Flow cytometry of lymph node aspirate can effectively differentiate the reactive lymphadenitis from the nodal non-Hodgkin lymphoma

The purpose of the present study was to compare the routine cytology, histology, immunohistochemistry and flow cytometry in the diagnosis of nodal lymphoma cases. Thirty five cases of clinically suspected lymphoproliferative disorder were included in this study. After preparation of smears from the fine needle aspirates on the glass slides for cytology, the residual material was processed for flow cytometric immunophenotyping accordingly. Subsequently, histopathology and immunohistochemistry findings of selected cases of lymph node biopsy were correlated to confirm and compare the diagnosis. Flow cytometric immunophenotypes of most of the cases corresponded to the histological and immunohistochemical diagnoses, five cases showed marked shift in diagnosis (e.g. aggressive NK cell leukemia) which were later validated by clinical outcomes. Flow cytometry immunophenotyping had shown high sensitivity (96.4%) and specificity (100%) if we considered both histopathology and immunohistochemistry combined as gold slandered, while in comparison to immunohistochemistry, flow cytometry immunophenotyping had shown 100% sensitivity and specificity. However, despite the improved diagnostic capacity provided by flow cytometry, morphologic analysis obtained from histopathology remains the cornerstone in the diagnosis of lymphoma.


Introduction
Lymphoma is a common malignancy worldwide affecting both children and adults.In Bangladesh, non-Hodgkin lymphoma (NHL) is relatively common and contributing to 7% of total cancer reported 1 as compared to 4% in USA. 2 Nodal lymphoma is rather prevalent in Bangladesh; for instance, lymphoma constitutes about 6% of all the cases of cervical lymphadenopathy persisting for more than three weeks. 3lthough fine needle aspiration cytology can diagnose almost all the cases of reactive lymphadenitis, only up to about 86% cases of NHL can be correctly identified by cytology alone. 4ne needle aspiration cytology is accepted in recurrence lymphoma; primary diagnosis and classification of NHLs are usually made by histological examination and till date, considered to be the gold standard.Worldwide, many centers have reported fine needle aspiration cytology show relatively high sensitivity and specificity in the diagnosis of lymphomas when used in conjunction with other ancillary techniques in last 15 years. 5, 68][9] Even though architectural and morphologic feature continues to be important parameters in World Health Organization's classifycation of hematopoietic and lymphoid tumors, lymphoma diagnosis is now routinely based on combined information from clinical manifestations, histomorphologic features, immunophenotype and genotype. 10,11,12 Fow cytometric immunophenotyping offers many advantages in the analysis of fine needle aspiration materials for lymphoma diagnosis.Moreover, it requires small number of cells and is also suitable for cells already in suspension.It is very sensitive immunophenotyping technique, and can detect aberrant cells even 1/10,000 cells. 13Thus, it almost replaced the classic immunocytochemistry. 14 As in immunocytochemistry only one antigen can be assessed per cell at a time, it is rather time consuming.Large number of specific monoclonal anti-bodies combining four or more fluorochromes can precisely define the cell profile and neoplastic nature of lymphoid cells make FCI is a more sensitive, specific and swift diagnostic tool than is immunocytochemistry.

