Utilization of the ClearLLab lymphoid screening tube for investigation of hematolymphoid neoplasias by flow cytometry
Wolfgang Kern1, Mike Keeney2 (retired), Joanne Luider3, Evelyn Karl1, Ben Hedley2, Lori Lowes2, Debbie Murphy3, Diana Careaga4, Guoyan Cheng4, Liliana Tejidor4
1Munich Leukemia Laboratory, Munich, DE, Germany,
2London Health Sciences Center, London, ON, Canada,
3Calgary Laboratory Services, Calgary, AB, Canada,
4Clinical Research, Beckman Coulter Inc., Miami, FL, United States
Flow cytometry immunophenotyping is an integral part of diagnostics for hematologic malignancies. The ClearLLab LS* tube contains CD8-FITC, Kappa-FITC, CD4-PE, Lambda-PE, CD19-ECD, CD56- PE-Cy5.5, CD10-PE-Cy7, CD34-APC, CD5-APC-A700, CD20-APC-A750, CD3-PB and CD45-KrO in dried format and should prove useful as a screening tube for hematolymphoid malignancies.
A blinded comparison between lab developed tests (LDTs) and ClearLLab LS was performed to identify pathologic cell populations and exclude hematological malignancy. 61 bone marrow (BM), 22 lymph node (LN) and 84 whole blood (WB) samples were assessed in 3 North American/European laboratories with their respective LDTs and in parallel to the single ClearLLab LS reagent panel.
77 samples with no hematological malignancy as analyzed by LDT were also negative for hematological malignancy by ClearLLab LS (100% agreement). Out of 90 samples with hematological malignancy and abnormal population within the sample tested by LDT, 86 were positive by ClearLLab LS resulting in a 95.6% agreement compared to LDT. Of the 4 cases which were not detected positive by ClearLLab LS, 3 were plasma cell neoplasms and 1 mature T cell malignancies with loss of CD7. The presence of CD34 in the tube allowed the detection of blast populations in several myeloid neoplasms that were tested.
The ClearLLab LS screening tube showed agreement with LDTs developed by 3 international laboratories.
The ClearLLab LS reagent is a comprehensive multicolor Leukemia and Lymphoma** (L&L) screening tube consisting of 12 antibodies and a 10-color cocktail reagent composed of antibodies directed against T, B, and NK lineage antigens.
Combinations are intended to identify the lymphocyte subpopulations that express these antigens alone or in specific co-expression patterns and are designed to work with both normal and abnormal specimens.
The Bethesda(1) consensus recommendations indicated that flow cytometry is indicated in the following clinical conditions: cytopenias, anemia, leukopenia and/or thrombocytopenia; elevated leukocyte count (lymphocytosis, monocytosis and eosinophilia); presence of atypical cells or blasts in peripheral blood (PB), bone marrow or body fluids; plasmacytosis or monoclonal gammopathy; and organomegaly and tissue masses.
In these clinical situations, flow cytometry immunophenotyping can provide a sensitive assessment for the presence of hematologic malignancy and aid in demonstrating the absence of disease.
Flow cytometry may provide diagnostic information but, more importantly, can direct other ancillary testing to diagnose hematolymphoid malignancy.
The ClearLLab LS tube is intended as a screening tube to aid in determining the presence or absence of an abnormal phenotype(s) in PB, BM or LN samples.
The ClearLLab LS will correctly identify the presence or absence of abnormal populations in peripheral blood, bone marrow, and lymph nodes when compared to the final diagnosis as determined by the laboratory developed test.
Comparison of the ClearLLab LS to the LDT in multiple laboratories to determine the accuracy of the ClearLLab LS in determining the presence or absence of abnormal populations in multiple types of patient samples.
Materials & Methods
Table 1. The ClearLLab LS is a 12 antibody 10-color cocktail screening tube provided in the DURAClone dry reagent format.
Figure 1. Scheme showing the breakdown of samples per site. Two sites collected 56 samples and one 55 samples. Samples were split 42% normal (no disease) and an abnormal population were 58% of the total number of samples.
Figure 2. Sample preparation and procedure for the ClearLLab LS tube. This representation is for peripheral blood, which was the same for bone marrow and bloody lymph nodes. Lymph nodes that were not visibly bloody did not require the addition of VersaLyse.
Figure 3. Flow chart showing how the study was performed at each site.
Figure 4. Of the 167 samples that were investigated for the presence of abnormal populations or to exclude hematological malignancy, 84 were peripheral blood, 61 were bone marrow, and 22 lymph nodes. Samples were collected in three North American and European laboratories with their respective LDTs and in parallel to the single ClearLLab LS reagent panel.
Figure 5A. Breakdown of the samples in T, B, Myeloid malignancies included in this study.
Figure 5B. Breakdown of the 90 malignant samples according to the number of acute, chronic, non-Hodgkin’s lymphoma, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and plasma cell neoplasms included in this study.
Figure 6A. ClearLLab LS phenotypic agreement to specimens without abnormal phenotype identified by LDT, 100%.
Figure 6B. ClearLLab LS phenotypic agreement to specimens with abnormal phenotype identified by LDT, this represents 95.6% of the abnormal specimens.
- Seventy-seven samples with no hematological malignancy as analyzed by LDT were also negative for hematological malignancy by ClearLLab LS.
- 100% agreement of normal samples compared to LDT.
- Of 90 samples with hematological malignancy and abnormal population within the sample tested by LDT, 86 were positive by ClearLLab LS.
- 95.6% agreement in samples with an abnormal population compared to LDT.
- Four cases were not detected positive by ClearLLab LS when compared to LDT.
- Three plasma cell neoplasms were not detected, as the ClearLLab LS tube does not contain any plasma cell antigen.
- One mature T cell malignancy was not detected in the ClearLLab LS tube. These populations had a loss of CD7 which is not present in the screening tube.
The authors would like to thank the members of the laboratories in Munich Leukemia Laboratory (MLL), London Health Sciences Center, and Calgary Laboratory Services who performed and analyzed all the LDT data used as a reference for this study.
- 2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications. Davis BH et al. Cytometry B Clin Cytom. 2007;72 Suppl 1:S5.
- CLSI H26-A2 Validation Verification and Quality Assurance of Automated Hematology Analyzers. June 2010.
- CLSI EP05-A3 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline.
- CLSI EP12-A2. User Protocol for Evaluation of Qualitative Test performance; Approved Guideline - Second Edition. 2008.
- Swerdlow, S, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues 4th Edition. . Lyon : International Agency for Research on Cancer, 2008.
*ClearLLab LS (Lymphoid Screen) is CE-IVD marked under Regulation (EU) 2017/746. For In Vitro Diagnostic Use. The Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.
**For Non-Hodgkin’s lymphoma only