scholarly journals Multi-color flow cytometric immunophenotyping for detection of minimal residual disease in AML: past, present and future

2014 ◽  
Vol 49 (9) ◽  
pp. 1129-1138 ◽  
Author(s):  
J M Jaso ◽  
S A Wang ◽  
J L Jorgensen ◽  
P Lin
Keyword(s):  
Minimal Residual Disease ◽  
Residual Disease ◽  
Color Flow ◽  
Flow Cytometric Immunophenotyping ◽  
Flow Cytometric ◽  
Minimal Residual

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.

Standardized 4 Color Flow Cytometric Minimal Residual Disease Detection Failed To Overcome Regeneration Problems but Identifies Early Blast Clearence Predictive of Molecular Remission after Induction in Childhood B Lineage Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1842-1842 ◽  
Author(s):  
Ester Mejstrikova ◽  
Eva Fronkova ◽  
Drago Batinic ◽  
Klara Dubravcic ◽  
Flora Kiss ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Residual Disease ◽  
Univariate Analysis ◽  
Molecular Response ◽  
Color Flow ◽  
Time Points ◽  
Flow Cytometric ◽  
Therapeutic Decisions ◽  
B Lineage ◽  
Minimal Residual

Abstract Several studies showed a good correlation between minimal residual disease (MRD) obtained by flow cytometry (FC) and by PCR (Ig/TCR rearrangements). However, before FC is widely applied for therapeutic decisions we need exact and standardized criteria. Therefore, we pre-defined B-lineage cell subsets using 4 or 3-color FC and we established background levels for each subset in pre-defined time-points on therapy. Methods. Children with ALL were treated by ALL IC BFM2002 protocol. This international flow cytometric trial MiniMini contained 4 laboratories (Croatia 36 patients, Israel 48, Hong Kong 33 and Czechia 136). 442 samples were measured simultaneously by PCR and 4color FC. Pre-defined subsets (28 values) were measured at 5 time points (d0, d8, d15, d33 and week 12). Cut off for positivity and negativity of individual subpopulations were assessed as follows: 1) negative samples by PCR for respective time point were randomly divided into training and testing cohort 2) 98.5 percentile of individual subpopulation in training cohort was selected as a cut off for positivity and negativity 3) cut off values were tested on testing cohort and PCR positive samples (total 337 samples), sample was considered positive when having at least one value above cut off (when more values were above cut off, highest value was considered as MRD). Results. When all time points were analyzed together, sensitivity of FC MRD was 82% and specificity 88% There are no negative patients at day 8 in bone marrow by FC or by PCR. 5 samples at day 15 were positive by PCR (<=10^−4) and negative by FC, no sample was false positive by FC, sensitivity was 98%, no sample was negative by both methods. At day 33 and at week 12, sensitivity dramatically dropped to 34 and 13%, specificity was 80% and 100%. We next asked whether the early blast clearance assessed by FC MRD predicts positivity by PCR after induction. Univariate analysis showed that the blast clearance by FC both at days 8 and 15 predicts the PCR-based molecular response at day 33 and/or week 12. Patients with PCR-positive MRD at day 33 and at week 12 had more often over 50% blasts at day 8 BM (Fischer test p=0.0013 and p=0.04) and/or more than 5% blasts at day 15 by FC (p=0.0015 and p=0.0036). Conclusion. With a minimized subjective bias and exact background values set for each cellular subset and timepoint, we did not reach satisfactory sensitivity at day 33 and at week 12. The FC-assessed leukemia clearance at days 8 and 15 predicts MRD by PCR at day 33 and at week 12. Since a non leukemic background influences sensitivity of FC MRD at day 33 and at week 12, complex approach using 6 and more color combinations is needed for precise distinction between leukemic and non leukemic B cells.

scholarly journals Combination chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab shows significant clinical activity with low accompanying toxicity in previously untreated B chronic lymphocytic leukemia

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 405-411 ◽  
Author(s):  
Neil E. Kay ◽  
Susan M. Geyer ◽  
Timothy G. Call ◽  
Tait D. Shanafelt ◽  
Clive S. Zent ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Hematologic Toxicity ◽  
Color Flow ◽  
Significant Clinical Activity ◽  
Minimal Residual

Abstract Building on the prior work of use of pentostatin in chronic lymphocytic leukemia (CLL), we initiated a trial of combined pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) for 65 symptomatic, previously untreated patients. Of 64 evaluable patients, 34 (53%) were high Rai risk, 71% were nonmutated for the immunoglobulin heavy-chain variable region gene, 34% were CD38+, and 34% were ZAP-70+. Thirty patients (52%) had one anomaly detected by fluorescence in situ (FISH) hybridization, and 21 (36%) had complex FISH defects. Thirty-eight patients (58%) had grade 3+ hematologic toxicity but minimal transfusion needs and no major infections. Responses occurred in 58 patients (91%), with 26 (41%) complete responses (CRs), 14 (22%) nodular partial responses (nodular PRs), and 18 (28%) partial responses (PRs). Many patients with a CR also lacked evidence of minimal residual disease by 2-color flow cytometry. Examination of prognostic factors demonstrated poor response in the 3 patients with del(17p). In contrast, we found this regimen was equally effective in young versus older (> 70 years) patients and in del(11q22.3) versus other favorable prognostic factors. Thus, this novel regimen of pentostatin, cyclophosphamide, and rituximab for previously untreated patients with CLL demonstrated significant clinical activity despite poor risk-based prognoses, achievement of minimal residual disease in some, and modest toxicity.

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