Leukemia and lymphoma: an example of how flow cytometry aids diagnosis

An expanded portfolio of standardized in vitro diagnostic reagents has enhanced the work of the clinical flow cytometry laboratory, as published examples in a Beckman Coulter ClearLLab Casebook illustrate.

Flow cytometric immunophenotyping (FCI) techniques are an important element in aiding the diagnosis and follow-up monitoring of leukemia and non-Hodgkin’s lymphoma (NHL). The overriding benefit over immunohistochemistry (IHC) techniques is the ability to assess numerous markers simultaneously.

Flow cytometry’s ability to show even subtle differences in antigen density makes it possible to distinguish and characterize aberrant cells that may be present in minute quantities.1 Flow cytometry is already used for patients presenting with disease in the bone marrow and/or peripheral blood, and increasingly used to study lymph nodes.

Characterizing normal and aberrant cells

Evidence of flow cytometry’s ability to characterize both normal and aberrant cells was further strengthened when clinical studies by Beckman Coulter Life Sciences led to US clearance of the company’s multicolor dry pre-mixed panels of immunophenotyping reagents for both lymphoid and myeloid lineages. First came the ClearLLab five-color system in 2017, for use in the routine clinical setting on the FC 500 flow cytometer, and later, in 2019, the company’s ClearLLab 10-color panels were cleared for use on the Navios and Navios EX flow cytometers.2

Another key advantage of flow cytometry is the speed at which samples can be analyzed, usually providing a result within 1–2 hours, therefore avoiding issues over antigen degradation or retrieval. When in vitro diagnostic (IVD) panels consist of dry, preformulated reagents, there is no need to spend time manually pipetting antibodies, and the reagents themselves can be stored at 18–30˚C without degrading.3, 4 Working with dry pre-formulated and standardized IVD panels across different locations, and as part of the expanding NHS pathology networks, will enable laboratories to have greater confidence in the consistency and reliability of their results.

Standardized panels provide the opportunity to replace the error-prone manual method of laboratory-developed tests, which are time consuming to create

Standardized panels provide laboratories with the opportunity to confidently replace the error-prone manual method of laboratory-developed tests (LDTs), which are time-consuming to create. They also require the same rigorous assay validation even when replicated in another laboratory. Further, it is common for laboratories to deviate from the original LDT. While IHC can generally assess only one or two markers at a time, commercially available clinical flow cytometers can test tissue and body fluids, "gate out" cells from analysis, and examine 10 or more intracellular or surface antigens simultaneously.

ClearLLab Casebook aids pattern recognition

Flow cytometric immunophenotyping for leukemia and lymphoma relies on trained professionals being able to recognize the significance of differing patterns. Beckman Coulter has developed a ClearLLab 10C Casebook to help professionals become more familiar with the different staining patterns for both normal and abnormal immunophenotyping. It has 24 patient profiles, with a mix of normal specimens and common hematolymphoid malignancies.

The casebook enables the flow cytometry laboratory to become more familiar with different patterns to help identify even minuscule deviations in patient samples that might indicate a malignancy, or call for further investigation, an example of which may be found later in this article.

ClearLLab 10C users should expect to spend less time on error-prone manual tasks and more on value-added work, such as data analysis and interpretation. Patient data, even if assessed remotely, or in different laboratories in different places, will generate comparable results. While immunochemistry still has an important role, especially when bone marrow aspiration reduces the quality of the sample, the process of decalcifying and embedding bone marrow in paraffin wax requires additional preparation time.

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Workflow efficiency

ClearLLab 10C users should expect to spend less time on error-prone manual tasks and more on value-added work, such as data analysis and interpretation

The time spent validating different antibody clones, preparing, and validating cocktails, and ensuring that cocktailed reagents do not degrade is significant. The ClearLLab 10C system standardizes the flow cytometry approach from instrument set-up, quality control, data acquisition and data analysis.

With the ClearLLab 10C system, workflow is reduced to four straightforward, standardized steps – sample processing, sample acquisition, reporting and validation (Fig 1).

