scholarly journals Towards a Standardization and Characterization of Clinical Grade Adipose-Derived Stromal Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5013-5013
Author(s):  
Chiara Cugno ◽  
Rita Calzone ◽  
Giusy Gentilcore ◽  
Heba Sidahmed ◽  
Asma Al-Sulaiti ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Cell Yield ◽  
Population Doubling ◽  
Color Flow ◽  
Single Donor ◽  
Adipose Derived Stromal Cells

INTRODUCTION Mesenchymal Stromal Cells (MSCs) are multipotent cells with regenerative, anti-inflammatory, immunomodulatory and anti-tumorigenic properties, which are readily isolated from aspirates of adipose tissue (AT) due to their high availability. Compared with alternative sources, AT provides high cell yield. Despite the numerous clinical trials in autologous and allogeneic settings for various diseases worldwide, there are no US FDA-approved MSC-based products. Challenges for clinical translation of MSC-based therapies largely lie in the sourcing, production and basic characterization of such products (Camilleri et al, 2016; Mendicino et al, 2014). Given the known donor-related variability (sex, age, liposuction sites, BMI, etc.), there is still ample room to: better standardize in vitro manufacturing processesrefine the characterization of clinical products. METHODS Seven samples of lipoaspirate were processed. AT manual digestion was compared with an automated collagenase-based enzymatic digestion by the Cytori Celution instrument, a closed system regulated as a medical device by EC for the production of adipose-derived stem cells-enriched products. Total cell yield, cell harvest at P0, P1 and P2, Population Doubling Level (PDL) and Doubling Time (DT) were evaluated. The obtained Adipose-derived Stromal Cells (ADSCs) were characterized for: Expression of 361 surface markers (BioLegend LegendScreen Human PE Kit), including 117 additional antigens compared to other reported screens. Cytori-isolated ADSCs from three donors for 6 total conditions (3 P2, 2 P3 and 1 IFN-g-primed P2) were screened. A broader characterization of fresh isolates (P0) as well as (P3) from the same donors was performed with a 20-color flow cytometric panel. Data were analyzed with FlowJo software.miRNA expression was evaluated using the 800 human miRNAs Nanostring panel. Nanostring nCounter Analysis System generated data, then Partek GS software was used for secondary analysis. RESULTS Cytori processing resulted in a higher cell yield at isolation (p= 0.0156), and ADSCs exhibited higher proliferation in vitro at P0 (n. of days at P0, p=0.0313), higher cell yield at P1 (p=0.0313), and higher PDL1 (p=0.0469). Out of the 361 markers examined, in every donor at each passage, at least 94 were found positive (expression >1%), including 30 within the newly screened 117 markers. Altogether, 27 markers were expressed at more than 85%, including 6 from the newly screened markers. Our analysis identified 50 donor-dependent markers, 50 passage-modulated markers (P2 vs P3) and 45 IFN-g-modulated marker, 41 increased and 4 decreased. We were able to identify the ADSCs progenitors markers CD146 and CD271 on cell as in P0 and P3, we observed a transition from a dominant expression in P0 of CD271 (34.05%) vs CD146 (1.62% ) to a dominant expression of CD146 in P3 (52%) vs CD271 (0.7%) in a single donor. The frequency of double positives CD146+CD271+ was 1% and 0.6% in P0 and P3 respectively. miRNA expression analysis by ANOVA revealed that (i) IFN-g-priming modulated 19 miRNAs, that 4 miRNAs were modulated by passage number and donors' origin. CONCLUSION Our study shows that the standardized Cytori processing advantageously substitutes AT manual digestion by enabling higher cell yield and potentially providing multiple ADSC doses from a single donor. The isolation procedure is standardized, the operator-dependent variations are minimized, and less prone to contamination compared to the lengthy, multistep process of manual digestion. The broad cell characterization with flow cytometry and miRNA expression revealed the expression and modulation of new markers. We believe that increasing the dimensionality of the ADSC characterization, beyond the traditional markers, could reveal markers that describe specific functional abilities of clinical ADSC product. Proper functional studies are necessary to validate our hypothesis. Disclosures No relevant conflicts of interest to declare.