Materials and Methods
This

Results
Among the 35 cases, 28 were males and 7 were females.The average ages of male and female patients were 47.7 ± 17.7 and 31.9 ± 13.3 years, res-pectively (p = 0.013), while the average age regardless of sex was 44.5 ± 17.9 years.Cervical lymph node was the most frequent (66%) site of sample collection.FNAC diagnosis revealed lymphoproliferative disorder as the most frequent (46%) entity among the samples under study.
Forty three percent cases were diagnosed as non-Hodgkin lymphoma without further lineage-specific subclassification, 17% cases as lymphoma of B cell lineage, 6% cases as lymphoma of B cell lineage, 6% cases as suspected lymphoma and 14% cases as other than lymphoma by histopathology alone.Histopathology reports or required tissue sample of 14% cases were not available.When the histological data were combined with immunohistochemistry, it revealed 34% and 11% cases of non Hodgkin lymphoma with B and T cell lineages, respectively, while 6% cases were still indeterminate regarding lineage.Besides, 9% cases were diagnosed as something other than lymphoma but combined histological and immunohistochemical inference were not available in 40% cases.Finally, flow cytometry subclassified 51% and 32% cases of non-Hodgkin lymphoma into B and T cell lineage, respectively, and left none of the cases non-specific of lineage.17% cases, however, were found to be inconsistent with lymphoma.
It is to be noted that, among the 35 cases 91% cases were primary and 9% cases were recurrent cases of lymphoma.Among the recurrent cases two were Bcell type non-Hodgkin lymphoma and one was Hodgkin lymphoma.All of those three recurrent cases were diagnosed definitely as positive for lymphoma by flow cytometry.
The changes of diagnoses across histopathology only to combine histological and immunohistochemical inference to ultimately flow cytometry found in this study are depicted in the Table IV.The diagnoses of some of the cases, i.e. follicular lymphoma and small lymphocytic lymphoma, remained unchanged across the aforementioned three platforms while radical changes in diagnosis was observed in a few cases, e.g. a case of non-Hodgkin lymphoma diagnosed initially by histopathology later diagnosed as mixed cellularity classical Hodgkin lymphoma by immunohisto- Proliferative index marker Ki67 Not applicable chemistry; but reverted to angioimmunoblastic T cell lymphoma by flow cytometry (Figure 1).The treatment response validated the diagnosis.Shifts of diagnoses towards gradual subclassification are also seen, i.e. two cases diagnosed as non-Hodgkin lymphoma by histopathology were subcategorized as non-Hodgkin lymphoma of B cell lineage using immunohistochemistry in combinations which were then further specified as diffuse large B cell lymphoma by flow cytometry.

Discussion
The Multiparameter flow cytometric immunophenotyping has significantly enhanced the diagnostic role of FNA, particularly in the case of hematolymphoid malignancies, playing a fundamental role in the differential diagnosis between reactive processes and lymphomas.This study showed that specific and sensitive identification of neoplastic cells, their accurate enumeration, and  a not otherwise specified subclassification can be effectively provided by modern flow cytometry techniques.The ability to gain more information from a smaller amount of tissue, with high-level multicolor FC, should reduce the number of inadequate results on specimens in which the number of cells is a limiting factor, such as fine-needle aspirates.

