High Frequency of Aberrant Phenotype in Multiple Myeloma Patients Detected by Multiparametric Flow Cytometry.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4862-4862
Author(s):  
Luiz Arthur Calheiros ◽  
Eliza Y.S. Kimura ◽  
Manuella S.S. Almeida ◽  
Maria de Lourdes L.F. Chauffaille ◽  
Jandey G. Bigonha ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
High Frequency ◽  
Plasma Cells ◽  
Residual Disease ◽  
Lymphoproliferative Disease ◽  
Multiparametric Flow Cytometry ◽  
Normal Plasma ◽  
Aberrant Phenotype

Abstract Multiple Myeloma (MM) is a B cell lymphoproliferative disease with clonal plasma cell accumulation in bone marrow. Multiparametric flow cytometry (MFC) is an usefull tool to distinguish MM cells from normal plasma cells. Normal plasma cells are characterized by the expression of CD19+, CD45++, CD38++, CD138++, cytoplasmic immunoglobulin light chains (κ and λ) and CD56- while most MM plasma cells lose CD19, CD45 and gain CD56. In addition, many other antigens may be expressed by myeloma cells such as myeloid or lymphoid lineage associated antigens and these abnormal antigen expression is known as aberrant phenotype (AP). We studied 29 MM patients at diagnosis, in attempt to evaluate AP, it’s frequency and relation to prognostic parameters. The following monoclonal antibodies were used: CD45, CD38, CD138, CD56, CD19, CD20, CD22, CD10, CD13, CD14, CD33, CD117, CD28 and CD40, conjugated to FITC, PE, PerCP and APC) and acquisition / analysis were done through flow cytometer (FACS calibur, BD, San Jose) using CELL QUEST software (BD). Plasma cells were identified by the expression of CD38, CD138 and CD45 and the monoclonality confirmed by immunoglobulin light chain restriction. Our results showed presence of at least 2 AP in all cases : 2 AP (7 patients), 3 AP (12 patients), 4 AP( 5 cases), 5 AP (4 cases) and 8 AP in one case. The most frequent APs were CD45−, CD56+, CD117+, CD13+, CD33+, and were observed in 88% of patients. The most frequent AP association was CD56+/CD45− (40%), followed by myeloid antigen associated phenotypes (CD117, CD33, CD13). The lymphoid antigens expression was more observed in patients with large number of AP (>4 AP). CD56- patients presented serum β2-microglobulin and ionic calcium labeling levels higher than CD56+ patients (p=0,02) showing the usefulness of this antigen as prognostic marker. Morphological analysis showed that the majority (55%) of plasmablastic cases expressed >2 myeloid antigens against 18% of mature plasma cell morphology cases. These results allow us to conclude that MM express high frequency of AP, highlighting the importance of CD56 as a prognostic factor. MFC may be useful to the immunological detection of minimal residual disease in a great majority of MM patients and we suggest the panel CD45, CD56, CD117, CD33 and CD13 for this purpose, in addition to CD38 and CD138.

Multiparameter Flow Cytometry For Detection Of Minimal Residual Disease In Multiple Myeloma After T-Cell Depleted Allogeneic Stem Cell Transplant

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4647-4647
Author(s):  
Satyajit Kosuri ◽  
Katherine M Smith ◽  
Deborah Kuk ◽  
Sean M. Devlin ◽  
Peter G. Maslak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry

