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Do you know which disease fits this month’s case? Then test your knowledge in the quiz below!

Which reactive or neoplastic condition triggered this lymphocytosis? B-cell chronic lymphatic leukaemia (B-CLL)
Reactive lymphocytosis caused by an acute phase T-cellular immune reaction
Reactive lymphocytosis caused by a humoral B-cell immune response
Precursor B-cell acute lymphoblastic leukaemia (B-ALL)

Online version of this month case:

The correct answer to June´s quiz is:

B-cell chronic lymphocytic leukaemia (B-CLL)

Scattergrams and microscopy

Table

Interpretation and differential diagnosis

The answer can be inferred from…

  • Lymphocytosis: increased LYMPH# and LYMPH%
  • Absence of reactive lymphocytes: Re-LYMPH ≤ 5%
  • No ‘Atypical Lympho?’ flag
  • Presence of malignant lymphocytes: ‘Abn Lympho?’ flag

 

Case history
A routine blood test was performed for an apparently healthy 53-year old blood donor.

Case results
In addition to increased WBC counts, an absolute and relative lymphocytosis was found. Increased conspicuous monomorphic lymphocytic cells were detected in the WDF scattergram without increased Re-LYMPH or AS-LYMPH and this triggered a WPC reflex measurement. Abnormal lymphocytes were visible as a separate cell population above the lymphocytes in the SSC-SFL scattergram of the WPC channel and an abnormal ratio of the two lymphocyte populations was visible in the SSC-FSC scattergram: the ‘lower’ lymphocyte population (W1) with increased membrane lipids and low forward scatter was comparatively large, confirming the presence of abnormal lymphocytes. In case of reactive lymphocytes, a lower lymphocyte population would have been present in the WPC scattergram as well but always combined with an increased population of reactive lymphocytes in the WDF scattergram. The peripheral blood smear showed an increased population of monomorphic small lymphocytes, which are difficult to distinguish from normal lymphocytes – in particular in an early-phase B-cell chronic lymphocytic leukaemia (B-CLL) without an extreme leucocytosis. The diagnosis was confirmed by immune flow cytometry.

 

The following answers are incorrect for the described reasons

 

Reactive lymphocytosis caused by an acute phase T-cellular immune reaction
An acute viral infection triggers a cellular immune response by T-lymphocytes, resulting in a lymphocytosis and a large increase of reactive lymphocytes with high fluorescence in the WDF scattergram (Re-LYMPH > 5%). Like the presented case, antibody-synthesizing cells would be absent (AS-LYMPH ≤ 1%) and the population of lymphocytes with high lipid content in area W2 of the WPC SSC-FSC scattergram (activated T-cells with increased membrane lipids) would be large. However, Re-LYMPH is low here and the XN algorithms classified the conspicuous lymphocytes as malignant rather than reactive, triggering the appearance of the ‘Abn Lympho?’ flag and not the ‘Atypical Lympho?’ flag. A reactive lymphocytosis could therefore be excluded. 

 

Reactive lymphocytosis caused by a humoral B-cell immune response
The humoral immune response, predominant during late-stage viral infections, is associated with increased numbers of reactive lymphocytes (Re-LYMPH% > 5%) and antibody-synthesizing plasma cells (AS-LYMPH% > 1%). The antibody-synthesizing plasma cells have a low lipid content and would be visible in area W1 of the WPC SSC-FSC scattergram. This would have resulted in a bigger W1/W2 ratio than observed here and the subsequent appearance of the ‘Atypical Lympho?’ flag (suspicion of reactive lymphocytes). Since none of these findings were present here, this diagnosis could be excluded a well.

 

Precursor B-cell acute lymphoblastic leukaemia (B-ALL)
B-ALL is one of the less common lymphoid neoplasms. It is characterized by symptoms related to anaemia, thrombocytopenia, and neutropenia due to infiltration of the bone marrow with tumour cells. B-lymphoblasts present in B-ALL samples appear as an abnormal population between the lymphocytes and monocytes in the WDF scattergram and these B-lymphoblasts, which are larger than other lymphocytes, are also visible in the SSC-FSC scattergram of the WPC channel where they trigger the appearance of a ’Blasts?’ flag. There were no blasts in the presented sample and this patient was not anaemic, thrombocytopenic or neutropenic so a B-ALL could be excluded.