Characterization of the immunomodulatory effect in vitro of microvesicles isolated from mesenchymal stromal cells on immune cells

2013 ◽  
Vol 41 (8) ◽  
pp. S64
Author(s):  
Antonella Conforti ◽  
Marco Scarsella ◽  
Ezio Giorda ◽  
Simone Biagini ◽  
Nadia Starc ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Immune Cells ◽  
Stromal Cells ◽  
Immunomodulatory Effect

Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content

2018 ◽  
Vol 205 (4) ◽  
pp. 226-239 ◽  
Author(s):  
Marijana Skific ◽  
Mirna Golemovic ◽  
Kristina Crkvenac-Gornik ◽  
Radovan Vrhovac ◽  
Branka Golubic Cepulic
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Immunological Tolerance ◽  
Biological Properties ◽  
Platelet Lysate ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Significant Difference

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

scholarly journals Bioreactor Processed Stromal Cell Seeding and Cultivation on Decellularized Pericardium Patches for Cardiovascular Use

2020 ◽  
Vol 10 (16) ◽  
pp. 5473
Author(s):  
Roman Matějka ◽  
Miroslav Koňařík ◽  
Jana Štěpanovská ◽  
Jan Lipenský ◽  
Jaroslav Chlupáč ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Stromal Cells ◽  
Subcutaneous Fat ◽  
Adipose Derived Stromal Cells ◽  
Pulsatile Pressure ◽  
Dynamic Cultivation ◽  
Decellularized Tissue ◽  
Static Conditions

(1) Background: Decellularized xenogeneic tissues are promising matrices for developing tissue-engineered cardiovascular grafts. In vitro recellularization of these tissues with stromal cells can provide a better in vivo remodelling and a lower thrombogenicity of the graft. The process of recellularization can be accelerated using a cultivation bioreactor simulating physiological conditions and stimuli. (2) Methods: Porcine pericardium was decellularized using a custom-built decellularization system with an optimized protocol. Autologous porcine adipose-derived stromal cells (PrASCs), isolated from the subcutaneous fat tissue, were used for recellularizing the decellularized pericardium. A custom cultivation bioreactor allowing the fixing of the decellularized tissue into a special cultivation chamber was created. The bioreactor maintained micro-perfusion and pulsatile pressure stimulation in order to promote the ingrowth of PrASCs inside the tissue and their differentiation. (3) Results: The dynamic cultivation promoted the ingrowth of cells into the decellularized tissue. Under static conditions, the cells penetrated only to the depth of 50 µm, whereas under dynamic conditions, the tissue was colonized up to 250 µm. The dynamic cultivation also supported the cell differentiation towards smooth muscle cells (SMCs). In order to ensure homogeneous cell colonization of the decellularized matrices, the bioreactor was designed to allow seeding of the cells from both sides of the tissue prior to the stimulation. In this case, the decellularized tissue was recolonized with cells within 5 days of dynamic cultivation. (4) Conclusions: Our newly designed dynamic bioreactor markedly accelerated the colonization of decellularized pericardium with ASCs and cell differentiation towards the SMC phenotype.

scholarly journals Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1580
Author(s):  
Yvonne Roger ◽  
Laura Burmeister ◽  
Anika Hamm ◽  
Kirsten Elger ◽  
Oliver Dittrich-Breiholz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Population Doubling ◽  
Pcr Analysis ◽  
Inhibitory Influence ◽  
In Vitro Differentiation ◽  
Cell Surface Antigens

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages—a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

Exosome derived from murine adipose-derived stromal cells: Neuroprotective effect on in vitro model of amyotrophic lateral sclerosis