Figure 1. ClearLLab 10-color workflow.

Every tube contains CD34 and CD45 for the detection of blast populations and their cells of origin, and the tube design also allows maturation sequences to be clearly identified, a key method for detecting abnormal populations. For compensation, there are 10 separate antibodies, including CD4, CD8 and CD3, and the antibodies are routed to a compensation bead kit that enables separation between negative and positive populations.

Figure 2. ClearLLab Control Cells, a liquid preparation of normal and abnormal stabilized human erythrocytes and leucocytes.

The ClearLLab Control Cells, a liquid preparation of stabilized human erythrocytes and leucocytes, were the first application-specific IVD control cells cleared in the USA for leukemia and lymphoma immunophenotyping (Fig 2). They replicate specimen characteristics such as lysing, light scatter, antigen expression and antibody staining properties, and provide assay values for up to 27 markers for both normal and abnormal control.

Bethesda International Consensus Recommendations

ClearLLab 10C tubes use the company’s DURA Innovations dry reagent technology for the panels, which removes the need to pipette antibodies manually. They can be stored at 18–30°C for 12 months without degradation, making it possible to order and validate a year’s supply as a single lot. No further validation is required apart from routine quality control monitoring. Further, waste generated by liquid reagent handling is reduced. As laboratories can order as many or as few kits as they need, low- and high-volume laboratories can use the same system and scale appropriately as their volumes grow.

ClearLLab reagents follow the 2006 Bethesda International Consensus Recommendations on the Flow Cytometric Immunophenotypic Analysis of Hematolymphoid Neoplasia.5 They are compatible with the World Health Organization (WHO) 2016 revised classification of myeloid neoplasms and acute leukemia. In collaboration with the European Association for Hematopathology and the Society for Hematopathology, WHO recently made important changes to the classification of these diseases. These included new criteria for the recognition of some previously described neoplasms as well as clarification and refinement of the defining criteria for others.6

Additionally, the ClearLLab B-cell tube contains CD200. Studies show that this has potential as a discriminator between B-CLL and mantle cell lymphoma.7 Also, the addition of T-cell receptor (TCR) γδ to the T-cell tube may be useful for detecting mature T-cell malignancies that may express this receptor.8

Take me to the guidelines now

Increasing importance of immunophenotyping

Flow cytometric immunophenotyping was first developed around 40 years ago as a research tool for studying the immune system. However, it might never have been used to assess leukemia and lymphomas if the acquired immune deficiency syndrome (AIDS) epidemic had not required the urgent adoption of this technology to monitor CD4-positive T-helper cell counts in the mid-1980s.

ClearLLab Control Cells replicate specimen characteristics such as lysing, light scatter, antigen expression and antibody staining properties

For today’s patients, quick and accurate laboratory testing using flow cytometry techniques has obvious benefits for treatment options and quality of life.

For laboratories, the development of 10-color flow cytometers, and the ability to replace their own LDTs with standardized, dry pre-formulated reagent panels, certainly enables them to achieve fast, detailed and accurate immunophenotyping.

Laboratories are able to work more efficiently and maximize the use of valuable staff resources. A complete standardized system such as the ClearLLab system spans both instrument and compensation set-up to quality control and analysis protocols. This approach allows flow cytometry professionals to concentrate on analyzing and interpreting the data, rather than spending time manually mixing antibody cocktails or being concerned about critical pattern deviations between laboratories or quality control compliance issues.

Chronic lymphocytic leukemia and small lymphocytic lymphoma

One example from the ClearLLab 10C Casebook (Case 6) is of a 55-year-old male with indications of chronic lymphocytic leukemia (CLL), with lung cytosis for greater than three months. The incidence of CLL is higher among whites than blacks and more prevalent in males than females, with a male-to-female ratio of 1.7:1. Chronic lymphocytic leukemia is a disease that primarily affects the elderly, the median age being 72 years. In familial cases, the median age is 58 years.

Table 1. Complete blood count details.

