a low CD34-positive cell (0.91 (0.61, 1.37)). CONCLUSION: The positive association between handgrip strength and hypertension is limited to high CD34-positive cells. This result may help clarify the role of vascular homeostasis in maintaining muscle strength.

Repression of protocadherin 17 is correlated with elevated angiogenesis and hypoxia markers in female patients with breast cancer.

BACKGROUND: Altered cadherin expression plays a vital role in tumorigenesis, angiogenesis and tumor progression. However, the function of protocadherin 17 (PCDH17) in breast cancer remains unclear. OBJECTIVE: Our target is to explore PCDH17 gene expression in breast carcinoma tissues and its relation to serum angiopoietin-2 (Ang-2), carbonic anhydrase IX (CAIX) and % of circulating CD34+ cells in breast cancer patients (BCPs). METHODS: This study included Fifty female BCPs and 50 healthy females as control group. Cancerous and neighboring normal breast tissues were collected from BCPs as well as blood samples at diagnosis. PCDH17 gene expression was evaluated by RT-PCR. Serum Ang-2, CAIX levels were measured by ELISA and % CD34+ cells were assessed by flow cytometry. RESULTS: PCDH17 was downregulated in cancerous breast tissues and its repression was significantly correlated with advanced stage and larger tumor size. Low PCDH17 was significantly correlated with serum Ang-2, % CD34+ cells and serum CAIX levels. Serum CAIX, Ang-2 and % CD34+ cells levels were highly elevated in BCPs and significantly correlated with clinical stage. CONCLUSIONS: PCDH17 downregulation correlated significantly with increased angiogenic and hypoxia biomarkers. These results explore the role of PCDH17 as a tumor suppressor gene inhibiting tumor growth and proliferation.

Vascular Regenerative Capacity and the Obesity Paradox in Coronary Artery Disease.

[Figure: see text].

Peripheral blood stem cell grafts in allogeneic hematopoietic cell transplantation: It is not all about the CD34+ cell dose.

Allogeneic Hematopoietic Cell Transplantation is a curative approach in various malignant and non-malignant disorders. The majority of adult transplants in the current era are performed using mobilized stem cells, harvested from the peripheral blood by leukapheresis. Peripheral blood stem cell (PBSC) collections are designed to target a dose of stem cells that will result in safe engraftment and hematopoietic recovery; however, 99 % of the cells contained in a PBSC graft are not stem cells and a growing number of studies attempt to characterize the associations between graft composition and transplant outcomes. A better understanding of the impact of the quantity and quality of various cell types in PBSC grafts may lead to development of novel collection strategies or improved donor selection algorithms. Here we review relevant findings from recent studies in this area.

A multicenter report on the safety and efficacy of plerixafor based stem cell mobilization in children with malignant disorders.

BACKGROUND: Pleraxifor for peripheral blood stem cell (PBSC) mobilization in children with malignancies is often given following failure of standard mobilization (SM) rather than as a primary mobilizing agent. STUDY DESIGN AND METHODS: In this retrospective multicenter study, we report the safety of plerixafor-based PBSC mobilization in children with malignancies and compare outcomes between patients who received plerixafor upfront with SM (Group A) with those who received plerixafor following failure of SM (Group B). In the latter pleraxifor was given either following a low peripheral blood (PB) CD34 (<20 cells/cu.mm) (Group B1) or as a second collection process due to an unsuccessful yield (CD34 + < 2 × 106 /kg) (Group B2) following failed SM and first apheresis attempts. RESULTS: The study cohort (n = 47) with a median age of 8 (range 0.6-21) year, comprised 19 (40%) Group A and 28 (60%) Group B patients (B1 = 12 and B2 = 16). Pleraxifor mobilization was successful in 87.2% of patients, similar between Groups A and B (84.2% vs 89.2%) and resulted in a median 4-fold increase in PB CD34. Median number of apheresis attempts was 2 in Groups A and B1 but 4 in Group B2. In Group B2, median total CD34+ yield post-plerixafor was 9-fold higher than after SM (P = .0013). Mild to moderate transient adverse events affected 8.5% of patients. Among patients who proceeded to autologous transplant (n = 39), all but one engrafted. CONCLUSION: Plerixafor-based PBSC collection was safe and effective in our cohort and supports consideration as a primary mobilizing agent in children with malignancies.