2016 ◽  
Vol 340 (1) ◽  
pp. 150-158 ◽  
Author(s):  
Roberta Bonafede ◽  
Ilaria Scambi ◽  
Daniele Peroni ◽  
Valentina Potrich ◽  
Federico Boschi ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Stromal Cells ◽  
In Vitro Model ◽  
Neuroprotective Effect ◽  
Adipose Derived Stromal Cells ◽  
Vitro Model ◽  
Lateral Sclerosis

Characterization of Receptors Involved in Heparin Antibody Complex Mediated Induction of Tissue Factor Expression in Monocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 224-224 ◽  
Author(s):  
Sam Glover ◽  
Nigel S. Key ◽  
Gowthami M Arepally ◽  
Nigel Mackman ◽  
Raj S. Kasthuri
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Platelet Factor ◽  
Antibody Complex

Abstract Abstract 224 Introduction: Heparin-induced thrombocytopenia (HIT) is a major cause of drug-induced thrombocytopenia and occurs in 1–5% of individuals exposed to heparin. Paradoxically, 30–50% of individuals with HIT develop thrombosis. The mechanism of thrombosis in HIT is poorly understood. We recently reported that HIT antibody complexes induce tissue factor (TF) expression in monocytes and result in the release of TF-positive microparticles (MPs). The mechanism by which HIT antibody complexes induce monocyte TF has not been established. The objective of this study is to characterize the receptors involved in HIT antibody complex mediated induction of TF expression in monocytes. As HIT antibody complex mediated activation of platelets is dependent on the FcgRIIA receptor, we evaluated the role of the FcgRII receptor in the induction of monocyte TF by HIT antibody complexes. We also evaluated the role of toll like receptor-4 (TLR4) and the platelet factor 4 (PF4) chemokine receptor CXCR3 in this process. Methods: The combination of heparin, PF4 and the murine monoclonal PF4/heparin-specific antibody KKO has been shown to cause activation of platelets and monocytes, and mimic HIT in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were pre-incubated for 30 min at 37°C with an inhibitory antibody to the FcgRII receptor (IV.3); anti-CXCR2, 3, or 4 antibodies; anti-TLR4 antibody; or mouse-IgG (mIgG) control. Following pre-incubation with antibodies for 30 minutes, heparin (1U/mL), PF4 (10μg/mL), and KKO (100μg/mL) – together referred to as the HIT antibody complex – were added. Heat-aggregated mIgG and LPS were used as positive controls for the FcgRII and TLR4 receptors, respectively. Following a 6-hour incubation, PBMCs were pelleted by centrifugation and MPs were isolated from the supernatant. The procoagulant activity (PCA) of PBMCs and MPs was measured using clotting assays performed in the presence of the anti-TF antibody HTF-1 or control antibody. TF dependent PCA was calculated by reference to a standard curve generated using relipidated recombinant TF. Results: Incubation of PBMCs with heat aggregated mIgG for 6 hours resulted in significant induction of cellular TF (345 +/− 36 pg/106 cells) which was blocked by 30 min pre-incubation with the antibody IV.3 (146 +/− 17 pg/106 cells, N=3, p<.003). However, pre-incubation with IV.3 had no significant effect on TF induction (140 +/− 5 pg/106 cells) associated with the HIT antibody complex when compared to control mIgG (110 +/− 18 pg/106 cells, N=3, p<0.11). PBMCs incubated with HIT antibody complexes in the presence of a TLR-4 antibody showed less TF activity (52 +/− 4 pg/106 cells) compared to control mIgG (80 +/− 10 pg/106 cells N=3, p<0.025). A similar, partial inhibition of TF activity was also observed in PBMCs incubated with LPS in the presence of an anti-TLR4 antibody (121 +/− 3 pg/106) compared with a control antibody (89 +/− 2 pg/106, N=3, p<.0013). Experiments with a more effective inhibitor of TLR4 are in progress. PBMCs incubated with the HIT antibody complexes in the presence of an anti-CXCR3 antibody showed less TF activity (36 +/− 7 pg/mL) compared to control mIgG (118 +/− 15 pg/106 cells, N=3, p<0.004). Antibodies against CXCR2 and CXCR4 did not have any significant effect on TF induction. Measurement of MP TF activity mirrored the results described above. Using flow cytometry and an anti-CXCR3 antibody labeled with FITC, we found that 5% (± 0.5%) of monocytes expressed CXCR3 (N=3), which is consistent with the reported literature. Conclusions: These data suggest that induction of TF in monocytes by HIT antibody complexes is not mediated by the FcgRII receptor. This is contrary to the mechanism of platelet activation by these antibody complexes, which is an FcgRIIa dependent process. We found that TLR4 plays a role in HIT antibody complex mediated induction of TF in monocytes and blocking TLR4 led to a 30% decrease in TF activity. On the other hand, CXCR3 appeared to play a more significant role with blockade of CXCR3 leading to a 70% decrease in TF activity. Further characterization of the role of these receptors in HIT antibody complex mediated induction of TF expression in monocytes is required. We speculate that the extent of CXCR3 and TLR4 expression in monocytes may influence the susceptibility to developing thrombotic complications in HIT. Disclosures: No relevant conflicts of interest to declare.