Underlying disease

B-cell chronic lymphocytic leukaemia (B-CLL; 1)

B-CLL is a chronic lymphoproliferative disorder that is probably caused by auto-antigens promoting cell division of B-cell precursors. It is characterised by the presence of uniformly round to somewhat irregular CD5- and CD23-positive B-cells in the peripheral blood. The designation small lymphocytic lymphoma is used when lymphadenopathy is observed, lymphocyte counts are below 5 x 109/L and no cytopenias are observed as a result of bone marrow infiltration. In the presented case the lymphocyte counts are below 5 x 109/L but a thrombocytopenia with reduced megakaryopoiesis was also found, suggesting bone marrow infiltration. Environmental risk factors for B-CLL are not known: risk factors for other types of leukaemia have no proven association with this condition. Genetics, however, is believed to play an important role in B-CLL, which affects Western populations to a much greater extent than far Eastern populations and has a male predominance of 1.5-2 to 1.

 

Classification of lymphoid tumours

Lymphomas are diverse, biologically complex neoplasms of the immune system and comprise approximately 4% of new cancers. Historically, several lymphoma classification schemes have been developed based exclusively on morphologic features but this limited approach has proved unreliable and immunophenotyping, by flow cytometry and/or immunohistochemistry, has emerged as a valuable addition to morphologic diagnosis. By combining light scatter characteristics, patterns of antigen expression and DNA content, flow cytometry provides information that is useful for making a diagnosis and subsequently assessing a prognosis.

 

Lymphocytes (2)

To fully understand the pathophysiology of lymphomas, understanding of normal lymphocyte morphology and activity is necessary.

 

Morphological classification

Small lymphocytes: these cells are 7-10µm in diameter with a uniform, round, intensely stained, condensed nucleus and only a thin rim of agranular cytoplasm containing a few ribosomes and organelles. In healthy individuals, the great majority of small lymphocytes are resting in the G0 phase of the cell cycle.

Large granular lymphocytes: these cells, which comprise 5-10% of total peripheral leucocytes, are approximately 20µm in diameter and possess granular cytoplasm.

 

Immunological classification

Lymphocytes are subdivided into three types: B-lymphocytes (B-cells), T-lymphocytes (T-cells) and natural killer (NK) cells. Small lymphocytes are divided immunologically into two major categories: T-cells (60-70%) and B-cells (10-30%). T-cells and B-cells derive from a common precursor and there are no morphological differences between these two cell types but they have differing ontogenies and are functionally distinct. Activated T-cells perform a range of functions, predominantly cytokine production and cellular cytotoxicity, while B-cells produce antibodies.

 

T-cells: lymphocytes that mature in the thymus are called T-cells. They are subdivided into CD4-positive T-cells, also called helper T-cells or Th cells, and CD8-positive cells, which are cytotoxic T-cells. Activated Th cells serve three main functions mediated by cytokines:

1. Assist B-cell activation (Th2 cells secreting IL-4 and IL-5)
2. Activate CD8-positive cytotoxic T-cells (Th1 cells secreting IL-2 and IFN-gamma)
3. Induce delayed type hypersensitivity (Th1 cells secreting IL-2 and IFN-gamma)

Activated cytotoxic T-cells induce cell death by inserting the pore-forming protein perforin into the cell membrane of the recipient cell, most commonly in virus-infected, tumour and allograft cells. Some CD8-positive T-cells suppress certain cell functions and are called suppressor cells. CD4-positive cells amount to approximately 65% of peripheral T-cells and CD8-positive cells to about 35%.

 

B-cells: B-cells do not require the thymus for maturation and exist in germinal centers of the lymph nodes, in the spleen, in the bone marrow and in mucosa-associated lymphoid tissue. They differentiate from pre-pre-B-cells to pre-B-cells and then to B-cells. After stimulation by an appropriate antigen, B-cells undergo clonal expansion and mature into immunoglobulin-secreting plasma cells. After an infection some of the plasma cells persist as memory cells, which can rapidly respond to a recurring infection by the same pathogen. B-cells possess characteristic cell surface markers that can be used for their identification by flow cytometry with fluorochrome-labelled antibodies. Approximately 30% of peripheral lymphocytes are B-cells.

