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

What causes this mild lymphocytosis and thrombocytopenia? Healthy individual
Post-infectious lymphocytosis
Idiopathic thrombocytopenic purpura (ITP)
B-cell chronic lymphocytic leukaemia (B-CLL)

Online version of this month`s case:

THE CORRECT ANSWER TO DECEMBER’S QUIZ IS:

B-cell chronic lymphatic leukaemia (B-CLL)

Scattergrams and microscopy

Patient history: a 61-year old male patient with a mild lymphocytosis and thrombocytopenia.

WDF scattergram
WPC (SSC-SFL) scattergram
WPC SSC-FSC scattergram
PLT-F scattergram
Peripheral blood smear
Peripheral blood smear

Table

Interpretation and Differential Diagnosis

The answer can be inferred from…

  • Lymphocytosis: increased LYMPH# and LYMPH%
  • Presence of malignant lymphocytes: ‘Abn Lympho?’ flag
  • Absence of reactive lymphocytes: no ‘Atypical Lympho?’ flag, no high fluorescence lymphocytes (HFLC)
  • Impaired megakaryopoiesis: mild thrombocytopenia combined with a low immature platelet count (IPF#)

 

Case history

A 61-year old man visited his physician for a routine health check. When the physician detected enlarged lymph nodes she ordered a complete blood count.

Case results

Although WBC counts were normal, an absolute and relative lymphocytosis was observed. In addition, conspicuous lymphocytic cells were detected between the lymphocyte and monocyte populations in the WDF scattergram. In the absence of a WPC channel these cells would have triggered the ‘Blasts/Abn Lympho?’ and ‘Atypical Lympho?’ flags but in this case a WPC reflex measurement was triggered. Blasts were not detected in the flagging areas of the WPC scattergrams but an abnormal ratio of the two lymphocyte populations was visible in the SSC-FSC scattergram: the ‘lower’ lymphocyte population with increased membrane lipids and low forward scatter was comparatively large, indicating the presence of abnormal lymphocytes. This triggered the appearance of the ‘Abn Lympho?’ flag and not the ‘Atypical Lympho?’ flag. In case of reactive lymphocytes, a lower lymphocyte population would have been present in the scattergram as well but always combined with an increased population of reactive lymphocytes in the WDF scattergram, which was not the case here.The peripheral blood smear showed the presence of small, abnormal lymphocytes but these cells are very difficult to recognise in an early-phase B-cell chronic lymphocytic leukaemia (B-CLL). No reactive neutrophils or monocytes were observed. Immunotyping using flow cytometry showed a monoclonal CD19 kappa-positive cell clone.

The following answers are incorrect for the described reasons

Healthy individual

Most haematological parameters are normal in this patient and even the mild lymphocytosis may have been induced by a past viral infection. However, the mild thrombocytopenia and low absolute IPF indicate impaired bone marrow activity, and the presence of abnormal, malignant lymphocytes, visible between the lymphocyte and monocyte populations in the WDF scattergram, clearly indicates a pathological condition so this individual is unquestionably sick.

Post-infectious lymphocytosis

The mild lymphocytosis observed here could have been induced by a past infection from which the patient had already recovered. Viral infections trigger an acute phase cellular immune response by the activation of CD8-positive cytotoxic T-cells, CD4-positive T helper 1 cells and natural killer cells as the first cellular defence. This results in a highly increased fluorescence intensity of the lymphocyte population. In the presented case the fluorescence intensity of the lymphocytes is only slightly increased, so both the ‘Blasts/Abn Lympho?’ and ‘Atypical Lympho?’ flags would have been shown if only a WDF measurement (without WPC) would have been performed. Furthermore, the predominant lymphocyte populations in a post-infectious lymphocytosis are CD4-positive T helper 2 cells and activated B-cells (plasma cells), which are activated during the humoral immune response. This would have resulted in a normal fluorescence intensity of the lower lymphocyte population containing T helper 2 cells, and a separate highly-fluorescent population in the WDF scattergram containing the plasma cells. In addition, the population of lymphocytes with low lipid content (the ‘upper’ lymphocyte population in the SSC-FSC scattergram of the WPC channel) would have been bigger. The ‘lower’ lymphocyte population in this patient is comparatively large, so a post-infectious lymphocytosis is unlikely. The presence of abnormal, monomorphic lymphocytes in the presented patient (rather than reactive, atypical lymphocytes) also makes a reactive condition such as a post-infectious lymphocytosis unlikely.

Idiopathic thrombocytopenic purpura (ITP)

ITP is an autoimmune haematological disorder in which autoantibodies against platelet antigens induce accelerated platelet destruction, leading to a reduction in peripheral blood platelets. ITP causes a characteristic purpuric rash and a tendency to bleed, for example from the nose or periodontal gums. It is difficult to distinguish ITP from other causes of thrombocytopenia so diagnosis is a process of exclusion. The presented patient has a mild thrombocytopenia but the normal immature platelet fraction (IPF) and low absolute IPF count (IPF#) indicate a production problem rather than accelerated platelet destruction. In addition, ITP is not associated with the presence of abnormal lymphocytes so it can be excluded here.

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 leukocytes, 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
  • d)     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

Advanced clinical parameters

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