Introduction Multiparameter flow cytometry (MFC) has been shown to be a sensitive, reproducible and broadly applicable method for the early detection of minimal residual disease (MRD) in the bone marrow (BM) of pts with multiple myeloma (MM) following induction chemotherapy and/or autologous stem cell transplantation. In this study, we were interested in assessing the potential of MFC as a reliable and potentially predictive marker in pts with multiple myeloma who have undergone T-cell depleted allogeneic hematopoietic stem cell transplantation (TCD HSCT). Methods We analyzed the results of MFC obtained in 35pts with multiply relapsed MM, who also have high-risk cytogenetics undergoing allo TCD-HSCT from HLA compatible related (n= 15) and unrelated (matched (n=8), mismatched (n=12) donors. We compared these results to standard myeloma markers obtained from the blood and marrow of these pts at days 30, 60-90, 120-180, 12 and 24 months routinely and as clinically indicated thereafter post TCD HSCT. Disease evaluation included serologic immunoglobulin levels, serum protein electrophoresis/immunofixation, and serum analysis of free light chains, bone marrow biopsy and aspirate. Bone marrow specimens from each time point were also analyzed by MFC with a panel including CD38, CD56, CD45, CD19, CD138, cyKAPPA, and cyLAMBDA by gating on distinct populations of bright CD38+/CD45- plasma cells at 200,000 acquired events total or at least 100 gated plasma cell events. Malignant plasma cells (MPC) were defined as CD38+/CD138+/CD56+/CD45- and/or positive for light chain clonal excess. MPC were detected in the BM sample at the MFC sensitivity of 10-4(>1 MPC in 104normal cells). Results Thirty-five pts with multiply relapsed MM undergoing allo TCD HSCT were analyzed over median follow up of 27 months (range 6.2 – 53.3). Eighteen/35 pts did not relapse during the follow up period and none of these pts had a detectable CD38+/CD138+/CD56+/CD45- cell population by MFC. Seventeen/35 pts developed relapsed disease at a median of 12.5 months (range 3.2 – 52.5) post allo TCD-HSCT by standard serologic markers and all pts were found to be positive by MFC. The percentages of bright CD38+/CD45- cells in these pts ranged from 0.01% to 16.05% at time of first detection. In 14/17 pts, MFC became positive concurrently with standard serologic myeloma markers at relapse. In 3/17 pts, MFC detected a malignant plasma cell population with aberrant phenotype of 0.068%, 0.043% and 0.012% at 48.2, 24 and 25.4 months, respectively, post TCD HSCT in the absence of other positive markers in blood and bone marrow. These pts were also immunofixation (IF) negative at conversion to MFC positivity. Subsequent follow up of studies of these 3 pts lead to detection of recurrence by IF and/or M-spike/ aspirate at 3.8, 1.8 and 8.7 months with median follow up of 150 days after first MFC detection. The populations of MPC initially detected by MFC had increased upon relapse to higher levels. Interestingly, in 2 pts we detected 6 and 8% plasma cells by bone marrow aspirate at 90 days and 180 days, respectively, post TCD HSCT, while flow cytometry detected only CD138+/CD56-/CD45+ cells. These 2 pts never relapsed and continued to remain in CR without further intervention. Conclusions These analyses demonstrate that MFC performed on marrow specimen of pts with relapsed MM who underwent a TCD HSCT provides additional important results to assess the overall disease status. A negative MFC indicated non relapse 100% of the time attesting to its negative predictive value. In all of our patients diagnosed with relapsed MM by traditional parameters, MFC was concurrently positive. Importantly, in 3/17 pts (18%) MRD detected MPC prior to overt relapse. Interestingly, MFC was able to detect false positive marrow relapses as well. Therefore, MFC permits the detection of MRD preceding frank relapse and can distinguish a malignant plasma cell population from proliferating recovering marrow post transplant. In the post allo TCD-HSCT setting MFC may serve as an early marker which can help formulate the timing of therapeutic interventions, such as adoptive immunotherapeutic approaches, as MFC detection provides a window of several weeks to initiate treatment before disease recurrence by serology. Disclosures: No relevant conflicts of interest to declare.

Evolution of Multiparametric Flow Cytometry Testing for Minimal Residual Disease Assessment in Multiple Myeloma and Its Impact on Clinical Outcomes: A Single Institution Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2274-2274 ◽  
Author(s):  
Nischala Ammannagari ◽  
Paul K. Wallace ◽  
Theresa Hahn ◽  
Yali Zhang ◽  
Christine M. Ho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Clinical Outcomes ◽  
Research Funding ◽  
Plasma Cells ◽  
Residual Disease ◽  
Advisory Committees ◽  
Multiparametric Flow Cytometry ◽  
Minimal Residual

