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.

scholarly journals Identification and Characterization of Bacillus anthracis Spores by Multiparameter Flow Cytometry

2008 ◽  
Vol 74 (16) ◽  
pp. 5220-5223 ◽  
Author(s):  
William C. Schumacher ◽  
Craig A. Storozuk ◽  
Prabir K. Dutta ◽  
Andrew J. Phipps
Keyword(s):  
Flow Cytometry ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Multiparameter Flow Cytometry ◽  
Flow Cytometric Assay ◽  
Color Flow ◽  
Flow Cytometric ◽  
Bacillus Anthracis Spores ◽  
Identification And Characterization

ABSTRACT In response to the need for methods that can rapidly detect potentially virulent Bacillus anthracis spores, we developed a two-color flow cytometric assay capable of simultaneously identifying B. anthracis spores and the presence of spore-associated protective antigen, a virulence marker for strains harboring the pXO1 plasmid.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

Leukoreduction Decreases Alloimmunogenicity of Transfused Murine HOD RBCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 640-640 ◽  
Author(s):  
Jeanne Hendrickson ◽  
Eldad A. Hod ◽  
Steven L. Spitalnik ◽  
Christopher D. Hillyer ◽  
James C. Zimring
Keyword(s):  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
White Blood Cells ◽  
Specific Antigen ◽  
Hla Antigens ◽  
Danger Signal ◽  
Specific Expression ◽  
Flow Cytometric ◽  
Post Test

Abstract Abstract 640 Introduction: Alloimmunization against transfused red blood cell (RBC) antigens can lead to hemolytic transfusion reactions as well as difficulties in locating compatible units of blood for future transfusions. Despite the clinical significance, factors influencing rates of RBC alloimmunization are not clearly defined. Leukoreduction decreases rates of HLA alloimmunization, because HLA antigens are carried on white blood cells (WBCs). However, most human RBC antigens are not carried on WBCs, and the effect of leukoreduction on RBC alloimmunization is not known. Many potential variables, including the large number of RBC antigenic differences between donor and recipient, make this question difficult to answer in the setting of human transfusion medicine. We hypothesized that WBCs in transfused RBC units would increase alloimmunization to an RBC specific antigen, and used a reductionist murine model to investigate this issue. Materials and Methods: Transgenic HOD donors, with RBC specific expression of the model antigen hen egg lysozyme (HEL) linked to a multi-pass human Duffy antigen (Fyb), were bled into CPDA-1 to give a final storage medium concentration of 14%. Leukoreduction was performed over a Pall neonatal leukoreduction filter, with residual WBCs quantified by propridium iodide and Trucount beads. Expression of HEL and Fyb on HOD RBCs was assessed by flow cytometry prior to transfusion. Leukoreduced or non-leukoreduced pRBCs (75 μL) were transfused via lateral tail vein, and post-transfusion survival of HOD RBCs was determined by analysis of HEL and Fybexpression at 10 and 30 minutes, 2 and 24 hours following transfusion. Alloimmunization was assessed 2 weeks later by HEL-specific ELISA and flow cytometric crossmatching (using HOD or control FVB RBCs). Statistical significance (p<0.05) was determined using PRISM software and a two-way ANOVA with a Bonferroni post-test. Results: Alloantibodies against HOD RBCs were detectable by HEL-specific ELISA in recipients following a single transfusion of non-leukoreduced or leukoreduced RBCs. In 4 of 5 experiments (n=50 mice total), non-leukoreduced HOD RBCs were statistically significantly more immunogenic than leukoreduced HOD RBCs (sera diluted 1:50). Alloantibodies against HOD RBCs were detected in a minority of mice by flow cytometric crossmatching, which is less sensitive than ELISA. By histogram overlay of sera crossmatched with HOD RBCs compared to control FVB RBCs, 6/25 recipients of non-leukoreduced RBCs and 2/25 recipients of leukoreduced RBCs had a detectable anti-HOD response. In each experiment, there was a >3 log10 decrease in leukocytes after leukoreduction. HEL and Fyb expression was comparable as determined by flow cytometry in samples pre- and post-leukoreduction. Mean 24 hour post-transfusion survival was 98.7% (95% CI 97-100%) and 97.9% (95% CI 95.4-100%) in recipients of non-leukoreduced and leukoreduced RBCs, respectively. Conclusions: Leukoreduction of murine HOD RBCs decreases alloimmunogenicity of the RBC specific HEL antigen. This decrease is subtle and is better detected by HEL-specific ELISA than by less sensitive flow cytometric crossmatching. Although one explanation for these findings is that the WBCs themselves are contributing to the increased immunogenicity by providing a danger signal, it is also possible that non-specific alloreactivity of transfused WBCs or other cells is playing a role. Furthermore, it cannot be ruled out that changes in platelets or RBCs induced by the leukoreduction filter are involved. However, the observed changes in immunogenicity cannot be explained by a decrease in HEL expression post-leukoreduction or by altered clearance of leukroreduced RBCs after transfusion, as HEL and Fyb expression, as well as 24 hour post-transfusion survival of HOD RBCs, were similar in both groups. If the immunogenicity of other RBC antigens is also decreased after leukoreduction, then patients at highest risk of RBC alloimmunization may benefit from receiving solely leukoreduced blood products. Furthermore, the development of higher efficiency and/or modified leukoreduction filters may be warranted. Disclosures: No relevant conflicts of interest to declare.