Anti-Inflammatory Curcumin Inhibits Activation-Induced Cytosine Deaminase (AID) within Cycling Normal and CLL B Lymphocytes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4606-4606
Author(s):  
Shabirul Haque ◽  
Bukhtawar Waqas ◽  
Hyunjoo Lee ◽  
Piers EM Patten ◽  
Jonathan E Kolitz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
B Lymphocytes ◽  
Messenger Rna ◽  
Conflicts Of Interest ◽  
Curcuma Longa ◽  
Cell Populations ◽  
Color Flow ◽  
Anti Inflammatory

Abstract Abstract 4606 Curcumin is a natural phenolic compound within the spice, Curcuma longa. It has noted anti-inflammatory effects, in large part due to its potent suppressive effect on the NF-kB signaling pathway. AID is a NF-kB-regulated enzyme, essential for B cell Ig class switch recombination and somatic hypermutation and recently shown to promote oncogenic transformation within both B cell and non-B cell lineages. This study has examined the effect of curcumin on the division-linked upregulation of AID protein and mRNA within several human B cell populations: in vitro-activated normal and CLL B lymphocytes and the AID-positive, pre-germinal center B cell line, CL-01. CFSE-labeled, IgM+ human B2 cells isolated from spleen/tonsil were pre-activated for 4–5 days with stimuli likely encountered in sites of inflammation, i.e. limiting surrogate C3dg-coated antigen (anti-IgM: anti-CD21: dextran) + IL-4 + BAFF. Peripheral blood B-CLL cells were activated with TLR-9 ligand, ODN-2006, + IL-15. Curcumin at doses from 6 to 50 μ M, and parallel DMSO vehicle controls, were pulsed into dividing B cell cultures (day 3, 4, or 5 of activation), and AID mRNA and protein assessed after 1 to 2 days. In experiments with CL-01 B cells, the kinetics of curcumin-induced AID suppression was further analyzed. Messenger RNA was monitored by both quantitative and qualitative RT-PCR; AID protein was assessed by two-color flow cytometry of CFSE-labeled cells and immunoblotting. The above experiments revealed that curcumin can significantly down-regulate AID mRNA and protein, in dose dependent fashion within each of the above B cell populations. Following a 16h pulse of curcumin (25 μ M), AID mRNA within CL-01 cells was inhibited by 60% (p=0.001), and accompanied by ~ 60% decrease in AID protein. Within cultures of replicating normal human B lymphocytes, a similar pulse of curcumin reduced total culture AID mRNA by an average of 70% in 3 experiments. AID protein in blasts representing 3–4 divisions was reduced by 79%, and in those representing 1–2 divisions by 58%, within a representative experiment. AID mRNA was evident within all in vitro-activated B-CLL clones tested (total = 6 clones at the time of submission). This was significantly reduced by a 15–24 hr pulse with curcumin (20-25 μ M): 42% inhibition (p=0.02)). The inhibitory effects of curcumin were evident in both IgHV mutated and unmutated clones. Within stimulated B-CLL assessed for AID protein (4 total clones, of which 2 were positive), a 20 hr pulse of curcumin at 40 μ M and 20 μ M reduced AID expression in one clone by 80% and 40%, respectively. In the other clone, a maximal tested dose of 20 μ M curcumin reduced AID protein by only 12%. Suppression of AID mRNA in the CL-01 cell line was noted as soon as 3 hours following exposure to curcumin and was preceded by inhibition of NF-kB activation, both baseline and BAFF-induced. The latter was determined through monitoring intracellular levels of phospho-p65-Ser(529) – an active phosphorylated form of NF-kB RelA. Taken together, these findings suggest that NF-kB- and AID-suppressing curcumin may be useful in reducing the risk of malignant transformation and B-CLL progression into more malignant subclones, as well as treating B cell autoimmune diseases driven by pathogenic, somatically-mutated IgG autoantibodies. Disclosures: No relevant conflicts of interest to declare.