Increased circulating endothelial progenitor cells (EPCs) in prepubertal children born prematurely: a possible link between prematurity and cardiovascular risk.

BACKGROUND: Endothelial progenitor cells (EPCs) ensure vascular integrity and neovascularization. No studies have investigated EPCs in preterm-born children beyond infancy. METHODS: One hundred and thirty-six prepubertal children were enrolled: 63 preterm and 73 born at term (controls). Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were measured in preterm-born children compared to controls. Body mass index (BMI), waist-to-hip ratio (WHR), neck circumference, systolic and diastolic blood pressure (SBP and DBP, respectively), fasting glucose, insulin, lipid profile, common carotid and abdominal aortic intima-media thickness (cIMT and aIMT, respectively), endothelium-dependent brachial artery flow-mediated dilation (FMD), and echocardiographic parameters were also assessed. RESULTS: Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were significantly higher in preterm-born children compared to controls (p < 0.001 and p < 0.001, respectively). In total study population and in the preterm-born group, EPCs were significantly lower in children born to mothers with gestational diabetes compared to non-diabetic mothers. Prematurity was associated with higher WHR, neck circumference, SBP, DBP, cIMT, aIMT, mean pressure, and velocity of pulmonary artery; the peak velocity of the brachial artery was significantly lower in children born prematurely. In multiple regression analysis, preterm birth and maternal gestational diabetes were recognized as independent predictors of EPCs. CONCLUSIONS: Circulating EPCs were increased in prepubertal preterm-born children in comparison with peers born full-term. Maternal gestational diabetes was associated with a decrease in EPCs. IMPACT: Mounting evidence supports the adverse effect of prematurity on cardiovascular health. However, the underlying mechanisms that could lead to endothelial dysfunction in preterm-born individuals are not fully understood. Endothelial progenitor cells (EPCs) ensure vascular integrity, normal endothelial function and neovascularization. No studies have investigated the EPCs counts in peripheral blood beyond infancy in children born prematurely. Circulating EPCs were significantly higher in preterm-born prepubertal children compared to controls, thus indicating that prematurity is possibly associated with endothelial damage. In total study population and in the preterm-born group, maternal gestational diabetes was associated with decreased EPCs concentrations.

Endothelial Progenitor Cell CD34+ and CD133+ Concentrations and Soluble HLA-G Concentrations During Pregnancy and in Cord Blood in Undifferentiated Connective Tissue Diseases Compared to Controls.

The objective of this study is to evaluate endothelial progenitor cells (EPCs) CD34+ CD133- and CD34+ CD133+ and soluble HLA-G (sHLA-G) concentrations among undifferentiated connective tissue disease (UCTD) subjects, compared to controls, during pregnancy and in cord blood. This is a case-control study including 29 controls and 29 UCTDs. CD34+ CD133-, CD34+ CD133+, and sHLA-G concentrations were detected in maternal plasma and in cord blood. This study was approved by the Medical-Ethical Committee of our Institution (Current Research Project N. 901-rcr2017i-23 of IRCCS Foundation Policlinico San Matteo of Pavia). Circulating CD34+ CD133- and CD34+ CD133+ counts and sHLA-G (soluble human leucocyte antigen G) concentrations in maternal peripherical blood were higher in UCTD compared to those in controls in first and third trimester of pregnancy and at delivery (p < 0.001). Maternal CD34+ CD133- and CD34+ CD133+ counts were strongly and significantly correlated in UCTD (Spearman's rho = 0.79, p < 0.0001) but not in controls (Spearman's rho = 0.10, p = 0.35). Cord blood CD34+ CD133- and CD34+ CD133+ median counts and median sHLA-G concentrations were higher among UCTD subjects than in controls (p < 0.001). Cord blood CD34+ and CD133+ counts were inversely and significantly correlated with sHLA-G concentrations among UCTDs, but not in controls. Early UCTD is characterized by increased EPC levels in maternal plasma and in cord blood and higher levels of sHLA-G, compared to controls. Data suggest that fetoplacental unit plays an independent role in the EPC response to a systemic autoimmune disease.

Correlation between peripheral blood neutrophil-lymphocyte ratio and CD34 expression in prostate cancer.

