The ClearLLab 10C Casebook

Introduction

This casebook has been designed to assist in the analysis of flow cytometric immunophenotyping data generated using Beckman Coulter’s ClearLLab 10C Panels IVD marked reagent for Leukemia and Lymphoma analysis on the Beckman Coulter Navios and Navios EX flow cytometers. Sample cases with characteristic findings typical of various lymphoid and myeloid neoplasms are included, as are cases from patients with clinical and/or laboratory findings that suggest an underlying neoplastic process, but in which no immunophenotypic abnormality is identified. Specimen types include peripheral whole blood, bone marrow, and lymph nodes. Each case includes a clinical vignette that describes the patient demographics and clinical history, case-specific listmode data files for reanalysis by the user of this casebook, ClearLLab 10C specific analysis protocols to be used with the listmode data, and a report showing the analysis with provided protocols. Each report includes analysis notes that highlight the immunophenotypic findings as well as potential pitfalls.

NOTE: Casebook examples are provided for illustrative purposes only, and not all categories of hematolymphoid neoplasms may be represented, nor are all possible immunophenotypic variants described or demonstrated.

Background

Flow cytometric immunophenotyping evaluates the presence and absence of specific antigens for each individual cell present in the specimen. When taken together, these results generate an immunophenotypic profile for each cell which is either consistent with an expected population (i.e. normal) or inconsistent with an expected population (i.e. aberrant) in that sample type. When evaluating samples from patients with suspected hematolymphoid malignancies, several steps are involved1:

  • Assessment of all cell populations in the sample.
  • Assignment of each cell population to either “normal” or “aberrant”.
  • Detailed characterization of the aberrant population according to the presence or absence of antigens as well as increased or decreased intensity of staining by fluorochrome-labeled antibodies.
  • Interpretation of the aberrant immunophenotype, incorporating where available additional information such as clinical history, histology, cytology, immunohistochemistry, and genotyping studies such as in situ hybridization, karyotyping, and molecular diagnostics.

Consensus recommendations for Immunophenotyping

Consensus recommendations for flow cytometric immunophenotyping of samples from patients with known or suspected hematolymphoid malignancies have emerged over the last two decades, and several guidelines have been published in the scientific literature. Flow cytometric immunophenotyping has been included in the WHO classification of Tumors of Haematopoetic and Lymphoid Tissues since 20082. Medical indications and flow cytometry assay validation including pre-analytic, analytic, and post-analytic details of testing are addressed in the 2006 Bethesda International Consensus Conference recommendations3,4,5 and the ICSH/ ICCS practice guidelines for cell-based fluorescence assays6,7,8.

ClearLLab 10C panels Intended use

The ClearLLab 10C Panels are intended for in vitro diagnostic use for qualitative identification of cell populations by multiparameter immunophenotyping on the Navios and Navios EX flow cytometers. These reagents are used as an aid in the differential diagnosis of hematologically abnormal patients having or suspected of having the following hematopoietic neoplasms: chronic leukemia, acute leukemia, non-Hodgkin lymphoma, myeloma, myelodysplastic syndrome (MDS), and/or myeloproliferative neoplasms (MPN). The reagents can be used with peripheral whole blood (collected in K2EDTA, Acid Citrate Dextrose (ACD) or Heparin), bone marrow (collected in K2EDTA, Acid Citrate Dextrose (ACD) or Heparin) and lymph node specimens. Interpretation of the results should be confirmed by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings.

These reagents provide multiparameter, qualitative results for the surface antigens listed below

  405 nm 488 nm 633 nm
ClearLLab 10C Panels PB1 Krome Orange FITC PE ECD PC5.5 PC7 APC APC-A7002 APC-A7503
B Cell Tube CD19 CD45 kappa lambda CD10 CD5 CD200 CD34 CD38 CD20
T Cell Tube CD3 CD45 TCRγδ CD4 CD2 CD56 CD5 CD34 CD7 CD8
M1 Cell Tube CD11b CD45 CD16 CD7 CD10 CD13 CD64 CD34 CD14 HLA-DR
M2 Cell Tube CD19 CD45 CD15 CD123 CD117 CD13 CD33 CD34 CD38 HLA-DR

1Pacific Blue. 2APC-Alexa Fluor 700. 3APC-Alexa Fluor 750.

