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October 2014 - Test your knowledge in the quiz!

Is this a malignant or a reactive lymphocytosis? Mantle cell lymphoma
Post-infectious lymphocytosis
Adult T-cell lymphoma
Infectious mononucleosis

Online version of this month`s case:

THE CORRECT ANSWER TO OCTOBER’S QUIZ IS:

Infectious mononucleosis

Scattergrams and microscopy

Patient history: a 19-year old man was examined by his general practitioner because of tiredness.

Although the WDF scattergram showed the presence of conspicuous lymphatic cells, no abnormal lymphocytes or blasts were found in the WPC scattergram so consequently only the ‘Atypical Lympho?’ flag was displayed.
No abnormal lymphocytes were visible in the WPC scattergram.
The SSC-FSC scattergram of the WPC channel showed a large population of lymphocytes with high lipid content, which have a low side scatter (SSC) and forward scatter (FSC).
Analysis of the RET scattergram revealed a reticulocytosis.
Peripheral blood smear
Peripheral blood smear

Table

Interpretation and Differential Diagnosis

The answer can be inferred from…

  • Severe lymphocytosis: high LYMPH# and LYMPH%
  • Presence of reactive lymphocytes: high HFLC count
  • Presence of ‘Atypical Lympho?’ flag, inferred from the combined results of the WDF and WPC channels (the ‘Blasts/Abn Lympho?’ flag was removed after measurement with the WPC channel)

 

Case history

A 19-year old man with fever, tiredness and frequent headaches visited his physician who ordered a complete blood investigation.

 

Case results

The most-noticeable findings in the complete blood count were a lymphocytosis and the presence of lymphocytes with increased fluorescence intensity, visible in the WDF scattergram as a population of cells with medium to high fluorescence intensity extending upwards from the lymphocyte population. The presence of these cells resulted in the ‘Blasts/Abn Lympho?’ and ‘Atypical Lympho?’ flags and triggered a measurement with the WPC channel, which showed that these were reactive and not malignant lymphocytes (the ‘Blasts/Abn Lympho?’ flag was removed). This caused the appearance of the ‘Atypical Lympho?’ flag rather than the ‘Abn Lympho?’ or ‘Blasts?’ flags. Microscopic differentiation confirmed a polymorphic lymphocytosis with the presence of atypical lymphocytes suspected reactive, while serology revealed the presence of antibodies against Epstein-Barr virus viral-capsid antigen (EB VCA) from type IgG and IgM while nuclear antigen 1 (EBNA-1) antibodies were absent, indicating an acute Epstein-Barr virus infection.

 

The following answers are incorrect for the described reasons

Mantle cell lymphoma

Mantle cell lymphoma is a mature B-cell neoplasm and comprises 3-10% of all non-Hodgkin lymphomas. It is most-prevalent in individuals of middle age and older and has a male predominance of 2:1 or greater. The primary genetic event is a translocation between chromosomes 11 and 14, which causes overexpression of cyclin D1. One of the defining characteristics of mantle cell lymphoma is the presence of small, abnormal lymphocytes called ‘mantle cells’. This would have resulted in an abnormally shaped lymphocyte population overlapping with the monocyte population in the WDF channel (but no HFLC cells), and would have subsequently triggered an ‘Abn Lympho?’ flag in the WPC channel instead of the ’Atypical Lympho?’ flag that was triggered here. In some rare lymphoma cases, the abnormal lymphocytes can interfere with the basophil population in the WNR scattergram. This would have resulted in a discrepancy in the basophil counts from the WNR and WDF channels and such a difference was not observed in the presented patient (not shown). In addition, the morphology observed in the blood smear showed polymorphic atypical lymphocytes, which are characteristic of a reactive rather than a malignant condition. Therefore, a mantle cell lymphoma can be excluded.

 

Post-infectious lymphocytosis

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 a first cellular defence. This results in an increased fluorescence intensity of the lymphocyte population as observed in this case. However, the predominant lymphocyte populations in a post-infectious lymphocytosis are CD4-positive T helper 2 cells and activated B-lymphocytes (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 so a humoral immune response or post-infectious lymphocytosis is unlikely. The presence of polymorphic atypical lymphocytes with blast-like characteristics (virocytes) also confirms a cellular lymphocyte response in an acute phase infection.

 

Adult T-cell leukaemia/lymphoma

An adult-T-cell leukaemia/lymphoma (ATL) is usually a highly aggressive non-Hodgkin's lymphoma and circulating lymphocytes with an irregular nuclear contour (leukaemic cells) are frequently seen. Several lines of evidence suggest that human T-cell leukaemia virus type 1 (HTLV-1) can cause ATL. This evidence includes the frequent isolation of HTLV-1 from patients with ATL and the detection of the HTLV-1 proviral genome in leukaemic cells. The presence of such leukaemic cells would have resulted in an abnormal shape of the lymphocyte population in the WDF channel, overlapping with the monocyte population and subsequently resulting in a ‘Blasts?’ flag in the WPC channel. In addition, leukaemic bone marrow infiltration would suppress production of other cell lines resulting in anaemia or thrombocytopenia, which were not observed here. In summary, the presented patient had reactive lymphocytes (visible in the WDF channel) but no abnormal lymphocytes or blasts (visible in the WPC channel and in the peripheral blood smear), and no signs of bone marrow suppression. Therefore, adult T-cell leukaemia/lymphoma is very unlikely.