Post-Transplant Immune Monitoring In Patients Receiving Allogeneic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5486-5486
Author(s):  
Silvia Park ◽  
Chul Won Jung ◽  
Jun Ho Jang ◽  
Eun Suk Kang ◽  
Kihyun Kim
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Immune Monitoring ◽  
Disease Relapse ◽  
Cell Secretion ◽  
Color Flow ◽  
Blood Samples ◽  
Post Transplant

Abstract Introduction There are still substantial morbidity and mortality caused by insufficient immunologic recovery after allo-HSCT. In this context, we attempt to evaluate the clinical relevance of immune monitoring in allo-HSCT recipients. Method Fifty five patients who underwent allo-HSCT between 2008 and 2012 were included. Peripheral blood samples were drawn from recipients before transplant, and on 4, 8, 12, 24, 36 and 48 weeks after transplant. Each blood samples were analyzed by multi-color flow cytometry for determining lymphocyte subsets. MNC were separated from blood specimen, and analyzed for the quantitation of Treg with the use of real-time PCR. We also examined T cell derived IFN-r by using in vitro culture, intracellular staining, and flow cytometry analysis. Results The median age was 43, and AML was the most common reason for transplantation (49.1%). Grade II or more aGVHD occurred in 36.4% of cases, and 49.1% exhibited moderate or severe cGVHD. The differences in the proportion (%) and the absolute number (/uL) of CD4+, CD8+ cells, CD4+ derived IFN-r (%), CD8+ derived IFN-r (%), and Treg (%) between the groups (Gr. II or more aGVHD (+) vs (-); moderate or severe cGVHD (+) vs (-)) were compared by Two sample t-test. Patients with Gr. II or more aGVHD showed decreased CD4+ count at 4, 8 and 12 weeks, but showed rather higher CD8+ count at 8 weeks after transplant. T-cell secretion function assessed by IFN-r (%), and Treg (%) was similar between two groups within 12 weeks after transplant. In case of cGVHD, both CD4+ and CD8+ count tended to be higher in patients with moderate or severe cGVHD, and the trends lasted for up to 48 weeks from allo-HSCT. Treg (%) was almost consistently lower throughout the period in these patients. There were 12 relapses within follow up period (median 36.1 months), and higher slope of post-transplant increase in CD8+ count and CD8 derived IFN-r were identified as protective factors for disease relapse. Conclusion In view of the results so far achieved, slow recovery of CD8 count and function might be associated with disease relapse. However, this is still a preliminary data, and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Absolute CD4 Counts Obtained by a Three-Color Flow-Cytometric Method without the Use of a Hematology Analyzer

1998 ◽  
Vol 5 (2) ◽  
pp. 266-269 ◽  
Author(s):  
Thomas S. Alexander
Keyword(s):  
Flow Cytometry ◽  
Color Flow ◽  
Cd4 Counts ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
Hematology Analyzer ◽  
Blood Specimens

ABSTRACT We evaluated the Ortho TRIO-Cytoronabsolute system for determining absolute CD4 counts. The CD4 counts in our blood specimens from 100 individuals ranged from 3 to 1,962; the percent CD4 ranged from 1.3 to 62.2, respectively. The TRIO system was biased toward lower absolute counts than a combination of flow cytometry and hematology but showed no bias in percent CD4 calculations.

scholarly journals Bortezomib, Dexamethasone, Thalidomide and Melphalan (VDT-Mel) Preparative Regimen in Autologous Stem Cell Transplant (ASCT) Results in a Very High Stringent CR (sCR) Rate in Multiple Myeloma (MM)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1971-1971
Author(s):  
Kalyan Nadiminti ◽  
Kamal Kant Singh Abbi ◽  
Annick Tricot ◽  
Allyson Schultz ◽  
Lindsay Dozeman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mortality Rate ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
High Dose ◽  
Cell Transplant ◽  
Color Flow ◽  
Preparative Regimen ◽  
Very High

