Correlation between flow cytometry and histologic findings : ten year experience in the investigation of lymphoproliferative diseases

objective: To demonstrate the advantages of correlating flow cytometry immunophenotyping with the pathology/ Trabalho realizado no Hospital Israelita Albert Einstein HIAE São Paulo (SP), Brasil. 1 Anatomia Patológica, Departamento de Patologia Clínica, Hospital Israelita Albert Einstein HIAE São Paulo (SP), Brasil. 2 Hematologia, Departamento de Patologia Clínica, Hospital Israelita Albert Einstein HIAE São Paulo (SP), Brasil. 3 Departamento de Patologia Clínica, Hospital Israelita Albert Einstein HIAE São Paulo (SP), Brasil. 4 Departamento de Imagem, Hospital Israelita Albert Einstein HIAE São Paulo (SP), Brasil. 5 Setor de Citometria de Fluxo, Departamento de Patologia Clínica, Hospital Israelita Albert Einstein – HIAE São Paulo (SP), Brasil. Autor correspondente: Nydia Strachman Bacal Setor de Citometria de Fluxo, Departamento de Patologia Clínica, Hospital Israelita Albert Einstein – HIAE – Avenida Albert Einstein, 627/701 Morumbi CEP 05651-901 São Paulo (SP), Brasil e-mail: nsbacal@einstein.br Data de submissão: 29/3/2011 Data de aceite: 20/5/2011 Conflito de interesse: nenhum

Descritores: Linfoma; Transtornos linfoproliferativos/diagnóstico; Imunofenotipagem; Citometria de fluxo; Imunoistoquímica intrODUctiOn Lymph node neoplasms often affect lymph nodes, the spleen, mucosa-associated lymphoid tissues, the skin, or non-lymphoid solid organs, resulting in tumors and enlarged organs.The diagnosis of lymphoproliferative diseases is made by pathology and immunohistochemistry of lymph nodes, which are the gold standard.These approaches demonstrate tissue structure and the classification of the type of lymphoma.These procedures, however, are time consuming and invasive; furthermore, some tissues are difficult to access.
Fine needle aspiration biopsy (FNAB) is often the first investigation (screening) for the differential diagnosis among benign/reactional and malignant tumors in cases of enlarged lymph nodes.It is a fast, simple, safe, and only slightly invasive technique for gathering well-preserved cells for studies.
FNAB may be used not only in tissue biopsies for pathology, but also as a technique for obtaining tissue specimens for other studies, including flow cytometry immunophenotyping, (1,2) because of its high positive predictive value.Several studies have been proposed for establishing the reliability of FNAB in the diagnosis of lymphoproliferative diseases (3)(4)(5)(6)(7)(8)(9) (Chart 1).
Immunological marker analysis of lymph nodes based on flow cytometry immunophenotyping has progressed rapidly in the last few decades, going from restricted use in research to routine use in laboratory diagnoses.This has become possible mainly by the wide availability of flow cytometers and the significant amount of monoclonal antibodies for rapidly detecting membrane and intracellular antigens in different cell suspensions (10,11) .
The immunophenotyping study of lymphoproliferative processes is used for distinguishing benign reactions and malignancies; it identifies monoclonality -mainly of B cellswhere there is restriction of one of the light immunoglobulin chains (Figure 1).Besides diagnosis, flow cytometry immunophenotyping is also applied for classifying the types and subtypes of lymphoproliferative diseases (12)(13)(14) .
The current classification of lymphoproliferative diseases (World Health Organization) (14) emphasizes histologic, clinical, cytologic, immunophenotypic, and genotypic aspects for diagnosing and defining the prognosis of lymphoproliferative diseases.Thus, new highly specific markers are described on an ongoing basis to improve the diagnosis and to yield information about the prognosis of these diseases; monoclonal antibody panels may include these new markers depending of the needs of clinical investigation (15)(16)(17) .
High quality smears are useful, since a differential diagnosis may be made based on the nature of cells (monomorphic; polymorphic; small, medium or large the diagnosis.A κkappa/λlambda proportion below 0.5 or over 3.0 suggests the presence of a clonal B cell population in peripheral blood, bone marrow, lymph nodes, the spleen, or other tissues with larger numbers of mature B lymphocytes (18) .