Underlying Disease

Infectious mononucleosis

Infectious mononucleosis is caused by the Epstein-Barr virus (EBV), which is a human-specific B-lymphotropic DNA virus of the Herpes virus group. It is easily transmitted via mucous membranes and the portal of entry is usually the lymphoepithelial cells lining the oral cavity (lymphoid tissue of the throat) where a persistent infection is established. Infection incidence correlates strongly with contact between mother and infant and post-adolescent relationships (“kissing disease”). The EBV is an extremely successful virus: the seropositive incidence in Europe is 70-80% in older children, 80-90% in adults and over 90% in the human population worldwide. The virus-host interaction is usually subclinical in young children, especially when the infant is still protected by maternal antibodies. However, in 30-50% of infected adults and adolescents clinical symptoms such as fever, lymph node swelling, hepatomegaly, splenomegaly and tonsillitis are present. These clinical symptoms were described 100 years ago as Pfeiffer’s glandular fever. While most EBV-infected B-lymphocytes undergo productive and lytic infection, some are latently infected and immortalised, and subsequently under life-long control by T-lymphocytes in immunocompetent humans. If an immune defect manifests in seropositive patients, the immortalised B-cell clone may develop into a lymphoma as often happens in patients with AIDS. Furthermore, a role for EBV has been described in several lymphomas and Hodgkin’s disease (1, 2).

Lymphatic reaction to EBV infection

 
EBV enters the lymphoid tissue of the oropharynx and infects B-lymphocytes by binding the complement-receptor CD21. Molecular analysis indicated that EBV mainly infects the germinal centre of the lymph node and/or memory B-cells, but also some naive B-cells. Clonal expansion of infected B-cells in the interfollicular region is (largely) restricted to germinal centre/memory B-cells and is not accompanied by somatic hypermutation. Occasionally, EBV-positive B-cells are detected in the germinal centre. These cells also undergo clonal expansion, but they do not participate in a normal germinal centre reaction. It is unclear whether EBV directly infects germinal centre B-cells, or whether EBV-infected memory B-cells enter germinal centres. Cells within clones of EBV-positive B-cells in the interfollicular region (not in the peripheral blood) show variation in morphology – small cells, blasts (centroblasts) and Hodgkin and Reed-Sternberg (HRS)-like cells – and presumably also in their EBV-gene expression pattern. Infected clonal B-cells differentiate to plasma cells and cause a temporary increase in the heterophile antibody titre (Paul-Bunnell test). Cytokine production by infected B-cells activates cytotoxic T-lymphocytes and natural killer cells, which eliminate the EBV-positive B-cells and suppress B-cell activation.

Cytology

The WBC count can be increased up to 20,000/µL with more than 60% mononuclear cells. The peripheral blood smear shows different developmental stages of lymphatic cells depending on the phase of the disease: small lymphocytes, lymphocytes with large granules (natural killer cells), large lymphoid cells of up to 20µm with irregular cytoplasm (T-suppressor cells) and T-immunoblasts. This diversity of lymphoid cells results in the typical “colourful” morphology in the peripheral blood smear. The heterogeneity of the mononuclear cells in the peripheral blood smear is the most important indication for the diagnosis of infectious mononucleosis.

Serology

Most patients with infectious mononucleosis have increased levels of lactate dehydrogenase (LDH), which originates from lytic B-lymphocytes. In addition, detection of typical (though not virus-specific), heterophile antibodies is possible with the Paul-Bunnell test; many commercial diagnostic kit variants are available. Unfortunately, the test frequently fails in young children because the stimulation of acute phase antibodies is not strong. Heterophile antibodies appear with the onset of disease and disappear again after recovery.

Virus-specific antigen tests

Virus-specific antigens formed in the course of infection can be demonstrated in the infected cells by different methods. Early antigens (EA), virus capsid antigens (VCA), nuclear antigen 1 (EBNA-1) and membrane antigens can be differentiated.
 

  • Antibody against nuclear antigen 1 (EBNA-1):
  • This antigen appears in the nucleus of all persistently infected cells. It is expressed relatively late. The formation of antibodies against EBNA-1 is induced only after weeks or months. A clear positive test result (second titre step) indicates a past infection.
  • Antibodies against the virus capsid antigen (VCA):

If VCA antibodies are not present, an EBV infection can be excluded because the VCA antigen is strongly immunogenic. Most patients produce IgM and IgG antibodies at the onset of disease.
If the VCA test is positive with an IgG titre of 1:160 or more, and the anti EBNA- 1 test is negative, this is an indication of an acute, fresh or reactivated infection. In most primary infections this can be confirmed by a positive IgM titre. Normally, IgM antibodies persist no longer than 10 weeks. However, 10-20% of infected young children are IgM-negative. Furthermore, in adults it is sometimes difficult to distinguish between a more dangerous primary infection in immunosuppressed patients and a usually harmless reactivation. In immunosuppressed patients with primary infections the VCA IgM test is frequently negative. The test for EA antibodies is more sensitive.

 

 

Literature

  1. Grywalska et al. (2013): Epstein-Barr virus-associated lymphoproliferative disorders. Postepy Hig Med Dosw (Online) 24(67): 481-490
  2. Küppers R (2003): B-cells under influence: transformation of B-cells by Epstein–Barr virus. Nat Rev Immunol 3(10): 801-812
  3. Fuchs R and Thomalla J (2005): Manual of “Mikroskopierkurs Hämatologie”: 103-104

Advanced clinical parameters

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