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

Can you explain the observed lymphocytosis? Acute phase of infectious mononucleosis
B-cell chronic lymphatic leukaemia (B-CLL)
Acute B lymphoblastic leukaemia (B-ALL)
Recovery phase of cytomegalovirus (CMV) infection


Online Version of this month´s case:

Correction

The ‘Abn Lympho?’ flag printed on the calendar is not correct. The ‘Atypical Lympho?’ flag was triggered by the analyser. Sorry for the confusion.

The correct answer to March´s Quiz is:

Recovery phase of cytomegalovirus (CMV) infection

Scattergrams and microscopy

Patient history: an unclear lymphocytosis was found in a 35-year old woman.

Table

Interpretation and Differential Diagnosis

The answer can be inferred from…

  • Lymphocytosis: increased LYMPH#
  • Presence of antibody-synthesizing lymphocytes (AS-LYMPH% >1%) in combination with an increased ‘upper’ lymphocyte population in the WPC scattergram
  • Presence of reactive lymphocytes: ‘Atypical Lympho?’ flag
  • Absence of anaemia: RBC, HGB and HCT normal
  • Normal megakaryopoiesis: low IPF# but normal PLT and IPF (%)

 

Case history

A 35-year old woman visited her physician for pre-pregnancy consultation. A blood sample was sent to a private medical services laboratory where a lymphocytosis was detected by the XN-series haematology analyser, suggesting the presence of either a reactive or malignant condition.

Case results

WBC analysis revealed an absolute lymphocytosis of 5,930/µL with slightly increased reactive lymphocytes (Re-LYMPH% = 5.1%), but at 3.8% the fraction of antibody-synthesizing lymphocytes (plasma cells) was clearly increased (AS-LYMPH% > 1%). Combined with the increased W1 lymphocyte / W2 lymphocyte ratio, observed in the WPC scattergram, this resulted in the flag ‘Atypical Lympho?’ (suspected reactive lymphocytes). These findings pointed to a late-phase viral infection (humoral immune response). Furthermore, the increased populations of microcytic RBC (MicroR = 10.3%) and hypochromic RBC (HYPO-He = 2.3%) pointed to a possible functional iron deficiency due to the ongoing infection. Besides this suspected mild functional iron deficiency, the patient had no anaemia and thrombopoiesis was normal (normal PLT and IPF), which make a reactive lymphocytosis more likely than a neoplastic lymphocytosis. Plasma cells and RBC of varying shapes and sizes were observed in the peripheral blood smear.

The following answers are incorrect for the described reasons

Acute phase of infectious mononucleosis

An acute viral infection results in a lymphocytosis and a large increase of reactive lymphocytes (Re-LYMPH), which have an increased fluorescence intensity. This is mainly due to the cellular immune response by T-lymphocytes, but it is not associated with an increase in antibody synthesizing cells (AS-LYMPH ≤ 1%). In addition, the population of lymphocytes with high lipid content (activated T-cells with increased membrane lipids), which occurs in the lower lymphocyte population W2 in the SSC-FSC scattergram of the WPC channel would have been bigger resulting in a lower W1/W2 ratio.

 

B-cell chronic lymphatic leukaemia (B-CLL)

B-CLL is a chronic lymphoproliferative disorder and lymphocyte counts in B-CLL patients are usually above 5 x 109/L (5.93 x 109/L here) and the WDF scattergram of samples obtained from B-CLL patients looks very similar to the one presented here. However, antibody-synthesizing lymphocytes (AS-LYMPH) are not present in B-CLL patients and the observed population of lymphocytes with low lipid content in the W1 area of the SSC-FSC scattergram of the WPC channel is not present in a B-CLL. Finally, the presented WPC scattergram doesn’t show cells in the high-fluorescence area above the lymphocytes and therefore the presence of abnormal lymphocytes could be excluded. B-CLL samples contain abnormal lymphocytes, which would have triggered an ‘Abn Lympho?’ flag rather than the observed ‘Atypical Lympho?’ flag.

 

Acute B 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. In addition, B-ALL was unlikely because antibody-synthesizing lymphocytes (AS-LYMPH) are not present in B-ALL patients unless they also have an infection.

Underlying Disease

Cytomegalovirus (CMV) infections

Infections with the cytomegalovirus (CMV, a DNA virus of the Herpes virus group) can be life threatening in immunocompromised individuals such as cancer patients receiving chemotherapy or transplant recipients. In immunocompetent individuals however, infection is often asymptomatic or resembling infectious mononucleosis and antiviral therapy is typically not necessary. Similar to Epstein-Barr virus infections, CMV-infected cells become latently infected and reactivation of latent CMV is most-likely after immunosuppression. The seropositive incidence for CMV is high, up to 100% in some populations, and increases with age (1). Transmission occurs via close contact with infected individuals or exposure to infected body fluids.

Cytology

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. In chronic infections or during the recovery phase, the predominant lymphocyte populations are CD4-positive T helper 2 cells and activated B-cells (plasma cells), which are activated during the humoral immune response (Figure 1). In case of CMV infections, this immune response leads to an absolute lymphocytosis, more than 50% mononuclear cells and the presence of atypical lymphocytes. In addition, CMV infects myeloid progenitors, megakaryocytes and myeloid dendritic cells that undergo lytic infection and in some patients this may result in mild anaemia or thrombocytopenia.

 

Diagnosis

The high seropositive incidence for CMV implies that CMV disease can only be diagnosed after taking into consideration a patient’s clinical history and symptoms, which may include fever and inflammation of various organs such as liver, lungs, colon and brain. The presence of CMV-specific IgM suggests a recent infection or reactivation while consistent IgG elevation over two to four weeks indicates a persistent infection. However, to confirm CMV disease the viral load has to be established because it is higher in patients with symptomatic CMV disease. Quantification of viral DNA can be done with PCR and a standardized test is available (2, 3).

Figure 1. Adaptive immune response to viral infections

Literature

  1. Staras et al. (2006): Seroprevalence of cytomegalovirus infection in the United States, 1988-1994. Clin Infect Dis 43(9): 1143-1151
  2. Kraft et al. (2012): Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin Infect Dis 54(12): 1793-1797
  3. Caliendo (2013): The long road toward standardization of viral load testing for cytomegalovirus. Clin Infect Dis 56(3): 374-375

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Correction:

The ‘Abn Lympho?’ flag printed on the calendar is not correct. The ‘Atypical Lympho?’ flag was triggered by the analyser. Sorry for the confusion.

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