Abstract Background: Melphalan 200mg/m2 is the standard preparative regimen in MM and addition of other cytotoxic drugs has not been found to result in superior activity. The novel agents have improved outcome in MM significantly, but data on their role in preparative regimens are scarce. The purpose of this study was to understand the toxicity and efficacy of triple therapy with VDT in combination with high-dose melphalan. Methods: An IRB approved retrospective analysis was performed on all patients who received an ASCT with the VDT-Mel during 2012-2014. Mel: 100 mg/m2 was given on days -4 and -1; V: 1 mg/m2 on days -4, -1, +2 and +5; T: 100 mg daily from -5 to +5; and D: 20 mg/day from -4 to -1 and +2 to +5. End points were treatment-related toxicity during the first 100 days and quality of response at 6 months post-transplant; 98 patients had follow-up ≥ 6 months. Patients in sCR were also minimal residual disease negative (MRD-) by 10-color flow cytometry with a sensitivity of 10-4. Results: 100 patients received 153 transplants; 47 patients underwent single and 53 had tandem transplants (TT); 64 patients received early (≤ 12 months of induction therapy) and 36 salvage transplantation. Median age was 61 y; median followup was 16.2 months. Only 1patient had achieved a sCR and 11 a CR prior to transplantation. Best responses at 6 months were 53% sCR (and MRD-), 24% CR, and 9% VGPR. The sCR rate after single transplant was 47% (overall) and 54% (early transplant) vs 59% and 60% after TT. Grade 3-5 non-hematologic toxicities were almost entirely related to infections (38% and 53% in single and TT, respectively); the 100-day mortality rate was 2.6% (4/153), 1.8% for early transplants and 4.5% for salvage transplants. Median time to ANC recovery > 500/µL was 12 days in both early and salvage transplantation. Conclusion: VDT-Mel is well-tolerated and resulted in minimal additional toxicity and a similar mortality rate when compared to historic data of MEL alone. Importantly, the sCR rate with MRD- by flow cytometry at 6 months in our study was very high compared to published reports. The ultimate sCR rate will be higher as at this time an additional 13 patients attained a sCR during further follow up past 6 months for a total of 66% sCR. Since both sCR and MRD- are proven early surrogate markers for progression-free and overall survival, it appears highly likely that this regimen will be superior to Mel alone and should become the new standard for ASCT in myeloma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

Flow Cytometric Analysis of Lymphomas: Current Status and Usefulness

2006 ◽  
Vol 130 (12) ◽  
pp. 1850-1858
Author(s):  
Zahid Kaleem
Keyword(s):  
Flow Cytometry ◽  
Hodgkin Lymphoma ◽  
Flow Cytometric Analysis ◽  
Molecular Diagnostic ◽  
Routine Practice ◽  
Becton Dickinson ◽  
Color Flow ◽  
Non Hodgkin Lymphoma ◽  
Flow Cytometric ◽  
Diagnosis And Classification

Abstract Context.—Immunophenotyping has become a routine practice in the diagnosis and classification of most cases of non-Hodgkin lymphoma, and flow cytometry is often the method of choice in many laboratories. The role that flow cytometry plays, however, extends beyond just diagnosis and classification. Objective.—To review and evaluate the current roles of flow cytometry in non-Hodgkin lymphoma, to compare it with immunohistochemistry, and to discuss its potential future applications in the molecular diagnostic era. Data Sources.—The information contained herein is derived from peer-reviewed articles on the subject published in the English-language medical literature during the years 1980 to 2005 that were identified using PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi, 1980–2005) search, various books and other sources on flow cytometry, and the author's personal experience of more than 10 years with flow cytometric analysis of lymphomas and leukemia using Becton-Dickinson (San Jose, Calif) and Beckman-Coulter (Miami, Fla) flow cytometers. Study Selection.—Studies were selected based on adequate material and methods, statistically significant results, and adequate clinical follow-up. Data Extraction.—The data from various sources were compared when the methods used were the same or similar and appropriate controls were included. Most of the studies employed 2-color, 3-color, or 4-color flow cytometers with antibodies from Becton-Dickinson, Beckman-Coulter, or DakoCytomation (Carpinteria, Calif). Results were evaluated from studies utilizing the same or similar techniques and flow cytometers. Only objective data analyses from relevant and useful publications were included for reporting and discussion. Data Synthesis.—Flow cytometry serves a variety of roles in the field of lymphoma/leukemia including rapid diagnosis, proper classification, staging, minimal residual disease detection, central nervous system lymphoma detection, evaluation of prognostic markers, detection of target molecules for therapies, ploidy analysis of lymphoma cell DNA, and evaluation of multidrug-resistance markers. It offers many advantages in comparison to immunohistochemistry for the same roles and provides uses that are either not possible or not preferable by immunohistochemistry such as multiparameter evaluation of single cells and detection of clonality in T cells. Conclusions.—By virtue of its ability to evaluate not only surface but also cytoplasmic and nuclear antigens, flow cytometry continues to enjoy widespread use in various capacities in lymphoma evaluation and treatment. Additional roles for flow cytometry are likely to be invented in the future and should provide distinctive uses in the molecular era.

Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

Sign in / Sign up

Export Citation Format

Share Document