Abstract Minimal residual disease (MRD) after autologous hematopoietic cell transplant (AHCT) in multiple myeloma (MM) has been shown to be an important predictor of clinical outcomes, suggesting that MRD negativity may be a new goal of therapy. Multiparametric flow cytometry (MFC) is a commonly used method for MRD assessment, however this technique is still evolving and efforts are underway to standardize this testing. The key factors which enable detection of residual malignant plasma cells by MFC remain an area of active investigation. We performed a retrospective review of 172 consecutive MM patients who received AHCT between 10/1/2007 and 5/31/2015 at our institution and had undergone MRD assessment by MFC at day +100 post-AHCT. Day +100 post-AHCT response was determined using the International Myeloma Working Group (IMWG) Uniform Response Criteria (URC) and was correlated with MRD assessment as well as progression free survival (PFS) and overall survival (OS). Data were collected on the specific MFC panel utilized, including the epitopes analyzed and the total plasma cell number (PCN) counted (normal and malignant PC). These variables were correlated with clinical outcomes including day +100 MM response, PFS and OS. Of 172 patients, 30 were MRD-positive, 133 MRD-negative, and 9 were equivocal at day +100 post-AHCT, the latter of which were excluded from further analyses. Day+100 MRD-negative status by MM response was: 31/37(84%) for VGPR, 35/41 (85%) for CR, and 42/42 (100%) for sCR. Patients who achieved a CR or sCR had improved PFS and OS rates compared with patients who achieved ≤VGPR: 3-year PFS: 61% (95% CI 49-74%) vs 46% (95% CI 32-59%), P=0.03; 3-year OS: 96% (95% CI 91-100%) vs 69% (95% CI 56-81%), P=0.005)). Patients with MRD-negative disease at day +100 post-AHCT had significantly superior PFS and OS compared to those with MRD-positive disease: 3-yr PFS 62% (95% CI 52-72%) vs 33% (95% CI 12-53%), P <0.0001) (Figure 1); 3-year OS 85% (95% CI 78-93%) vs 64% (95% CI 44-85%), P=0.004). There was no association between MRD status and age (<60 vs ≥60 years), sex, race (white vs other), performance status (KPS ≤80 vs ≥90), or subsequent transplant (P>0.1). The details of the four different MRD MFC panels are shown in Table 1. Panels C and D were compared, at a similar PCN level, but different epitopes tested, and found no significant difference in PFS or OS. Further analysis of PCN within the MRD-negative cohort revealed a trend towards improved 3-yr PFS rates with increasing numbers of PCN analyzed: 42% (95% CI 20-63%) for PCN<250,000, 68% (95% CI 52-83%) for PCN=250,000-500,000, 59% (95% CI 42-76%) for PCN >500,000-1,000,000 and 89% (78-100%) for PCN>1,000,000 (P=0.099) (Figure 2). The 3-yr OS rates for MRD-negative patients were higher for increasing PCNs analyzed, but the PCN categories were not statistically significantly different: 74% (95% CI 54-94%) for PCN<250,000, 88% (95% CI 77-99%) for PCN=250,000-500,000, 85% (95% CI 73-98%) for PCN >500,000-1,000,000 and 100% for PCN>1,000,000 (P=0.2). Sensitivity analysis revealed similar trends when a cut-off of above or below 500,000 or 1,000,000 was used. Our results confirm that achievement of MFC MRD negativity at day +100 post-AHCT is associated with improved PFS and OS. Factors such as the long-half lives of immunoglobulins, the quality of the bone marrow aspirate obtained, and the presence of occult extramedullary disease may account for the patients who were MRD negative but did not achieve a CR at day +100 post AHCT by IMWG URC. MRD assessment by MFC at our institution has evolved over time to include higher numbers of acquired and analyzed events. Notably, there was a trend towards improved outcomes with greater numbers of plasma cells analyzed, suggesting that continued development of MRD assessment by MFC should focus on increasing PCN analyzed in order to improve detection of residual MM clones. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holstein:Millennium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Minimal Residual Disease Analysis in Multiple Myeloma Patients By 6-Color Flow Cytometry: An Experience from Tertiary Care Center of North India

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5343-5343
Author(s):  
Praveen Sharma ◽  
Man Updesh Singh Sachdeva ◽  
Neelam Varma ◽  
Parveen Bose ◽  
Pankaj Malhotra
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Light Chain ◽  
Bone Marrow Aspirate ◽  
Plasma Cells ◽  
Residual Disease ◽  
Color Flow ◽  
Multiparametric Flow Cytometry ◽  
Minimal Residual