ClearLLab Compensation Kit

  405 nm 488 nm 633 nm
ClearLLab 10C Panels PB1 Krome Orange FITC PE ECD PC5.5 PC7 APC APC-A7002 APC-A7503
Compensation Kit CD4 CD8 CD4 CD4 CD3 CD4 CD4 CD4 CD4 CD4

1Pacific Blue. 2APC-Alexa Fluor 700. 3APC-Alexa Fluor 750.

The above reagent is provided in a standardized format to be used with reagents for sample preparation and cytometer set-up, along with software for data acquisition and analysis. ClearLLab 10C Panels meet recommendations for standardization as outlined by the Bethesda guidelines2. Additional information regarding ClearLLab 10C Panels is available here.

Case Selection & Interpretation

The data presented in this case book was generated following the procedure detailed within the ClearLLab 10C Panel Instructions For Use (IFU) available at beckman.com.

Representative cases were selected from clinical trial data and were reviewed, annotated, and interpreted by:

  • Brent Wood, MD PhD Professor, Laboratory Medicine and Pathology Division Head, Hematopathology Director, Hematopathology Laboratory and SCCA Pathology Medical Director, SCCA Laboratories University of Washington, Seattle, WA, USA
  • Xueyan Chen, MD PhD Assistant Professor, Laboratory Medicine Associate Director, Hematopathology Laboratory University of Washington, Seattle, WA
  • Yi Zhou, MD PhD Assistant Professor Department of Laboratory Medicine University of Washington, Seattle, WA

We wish to thank our Principal Investigators & clinical trial sites for their contribution to the clinical trial and to the development of this casebook:

  • Mike Keeney, retired, FCSMLS (D) London Health Sciences Center London, ON Canada
  • Joanne Luider, BSc ART MLT Calgary Laboratory Services Calgary, AB Canada
  • Wolfgang Kern, MD Munich Leukemia Laboratory Munich, Germany
  • Adrian Padurean, MD NeoGenomics Laboratory, Inc. Fort Myers, FL USA

References

  1. Flow Cytometric Immunophenotyping for Hematologic Neoplasms. F.E. Craig, K.A. Foon. Blood. 2008; 111; 3941-3967.
  2. Swerdlow SH, Campo E, Harris NL, Jaffe EA, Pileri SA, Stain H, Thiele J, & Vardiman JW (eds) (2008) WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. IARC Press: Lyon
  3. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Swerdlow SH, et al. Blood. 2016;127:2375-90.
  4. The 2016 revision of the World Health Organization classification of myeloid neoplasms and acute leukemia. Arber DA, et al. Blood 2016 127:2391-2405.
  5. 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia. Wood BL, Arroz M, Barnett D, DiGiuseppe J, Greig B, Kussick SJ, Oldaker T, Shenkin M, Stone E, Wallace P. Cytometry B Clin Cytom. 2007;72 Suppl 1:S14-22
  6. 2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications. Davis BH, Holden JT, Bene MC, Borowitz MJ, Braylan RC, Cornfield D, Gorczyca W, Lee R, Maiese R, Orfao A, Wells D, Wood BL, Stetler-Stevenson M. Cytometry B Clin Cytom. 2007;72 Suppl 1:S5-13
  7. 2006 Bethesda International Consensus Conference on Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasia. Stetler-Stevenson M, Davis B, Wood B, Braylan R. Cytometry B Clin Cytom. 2007;72 Suppl 1:S3
  8. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part III - analytical issues. Tanqri S, Vall H, Kaplan D, Hoffman B, Purvis N, Porwit A, Hunsberger B, Shankey TV; ICSH/ICCS Working Group. Cytometry B Clin Cytom. 2013 Sep-Oct;84(5):291-308. doi: 10.1002/cyto.b.21106
  9. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations. Barnett D, Louzao R, Gambell P, De J, Oldaker T, Hanson CA; ICSH/ICCS Working Group. Cytometry B Clin Cytom. 2013 Sep-Oct;84(5):309-14. doi: 10.1002/cyto.b.21107
  10. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part V - assay performance criteria. Wood B, Jevremovic D, Béné MC, Yan M, Jacobs P, Litwin V; ICSH/ICCS Working Group. Cytometry B Clin Cytom. 2013 Sep-Oct;84(5):315-23. doi: 10.1002/cyto.b.2110

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