פוסטרים להצגה בכינוס

Use of Flow Cytometry in Screening and Diagnosis of Neuroblastoma in children - 2 Case Reports

Eti Broide, Natasha Melinkov, Esther Paz and Marc Assous.
Microbiology and Immunology laboratory, Shaare Zedek Medical Center Jerusalem, Israel

Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor in infancy. This accounts for 7-10% of all pediatric malignancies. The clinical spectrum may be diverse and nonspecific, and therefore present a diagnostic challenge. Often the disease is not suspected, and is found on bone marrow evaluation for an unrelated disease. Bone marrow involvement occurs in approximately 40% of patients with neuroblastoma.

Flow cytometry is a quick and useful method for the detection and diagnosis of neuroblastoma cells in the bone marrow despite not being hematopoietic cells.

Neuroblastoma cells are typically negative for CD45 (Swertz, 2004) and express CD56, CD81. Using this combination of markers by flow cytometry, the NB cells can be detected in the bone marrow with a high sensitivity. In addition, the intensity of CD56 on NB cells is very high (Warzynski,2002), enabling it to be used as a screening marker for NB in routine peripheral blood and bone marrow samples being evaluated on the suspicion of other conditions. When high intensity of CD56 is detected on the CD45 negative population, a combination of CD81, CD56 and CD45 can be set up to confirm the diagnosis. Two children suffering from anemia and suspected as suffering from acute leukemia underwent bone marrow evaluation were diagnosed for neuroblastoma incidentally by the above described method.

Methods: Flow cytometry was done on bone marrow specimens according to our protocol for screening of new patients... CD3 , CD4, CD5, CD8,CD10 ,CD13 ,CD14, CD19, CD20,CD33, CD34, CD56 ,CD117,CD 235a,,CD71,HLA-DR, Ka/La ,CD138 were assayed. The cells were stained for 20 minutes, lysed, and then washed with PBS. / fetal calf serum. The cells were then resuspended in PBS/sodium azide, and 100,000 events per tube were acquired by the CantoII flow cytometer. After an aberrant expression of markers on the CD45 negative population was noted, the cells were stained for CD81.

Results: In two routine bone marrow aspirates we detected CD71/CD235a negative cells, which expressed a strong aberrant expression of CD56. In our first case, there was a 30% CD45 negative area, which was only positive for 10% nucleated red blood cells. After analysis of other markers routinely stained, we noted a strong CD56 expression, and restained with CD81 to confirm the presence of neuroblastoma cells. In the second case, over 65 % of the 10% CD45 negative population was positive for CD71/CD235a, which is not abnormal.
However, after looking at all markers which had been stained for the hematopoietic cell population we again noticed CD56 strong expressions. In this case, only 30% of the population expressed CD56, and CD81.
Immunohistochemistry reconfirmed the presence of neuroblastoma cells by positive staining for NSE, chromogranin and synaptophysin.

Discussion: We described two NB patients who were diagnosed by flow cytometry. The pathological population was CD45 negative with a low side scatter. Typically cells which are CD45 negative and have a low side scatter represent nucleated red blood cells, with varying expression of CD 71 and CD235a. In our first case, most of the cells in the CD45 negative area were also CD71/CD235a negative which led to suspicion of an aberrant population. However, in our second case, most of the CD45 negative population were positive for
CD71 and CD235a. The NB cells could easily be missed if this population was not analyzed with the other markers included in the screening panel. These cases emphasize the need for careful analysis of this population .CD56 is usually included in primary immunophenotyping screening panels in most clinical labs, making it an excellent marker for screening for NB in routine bone marrow specimens of children. We also detected CD15 on the NB cells, which has been documented (Pruszak, 2009) as a marker on neural stem cells.
In addition, flow cytometry can be used for sensitive residual testing, with a sensitivity of 1NB cell in 10⁴-10⁵ mononuclear cells, not possible by immunohistochemistry, and for differential diagnosis from other tumors when there is atypical morphology.