Abstract Therapeutic advances in multiple myeloma (MM) incorporating the use of high-dose melphalan, novel therapeutic immunomodulatory agents, proteasome inhibitors and supporting autologous stem-cell transplantation (ASCT) have improved response rates and overall survival. The detection of minimal residual disease (MRD) is recognized as a sensitive and rapid approach to evaluate treatment efficacy as a tool for predicting patient outcomes and guiding therapeutic decisions. MRD analysis is reflected by many different techniques, however, multiparametric flow cytometry is a sensitive, feasible and adequate method for monitoring residual disease. Studies from India related to this context are lacking. In the present study, we compare MRD levels in patients of multiple myeloma after chemotherapy/ASCT assessed by multiparametric flow cytometry, with M band status, immunofixation (IFE) and percentage of plasma cells on bone marrow aspirate. Seventeen patients of multiple myeloma were included in the study over a duration of one year, (Male=13, Female=4) with mean age of 56.8 years (range 44-80 years). MRD was analyzed using a dual laser 6 color-flow cytometer in 9 patients of ASCT (day 100) and 8 patients on chemotherapy alone (post-induction). Pre-titrated cocktail of CD38, CD138, CD19, CD45, cytoplasmic Kappa light chain, cytoplasmic lambda light chain, CD81, CD27, CD28 CD200 and CD10 were used in 6-color combination of three tubes for MRD analysis. MRD was detectable in 5 patients, mean of 0.61% (range of 0.07 - 6.44%). M band and IFE were positive in 2 patients, each. Bone marrow plasma cells ranged from 0 to 22%. MRD levels did not show significant correlation with percentage of plasma cells in bone marrow aspirate, however it had an statistical agreement with presence or absence of serum M-band and IFE. Patients are on regular follow up for their clinical and hematological response. Disclosures No relevant conflicts of interest to declare.

scholarly journals Significant Plasma Cell Loss (up to 90%) Despite Preserved Overall Cell Viability - Time Dependent Changes Observed in Multiparametric Flow Cytometry Analysis of Bone Marrow Samples in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4006-4006
Author(s):  
Tukten Rolfe ◽  
Quirine O'Loughlin ◽  
Heather Campbell ◽  
Jordan Barr ◽  
Fiona Shawyer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Yield ◽  
Relative Reduction ◽  
Sample Processing ◽  
Multiparametric Flow Cytometry

Abstract Multiparametric flow cytometry (MPFC) is a mainstream laboratory method used in the diagnosis of multiple myeloma. Minimal residual disease (MRD) assessment by EuroFlow next-generation flow cytometry allows assessment down to an assay sensitivity of 1x10 -5. Delayed sample processing remains a common challenge due to logistical limitations. Specialized tests performed in central pathology laboratories are frequently located a considerable distance from healthcare providers. Our study aims to evaluate the impact of delayed sample processing on plasma cell yield and bone marrow sample stability. There is little published data available. Plasma cell yield and bone marrow sample stability were investigated in patients with multiple myeloma who underwent bone marrow biopsy. Participants were included based on ³10% plasma cell burden by morphological quantification on the bone marrow aspirate smear. Bone marrow aspirates were collected in EDTA (with three samples also collected in lithium heparin) and stored at four degrees Celsius. Samples were analyzed by MPFC within four hours of collection, at 24 and at 48 hours after collection. CD138 and CD38 co-expression were used to identify plasma cells, and absence of 7-AAD to determine cell viability. Mean fluorescence intensity (MFI) of CD138 and CD38 was recorded. Statistical analyses were performed using two-tailed Wilcoxon signed-rank tests and repeated measures ANOVA with significance assigned at p&lt;0.05. Bone marrow aspirate samples of nine participants were evaluated. Significant reduction in plasma cell yield was observed over time (p&lt;0.001) while sample integrity remained unchanged (p&gt;0.05). The most marked reduction in plasma cell detection was seen between initial processing and 24 hours (median absolute reduction 9%, range 0 to 23% and median relative reduction 37%, range -8 to 90%, p&lt;0.01). Further significant reduction of plasma cells occurred after an additional 24 hours (p=0.025). At 48 hours, the median absolute reduction in plasma cell yield from initial testing was 12% (range 1 to 24%) and median relative reduction was 40% (range 18 to 90%). Sample integrity remained constant. The median viability at collection, 24 hours and 48 hours was 91%, 93% and 95% respectively. The most significant specimen deterioration observed was 13% viability reduction to 75% overall by 48 hours. Three of the participants had additional samples collected in lithium heparin anticoagulant media that were analyzed in parallel with their EDTA samples. Plasma cell yield remained similar across the two different anticoagulants with overall cell viability remaining high in lithium heparin (³90%). A trend of time-dependent reduction of CD138 MFI was observed with lithium heparin but not with EDTA. This study demonstrates the significance of time to processing as a pre-analytical variable in MPFC in multiple myeloma. The greatest loss of plasma cells occurs within the first 24 hours after collection but continues to fall significantly out to 48 hours. Reductions of up to 90% were observed in our small cohort and represent a potential 1 log reduction in yield. This decrease in plasma cell yield raises questions of reliability and validity of flow cytometry, whereby the sensitivity depth may be compromised if the sample cannot be processed on the same day of collection. It is a technical limitation of flow cytometry in comparison to polymerase chain reaction methods where sensitivity is unaffected by delays in processing. The overall viability of cells within the samples remained stable over time, despite the decline in plasma cells. A reduction in CD138 MFI is observed in lithium heparin storage medium that may impact on standardized gating techniques. Further validation studies are warranted to explore these phenomena. MRD monitoring in multiple myeloma is rapidly becoming an accepted standard of care in the evaluation of treatment response and represents an independent prognostic maker of progression free survival that can be used to guide further therapy. Our findings indicate the potential of false negative MRD results with delays in sample processing. This questions the current consensus guidelines that recommend samples can be processed up to 2 days after collection. These guidelines may need to be revised in the near future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A Single-Tube Seven-Colour Flow Cytometry Assay for Detection of Minimal Residual Disease In Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1688-1688
Author(s):  
Soraya Wuilleme ◽  
Nelly Robillard ◽  
Steven Richebourg ◽  
Marion Eveillard ◽  
Laurence Lodé ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Flow Cytometry Assay ◽  
Single Tube ◽  
Cell Identification ◽  
Colour Flow ◽  
Multiparameter Analysis ◽  
Minimal Residual