 

Natural Killer (NK) cells: these cells are sometimes called large granular lymphocytes (LGL). NK cells are cytotoxic lymphocytes, distinct from B-cells and T-cells, which participate in both innate immunity and adaptive immunity. They lack the classic cell surface markers of other lymphocytes except CD2 and CD16 and do not have rearranged T-cell receptor or immunoglobulin genes. For this reason they are often referred to as null, non-T- or non-B-cells. NK cells express CD56, which is shared by few other cells and can therefore be used to detect them. Their principal characteristics are nonspecific cytotoxicity, which is activated by cytokines, particularly interferons and IL-2. NK cells are designed to eliminate malignant cells and virus-infected cells by:

1. Eradicating cells to which antibodies have bound via a process called antibody-dependent cellular cytotoxicity
2. Destroying cells that lack MHC-I molecules on their surface

 

Classification of lymphomas (3, 4, 5)

The classification of the World Health Organisation (WHO; 3), which is based on the Revised European-American Classification of Lymphoid Neoplasms (REAL; 4) recognises four major categories of lymphoid malignancy based on morphology and cell lineage:

1. Precursor lymphoid neoplasms
2. Mature B-cell neoplasms
3. Mature T-cell and NK-cell neoplasms
4. Hodgkin lymphoma

Lymphomas as well as lymphoid leukaemias are included in this classification because solid and circulating phases are present in many lymphoid neoplasms and any distinction between them is artificial. Major differences between the WHO classification and the REAL classification are reviewed by Cogliatti and Schmid (6).

 

Within the precursor lymphoid neoplasm group, two subdivisions are recognised (3):

1. B lymphoblastic leukaemias/lymphomas
2. T lymphoblastic leukaemias/lymphomas

 

Within the mature B-cell neoplasm group, more than 25 subdivisions are recognised (3), such as:

1. B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma 
2. B-cell prolymphocytic leukaemia
3. Lymphoplasmacytic lymphoma
4. Mantle cell lymphoma
5. Follicular lymphoma (grade 1, grade 2, grade 3a, grade 3b)
6. Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma)
7. Nodal marginal zone lymphoma
8. Splenic B-cell marginal zone lymphoma
9. Hairy cell leukaemia
10. Plasma cell myeloma
11. Diffuse large B-cell lymphoma
12. Primary mediastinal (thymic) large B-cell lymphoma
13. Intravascular large B-cell lymphoma
14. Primary effusion lymphoma
15. Burkitt lymphoma

 

The mature T-cell and NK-cell neoplasms are also subdivided and the conditions include:

1. T-cell prolymphocytic leukaemia
2. T-cell large granular lymphocytic leukaemia
3. Aggressive NK-cell leukaemia
4. Mycosis fungoides
5. Sezary syndrome
6. Peripheral T-cell lymphoma, not otherwise characterised
7. Hepatosplenic T-cell lymphoma
8. Subcutaneous panniculitis-like T-cell lymphoma
9. Angioimmunoblastic T-cell lymphoma
10. Extranodal T-/NK-cell lymphoma, nasal type
11. Enteropathy-associated T-cell lymphoma
12. Adult T-cell leukaemia/lymphoma
13. Anaplastic large cell lymphoma, ALK positive/negative

 

Hodgkin lymphoma is subdivided into:

1. Nodular lymphocyte-predominant Hodgkin lymphoma
2. Classical Hodgkin lymphoma:
a) Nodular sclerosis Hodgkin lymphoma
b) Lymphocyte-rich classical Hodgkin lymphoma
c) Mixed cellularity Hodgkin lymphoma

Lymphocyte depletion Hodgkin lymphoma

Literature

  1. Müller-Hermelink HK, Montserrat E, Catovsky D et al (2007): Chronic Lymphocytic Leukaemia/Small Lymphocytic Lymphoma. In: Swerdlow SH, Campo E, Harris NL, et al (Editors): World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th Edition. Lyon, France. International Agency for Research on Cancer (IARC) Press: 157-166
  2. Shinton NK (2007): CRC Desk Reference for Hematology, second edition.
  3. Jaffe ES, Harris NL, Stein H et al (2007): Introduction and Overview of the Classification of Lymphoid Neoplasms. In: Swerdlow SH, Campo E, Harris NL, et al (Editors): World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th Edition. Lyon, France. International Agency for Research on Cancer (IARC) Press: 157-166
  4. Harris NL, Jaffe ES, Stein H et al (1994): A Revised European-American Classification of Lymphoid Neoplasms: a proposal from the International Lymphoma Study Group. Blood 84(5): 1361-1392
  5. The Non-Hodgkin’s Lymphoma Classification Project (1997): A clinical evaluation of the International Lymphoma Study Group classification of Non-Hodgkin’s Lymphoma. Blood 89(11): 3909-3918
  6. Cogliatti SB, Schmid U (2002): Who is WHO and what was REAL? Swiss Med Wkly 132(43-44): 607-617

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