BACKGROUNDS: The association of neutrophil-lymphocyte ratio (NLR) and CD34 expression level with PSA level, Gleason score, and clinical stage was investigated in patients with prostate cancer. The correlation between NLR and CD34 expression was also investigated to provide evidence supporting the use of NLR for predicting the prognosis of prostate cancer patients. METHODS: Clinical data of 75 patients diagnosed with prostate cancer by prostate aspiration biopsy were retrospectively analyzed. The correlation between NLR, CD34 expression, and clinicopathological characteristics was analyzed using the χ2 test and one-way analysis of variance. The correlation between NLR and CD34 was determined using the Pearson coefficient. Disease free survival was estimated by Kaplan-Meier analysis. RESULTS: Both NLR and CD34 expression were significantly positively correlated with PSA, Gleason score, and clinical stage (P < 0.05 both). Patients in the NLRHigh/CD34High group were characterized by high PSA level and Gleason score and late clinical stage. NLR was positively correlated with CD34 expression (r = 0.529, P < 0.001). CONCLUSIONS: Pretreatment NLR was a valuable marker of prognosis in prostate cancer. NLR is positively correlated with CD34 expression.

Short-Term exposure of umbilical cord blood CD34+ cells to human platelet lysate and cytokines enhances engraftment.

BACKGROUND: Intra bone marrow (IBM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic progenitor stem cell (HSPC) transplantation and thus increases the number of HSPC that engraft. Despite physical delivery into the bone marrow cavity, many donor cells are rapidly redistributed by vascular perfusion. Thus, the objective of our study was to evaluate the ability of human platelet lysates (hPL) to improve HSPC retention into the bone marrow and consequently to improve engraftment. STUDY DESIGN AND METHODS: HSPC were isolated from human umbilical cord blood. HSPC were seeded in the wells of a 24-well microplate and exposed to increasing concentrations of hPL with or without cytokines for 24 hours. Following priming, HSPC cells chemotaxis to rhSDF-1 was determined in vitro and engraftment in NSG mice was evaluated. RESULTS: Priming of cord blood CD34+ cells to a combination of hPL and cytokines resulted in a significant increase (up to 3-fold) in the expression of the CD34 antigen on HSPC. This effect was closely correlated to a significantly increased (up to 7-fold) migration toward a rhSDF-1 concentration gradient. In addition, IBM injection of CD34+ cells previously primed with hPL+cytokines into NSG mice showed significantly increased engraftment as measured by human platelet numbers, human CD45 and human CD34+ cells for unprimed and primed cells, respectively. CONCLUSION: The use of hPL + cytokines as a short-term priming treatment for UCB could be an advantageous strategy to improve clinical outcomes following IBM injection.

Prophylactic versus therapeutic role of the transplanted CD34+ Umbilical Cord Blood Stem Cells and Wharton Jelly Mesenchymal Stem Cells in early / acute hepatic S. mansoni granulomas reversal in mice; a novel approach.

PURPOSE: Praziquantel (PZQ) is the conventional antibilharzial agent. Nevertheless, no antibilharzial prophylactic agents or 100% curable therapy approved and no reported data about use of human CD34+ Umbilical Cord Blood Stem Cells (CD34+UCBSCs) or Wharton Jelly Mesenchymal Stem Cells (WJMSCs) in prevention and/or complete eradication of acute S.mansoni granulomas in liver. We aimed to study possible prophylactic vs therapeutic role of human CD34+UCBSCs and WJMSCs in acute hepatic bilharzial granulomas in pre vs post-infected mice. METHODS: Seventy mice were divided into 7 groups (10 mice each): Normal, S.mansoni-infected, post-infected PZQ-treated, CD34+UCBSCs pre and post-infected, WJMSCs pre and post-infected. Serological, parasitological, histopathological evaluation using OCT4 & TGFB immunohistochemistry and quantitative image analysis assessment of TGFB-stained fibrogenesis in liver granulomas performed. RESULTS: Histopathologically, surprisingly and significantly, the prophylactic pre-infection stem cells (CD34+UCBSCs and WJMSCs) & similarly the post-infection CD34+UCBSCs treatment revealed eradication/reversal of the entire granulomas and no fibrosis. Moreover, post-infection PZQ treatment showed fewer and significantly smaller granulomas than post-infection WJMSCs treatment. Nevertheless, post-infection WJMSCs exhibited non-significant less TGFB-stained fibrogenesis. CONCLUSION: CD34+UCBSCs exerted the best prophylactic and therapeutic roles in prevention and complete cure of acute hepatic S.mansoni granulomas over WJMSCs and PZQ. In contrast, only pre-infection WJMSCs exhibited similar preventive (prophylactic) effect. On the contrary, post-infection WJMSCs were the worst (incompletely reversed granulomas). Post-infection Praziquantel was overall better therapeutically than WJMSCs in this regard. Accordingly, when it comes to WJMSCs application, WJMSCs are better used as a pre-infection prophylactic and preventive tool rather than a post-infection therapy. Further studies are needed.