The patient’s complete blood count (CBC) results (Table 1) show the white blood cells are increased at 19x109/L, with well-preserved hemoglobin at 162 g/L and normal platelets, but evidence of lymphocytosis (lymphocyte count 14x109 /L). The morphology shows well-defined soccer ball type pattern of the mature small lymphocytes (Fig 3a) while there is clear evidence of smudge cells in the abnormal bunching of cells (Fig 3b).

Figure 3. Chronic lymphocytic leukaemia showing a) mature small lymphocytes, and b) evidence of smudge cells in the abnormal bunching.



What is of interest in the phenotyping profile (Fig 4) is the highly distinct patterning for the orange or B-cell population. The selected plots show intermediate to bright CD5 and CD19 and dim CD20. CD45 is bright, as is CD200, with CD38 low to absent. It is also negative for CD10. Looking at the kappa and lambda staining in this sample, there is dim surface lambda light chain expression. Kappa and lambda light chain restriction is the hallmark of clonal B-cell expansion and therefore vital for the diagnosis of B-cell malignancies.

Figure 4. Phenotyping profile.

The lambda versus kappa dot plot (Fig 5) shows all CD19-positive cells. Normal mature B cells are polyclonal, expressing either kappa or lambda light chain in a ratio of 1.4 (range 1–2). The CD19-positive cells (orange) predominantly have surface kappa light chain expression at a decreased level compared with normal mature B cells, indicating a clonal B-cell population. A small population of polyclonal B cells (also in orange) with higher (normal) levels of kappa and lambda light chain expression is present.

Figure 5. Lambda versus kappa dot plot showing all CD19-positive cells.

The CD200 versus side scatter dot plot (Fig 6) shows all viable cells. Studies have shown that CD200 expression may help in the differential diagnosis between CLL and other mature B-cell neoplasms (MBN).9 CD200 is typically expressed on B cells but is negative on some neoplastic B cells. It is especially useful in distinguishing mantle cell lymphoma (usually CD200-negative) from CLL/small lymphocytic lymphoma (usually CD200-positive). The CD19-positive population (orange) in this plot is CD200-positive.

Figure 6. The CD200 versus side scatter dot plot showing all viable cells.

ClearLLab Casebook

Sample cases with characteristic findings typical of various lymphoid and myeloid neoplasms are included in the ClearLLab Casebook, 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, 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.

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.

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  1. Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008; 111: 3941–67.
  2. US Food and Drug Administration. FDA allows marketing of test to aid in the detection of certain leukemias and lymphomas. 29 June 2017 (www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm565321.htm). PPATHOLOGY IN PRACTICE iP
  3. Rajab A, Axler O, Leung J, Wozniak M, Porwit A. Ten-color 15-antibody flow cytometry panel for immunophenotyping of lymphocyte population. Int J Lab Hematol 2017; 39 (Suppl 1): 76–85.
  4. Smallwood C, Galama L, Apoll L, Heinrich KH, Demers J, Buchanan S. Examining the economic impact of laboratory defined testing on flow cytometry immunophenotyping for hematologic malignancies: an analysis of heath resource utilization (Poster). International Society for Pharmacoeconomics and Outcomes Research (ISPOR), 18th Annual European Congress, Milan, Italy. November 2015.
  5. Wood BL, Arroz M, Barnett D et al. 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. Cytometry B Clin Cytom 2007; 72 (Suppl 1): S14–22.
  6. Vardiman JW, Thiele J, Arber DA et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114 (5): 937–51.
  7. Falay M, Afacan Öztürk B, Güneş K et al. The Role of CD200 and CD43 expression in differential diagnosis between chronic lymphocytic leukemia and mantle cell lymphoma. Turk J Haematol 2018; 35 (2): 94–8.
  8. Hodges E, Krishna MT, Pickard C, Smith JL. Diagnostic role of tests for T cell receptor (TCR) genes. J Clin Pathol 2003; 56 (1): 1–11.
  9. Spacek M, Karban J, Radek M et al. CD200 expression improves differential diagnosis between chronic lymphocytic leukemia and mantle cell lymphoma. Blood 2014; 124: 5637.

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