Treatment of Acute Graft-Versus-Host Disease by Means of Multipotent Mesenchymal Stromal Cells: Role of Immunomodulating Soluble Factors Expressed by These Cells in Vitro

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4554-4554
Author(s):  
Natalia A. Petinaty ◽  
Larisa A. Kuzmina ◽  
Irina N. Shipounova ◽  
Oxana A Zhironkina ◽  
Alexey Bigildeev ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Acute Gvhd ◽  
Conflicts Of Interest ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Expression Level ◽  
Relative Level ◽  
Hematopoietic Stem ◽  
Steroid Resistant ◽  
Graft Versus Host

Abstract Abstract 4554 Background Severe graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic-stem cell transplantation (allo-HSCT). Steroids are the first-line treatment for established GvHD with a response rate of 30–50%. The efficacy of multipotent mesenchymal stromal cells (MMSCs) in the treatment of steroid-resistant GvHD was reported to be 71–94%. Although it has been shown that the immunomodulatory effect of MMSCs mainly occurs through the secretion of soluble mediators, the exact mechanism of their action remains controversial. Aim The aim of the study was to investigate the influence of immunomodulating factors expressed by donor's MMSCs in vitro on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. IL-6, IL-10, colony stimulating factor 1 (CSF1), indoleamine 2, 3 dioxygenase (IDO1), prostaglandin E synthase (PTEGS) and complement factor H (CFH) were measured as factors playing substantial role in GvHD development. Methods Five patients with steroid resistant acute GvHD were treated with bone marrow derived MMSCs from hematopoietic stem cells donors. MMSCs were infused intravenously at the dose 1×106 per kg of body weight. The efficiency of GvHD therapy by means of MMSCs was scored as: complete response – 3, partial response - 2, clinical improvement – 1, no response – 0. MMSCs were cultured in aMEM with 4% human platelet lysate for 2–5 passages. Total RNA was extracted from MMSCs at 2–3 passages by standard protocol. Relative level of gene expression was estimated by the real-time PCR with previous reverse transcription in MMSCs from 31 donors and determined by normalizing the expression of each target gene to b-actin and GAPDH, calculated using ΔΔCt method for each MMSCs sample. Results Characteristics of patients, donors and graft MMSCs are shown in the table 1. Correlation between relative level of gene expression and efficacy of MMSCs infusion was investigated. The existence of inverse negative relationship between IL-6, CSF1 expression level and treatment score was revealed. The increased level of IL-6 in donor's MMSCs in patients with low and no response (treatment score 1 – 0) could be related to main functions of this factor in inflammation. Increased level of CSF1 in donors' MMSCs could further enhance macrophage activation resulting in GvHD progression instead of inhibition. The augmentation of PTGES expression could be associated with improvement of response to MMSCs therapy, but the correlation was not found. There were no relationship between the expression levels of IL-10, IDO1 and CFH and efficiency of MMSCs infusion. In a patient with no response the relative expression level of all studied factors was altered in comparison with average level of their expression in MMSCs from studied donors: IL-6 and CSF1 increased about 2 fold, while the expression level of IL-10, CHF and PTGES decreased 1.7, 12 and 11 fold correspondingly. So not all MMSCs fit to GvHD therapy that could be associated with their characteristics. Conclusions The data demonstrate the influence of IL-6, CSF1 and PTGES expression on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. As most clinical trials involve non-relative allogeneic MMSCs, analysis of expression level of IL6, CSF1 and PTGES in available MMSCs before infusion could predict the efficiency of acute GvHD therapy and permit the choice of the most proper samples. For successful clinical use of MMSCs further investigation of their properties on cellular and molecular levels are needed. Disclosures: No relevant conflicts of interest to declare. Disclosures: No relevant conflicts of interest to declare.