Expression of Ki-67 and CD34 on blood and bone marrow cells of CML patients with different response to imatinib and nilotinib therapy.

AIM: To assess the expression of Ki-67 protein and CD34 antigen on peripheral blood (PB) and bone marrow (BM) cells in chronic myelogenous leukemia (CML) patients with different response to tyrosine kinase inhibitors (TKI) imatinib (IM) and nilotinib (NI) therapy. PATIENTS AND METHODS: BM aspirate and PB samples from 41 CML patients treated with IM and NI were studied by cytogenetic, molecular genetic, and flow cytometry methods. According to the response to TKIs, the patients were distributed into the optimal response, warning, and treatment failure groups. RESULTS: The patients with optimal response to TKI therapy showed the lowest levels of Ki-67 expression in PB and BM compared with the patients from warning and falure treatment groups, however, Ki-67 expression was close to the reference values in PB (0.7 ± 0.3)%, only in NI-treated patients, The highest expression of Ki-67 in PB was observed in patients from treatment failure groups. In PB of patients who received NI and did not achieve optimal response, CD34+ cell count increased by almost 4 times compared with that in the optimal response group. The results indicated that CD34+ cell pool expanded in patients with poor response to both IM and NI. In patients with optimal response to NI therapy, CD34+ cell counts in PB were within the reference range ​​and did not exceed 0.5%. Similar results were observed for Ki-67 and CD34+ in BM hematopoietic cells. CONCLUSIONS: Ki-67 expression and CD34+ cell count in PB and BM of CML patients increased with the acquisition of clonal resistance to IM and NI. NI provides a deeper molecular response compared with IM.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin ß3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

A novel method to evaluate prethawing viability of cryopreserved CD34+ hematopoietic stem cells for autologous transplantation.

BACKGROUND: Cryopreservation of CD34+ hematopoietic stem cells (HSCs) is associated with variable loss of viability. Although postfreezing CD34+ cell viability can be assessed on the sampling tube (bag tail) directly connected to the main bag (mother bag), results often underestimate the actual viability observed when the mother bag is thawed and reinfused. We assessed a novel method to measure postfreezing CD34+ cell viability, based on small bag (minibag) samples; results were compared with those obtained on the corresponding mother bags and bag tails. STUDY DESIGN AND METHODS: Sixty-one apheresis procedures of 42 patients undergoing autologous HSC transplant were analyzed. Viable CD34+ cells were quantified with flow cytometry before controlled rate freezing (ICE-CUBE14M system, SY-LAB- IceCube, SIAD), after 10 days of storage (mini-bag and bag tail), and before reinfusion (aliquot from a thawed mother bag). Results were compared using Student's t test and Spearman's rho correlation test. RESULTS: The mean CD34+ cell viability before cryopreservation was 99.3% (confidence interval [CI], 98.94-99.65%); the mean amount of CD34+ cells, white blood cells and neutrophils in the mother bag was 0.8 ± 1.1 × 109 /L, 63.4 ± 23.5 × 109 /L, and 25.7 ± 15.5 × 109 /L, respectively. Mother bags postthawing CD34+ cell viability was 72.3% (CI, 67.74-76.85%; p < 0.01 compared to prefreezing); no difference was observed with respect to minibags (73.7%; CI, 69.80-77.59%; p = NS), whereas significantly lower values were found for bag tails (58.6%; CI, 54.19-63.00%; p < 0.01 vs. both mini- and mother bags). CONCLUSION: Compared to bag tails, minibags represent a more accurate tool to measure the CD34+ cell viability of the apheresis mother bag prior to reinfusion; in addition, minibags may could be of help for case-by-case calculation of the amount of apheresis to be infused to patients undergoing autologous HSC transplant.