Evolution of diagnosis across histopathology, immunohistochemistry and flow cytometry
In the case of deep abdominal or intrathoracic lesions, where a surgical biopsy is not possible, the combined use of FNA cytology and FC proved to be a very useful diagnostic tool.
On the other hand, to gain rapid and reliable diagnosis and treatment decisions by the FCI may represent a true cost-effective advantage in developing countries like Bangladesh, where economic resources are limited to include lymphnode biopsy in diagnostic procedures.
Diagnoses variations across different methods are usual due to the varying amounts of informa- Sometimes the change may refer to a subclassification of a broader category while occasionally there are recategorizations. 23This study discuss mainly towards possible explanations and potential implications of the observed shifts of diagnostic categorization across different platforms.
Due to pleomorphic appearance of T lymphocytes at the background, it is not uncommon to misdiagnose T cell lymphomas as Hodgkin lymphoma by histopathology.If the follicular dendritic cell proliferation and prominent high endothelial venules are not typical enough in conjunction with Epstein-Barr virus (EBV) positive immunoblasts masquerading as Reed-Sternberg like cells, angioimmunoblastic T cell lymphoma might mimic mixed cellularity classical Hodgkin lymphoma, as evidenced by two cases of the present study. 24,25 naplastic large cell lymphoma is regarded as another differential diagnosis of angioimmunoblastic T cell lymphoma, exemplified by a case in the present study due to the fact that both might manifest endothelial proliferation. 26, 27Peripheral T cell lymphoma is another mimicker of mixed cellularity classical Hodgkin lymphoma because the former often shows Reed-Sternberg like cells, as seen in at least two cases of the present study. 28It is to note that, the cases which were initially diagnosed as Hodgkin lymphoma by histopatholgy and/or immunohistochemistry but later diagnosed as peripheral T cell lymphoma by FCI, were unresponsive to the conventional ABVD therapy but responded promptly upon the administration of appropriate therapy for peripheral T cell lymphoma in the light of FCI diagnosis.
Mantle cell lymphoma can be difficult to differentiate from small lymphocytic lymphoma because of the vague nodularity and neoplastic small cells shared by both as well as their nearsimilar immunohistochemical profiles where both CD23 and CD10 might show positivity necessitating flow cytometric measurement to determine the cut-off of their expression levels in order to tell the difference. 29.Due to seemingly inconspicuous morphology albeit poor prognosis, it is often impossible to diagnose NK cell lymphoma/leukemia with histopathology alone which was shown in two cases of the present study those required the help of flow cytometry. 30mphomas may also mimic benign conditions like chronic nonspecific lymphadenitis which is not too uncommon in medical literature.Two of the cases in the present study were diagnosed as such at first by later revealed by flow cytometry as peripheral T cell lymphoma and precursor T lymphoblastic lymphoma, respectively.The initial under diagnosis was probably due to the difficulty to distinguish between the morphologies of lymphoma cells and that of lymphocytes with reactive atypia as well as between the effacement of nodal architecture and paracortical hyperplasia of the lymph node which often misleads the pathologists. 31,32  validity of the diagnoses provided by flow cytometry, especially in cases where Hodgkin lymphomas were reclassified as non-Hodgkin lymphoma and cases of chronic nonspecific lymphadenitis being recategorized as T cell lymphoma, were assured by the improved treatment response after the therapies were reshaped and redesigned in the light of the new diagnoses.Unfortunately, one of the cases which were recategorized by flow cytometry as aggressive NK cell lymphoma died shortly after, probably highlighting the higher diagnostic utility of the method.These represent the importance of incorporating flow cytometry as an integral part of frontline lymphoma diagnostics.
The discussion so far emphasizes the need to consider appropriate differential diagnosis of lymphoma and highlights the necessity of advanced immunophenotyping methods like flow cytometry.While conventional morphological methods like histopathology and immunohistochemistry remain at the core of the lymphoma diagnostic array, flow cytometry as an adjunct offers the required accuracy and precision to help achieve a higher degree of favorable outcome.

Conclusion
Flow cytometry immunophenotyping (FCI) is a sensitive and specific method in diagnosis and classification of NHL as well as in detection of monoclonality.FCI also can effectively differentiate reactive lymphadenitis from nodal NHL.The result of FNAC in combination with FCI is effective in the diagnosis of NHL with complete subclassification.We can avoid expensive surgical biopsies in majority of cases.
Date: 23.3.2016).Written and informed consent was taken from each of the study subjects or their lawful guardians (wherever appropriate) for publishing the study.

Figure 1 :
Figure 1: FCI scatter plots of the lymph node aspirate from a representative case angioimmunoblastic T-cell lymphoma

Bishnu Pada Dey, Saumitra Chakravarty and Mohammed Kamal
Copyright:The opyright of this arti le is retai ted y the author s [Atri utio CC-By .]Availaleat: .aglajol.ifoA Jour al of Ba ga a dhu Sheikh Muji Medi al U i ersity, Dhaka, Ba gladeshFlow cytometry of lymph node aspirate can effectively differentiate the reactive lymphadenitis from the nodal non-Hodgkin lymphoma

Table II Immunophenotypic criteria for classification of B-cell lymphoma
* K/L: kappa/ lambda light-chain ratio; ω LH: lymphocytic/histiocytic cell; RS: ¢ Reed-Strenberg cellTable IIIImmunophenotypic criteria for classification of T-cell lymphoma