A New Algorithm and Panel Construction for Pediatric Leukemia Immunophenotyping Using 10-Color Flow Cytometry

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4799-4799
Author(s):  
Bettina Keller ◽  
Markus P Radsak ◽  
Joerg Faber ◽  
Alexandra Russo
Keyword(s):  
Flow Cytometry ◽  
Childhood Leukemia ◽  
Conflicts Of Interest ◽  
High Reliability ◽  
Maturation Stage ◽  
Specific Information ◽  
Color Flow ◽  
Pediatric Leukemia ◽  
Flow Cytometric

Abstract Abstract 4799 Background: Rapid identification and quantification of abnormal cell populations in minimal specimen are crucial for diagnosis and longitudinal minimal residual disease (MRD) testing of childhood leukemia. So far, most standard immunophenotypic analyses are performed using antibody panels with up to five-colors and require high cell numbers. For infant and pediatric specimen, high-level multicolor analyses is highly desirable to gather sufficient data for initial diagnostic and follow up monitoring of pathologic populations. Objective: In this study, we aimed to establish a newly defined pediatric multicolor flow cytometric panel algorithm with high reliability yet minimal specimen requirement. Results: We defined a 10-color flow cytometric panel using the new violet laser dye “KromeOrange (KO)”. Applying CD45-KO/Side Scatter gating, combined with 2 additional backbone markers the panel is designed in two consecutive steps. In the first step, a single standardized 10-color-“screening tube” (FITC-HLA-DR, PE-CD15/CD56, ECD-CD5, PC5.5-CD33, PC7-CD13, APC-CD117, APC A700-CD34, APC A750-CD19, PB-CD3, KrO-CD45) is applied for initial orientation of specific lineage assignment. Based on results obtained with the screening tube, a specific multi-tube “classification panel” is used to complete detailed characterization of lineage specific malignancy and maturation stage. Suitable specimens include fresh blood, bone marrow and all body fluids. All samples are stained directly with monoclonal antibodies, followed by the lyses of erythrocytes and a short wash. Compared to standard five color panel previously used the application of greater numbers of informative antibodies in the screening tube and in the 2ndstep muti-tube classification panel is cost and time efficient and results in a more precise characterization of any single event. Conclusion: Our panel construction and algorithm definition for infant and pediatric leukemia immunophenotyping is one of the first 10-color flow cytometry panels described for this application. Advantages are the possibility to obtain highly specific information from minimal specimens with significantly improved laboratory efficiency. The overall performance is currently tested in a routine clinical setting. Disclosures: No relevant conflicts of interest to declare.

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