Abstract Abstract 1688 The eradication of minimal residual disease (MRD) in myeloma predicts for improved outcome. A number of different approaches to myeloma MRD detection are available; these vary widely in sensitivity and cost. Flow cytometric assessment of MRD may be preferable in practice because of lower cost and easier feasibility. Myeloma MRD flow cytometry requires at least three markers for plasma cell identification (CD38, CD138 and CD45) and combination of several additional markers to detect phenotypic abnormality including CD19, CD20, CD27, CD28, CD45, CD56 and CD117. Also, assessment of immunoglobulin light-chain restriction (cytoplasmic K and L) combined with myeloma-associated phenotypic plasma cell abnormalities, is very important. Four-tube four-colour flow cytometry combine markers CD38/CD138/CD45 with markers for plasma cell phenotypic abnormalities and clonality. Six –colour flow cytometry combines the same markers (markers for plasma cell identification) plus clonality markers; it potentially increases the sensitivity of the method through coincident multiparameter analysis. However, the single-tube six-colour flow cytometry, proposed by others studies, excludes the myeloma-associated phenotypic plasma cell abnormalities and consequently decreases specificity of the assay. We propose a new single-tube seven-colour flow cytometry, including plasma cell identification antigens, clonality markers and myeloma-associated phenotypic plasma cell abnormalities markers. In this new method, PCs are stained with antibodies: (i) CD38, CD138, CD45 used for identified plasma cells and percentage plasma cells to total leucocytes. (ii) CD19 and CD56+CD28 used to identify normal and abnormal plasma cells; and (iii) cy-IgK and cy-IgL, for confirm the plasma cells clonality. We analysed normal bone marrow provided from healthy individuals. Our results showed a presence myeloma-associated phenotypic plasma cell abnormalities at low levels in healthy individual. The monotypy studies confirm polyclonality of this normal plasma cells. Then we compared MRD assessement with single-six colour flow cytometry assay (plasma cells markers, clonality markers and exluding myeloma-associated phenotypic markers) and seven-colour flow cytometry assay (including myeloma-associated phenotypic markers). Six –colour flow cytometry has a better sensitivity and showed efficacy for quantification MRD in myeloma patients. However, the single-tube six-colour flow cytometry excluded the myeloma-associated phenotypic plasma cell abnormalities and in some cases the seven-colour flow cytometry will be more informative because it detected myeloma-asociated phenotypic marquers combined with clonality marquers. Finally, the single-tube seven colour flow cytometry assay provides reduction in antibody cost and increases sensitivity and specificity of the method through coincident multiparameter analysis. Disclosures: No relevant conflicts of interest to declare.

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.

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