OBJectiVe
The purpose of this study was to demonstrate the advantages of correlating flow cytometry immunophenotyping and pathology/immunohistochemistry of enlarged lymph nodes and/or nodules in the diagnosis of lymphoproliferative diseases.

MetHODS
A retrospective study was made of 157 biopsy or fine needle aspiration specimens of lymph nodes or nodules obtained from 142 patients from 1999 to 2009; the specimens were sent simultaneously to the Flow Cytometry Unit and the Pathology Unit of the Israelita Albert Einstein Hospital, São Paulo, SP.

Pathology/immunohistochemistry and cytology
Biopsies or FNAB of lymph nodes or nodules were done in all patients for histologic and immunohistochemical diagnosis.The hematologic and laboratory routine practices were not altered or interfered with for this study.
Pathologists at our hospital usually have three moments to evaluate fresh tissue samples: during ultrasonography for FNABs, during computed tomography for guided needle biopsies, and during intraoperative freeze sections in the surgical theater.
Pathologists promptly examined FNABs specimens of lymph node/masses to establish cell representativeness; before tissue fixation of the specimens in 95% alcohol), the material was transferred to a tube containing ethylenediaminetetraacetic acid (EDTA) and 2 mL of a RPMI culture medium (RPMI 1640, developed at the Roswell Park Memorial Institute).Flow cytometry immunophenotyping took place within 6 hours of obtaining the specimen, which precluded the need for fixation.
For the biopsies, pathologists selected representative samples of fresh specimens for tissue fixation (10% formaldehyde).The specimens were then transferred to a tube containing EDTA, and flow cytometry immunophenotyping was done similarly to the FNAB cases.
In the pathology laboratory, smears and cell centrifugates of FNAB specimens were routinely prepared sized).Attention should be given to significantly hemodiluted materials, because proliferative cases may be mistakenly diagnosed as reactional.
The immediate morphological evaluation of specimens after fine needle aspiration may lead to a second FNAB or to a lymph node biopsy to obtain a specimen with more adequate cellularity (1,6,16,18,19) .
B cells comprise about 40% and T cells comprise about 55% of normal lymph nodes.The subtype CD4 predominates among CD3+ cells, and the CD4/CD8 ratio is over 4. The frequency of natural killer cells in normal lymph nodes is very low (about 1%).On the other hand, the tonsils are lymphoid organs in which B cells (CD19+) predominate; the remaining cells are CD3+ with a predominance of the CD4 subtype, as in lymph nodes (20,21) .
B cell lymphomas are the majority among non-Hodgkin lymphomas; in such cases, establishing cell clonicity -by restriction of one of the κ (kappa) or λ (lambda) light chains -is generally the key to define Source: Laboratory of Special Techniques, Flow Cytometry Unit, Hospital Israelita Albert Einstein for cytology; the Papanicolaou and Giemsa fixation were used.Biopsies went through routine histologic preparation and the slides were hematoxylin-eosin stained.
Immunohistochemistry consisted of placing the specimens on glass slides previously prepared with a poly-D-Lysine adhesive (Sigma, St. Louis, MO, US, code P7886) and kept in an oven at 60 o C for 4 hours.Deparaffining was done with repeated xylol baths, absolute ethyl alcohol, and washing with a buffered saline solution, a phosphate buffer solution (PBS), and blockage of endogenous activity with a 3% H2O2 solution.Antigenic recovery was attained by heat or the enzyme method.After recovery of the epitopes, the slides were incubated with the primary antibodies for 12 to 18 hours at 4 o C at appropriate dilutions for each antibody.The slides were then washed again with PBS and incubated for 60 minutes with the respective secondary antibodies.Polymer detection systems were then applied.The slides were processed by a treatment with 3,3'-diaminobenzidine (DAB, Sigma, St. Louis, MO, US, code.D5637), H2O2 (final concentration = 0.2%), Mayer hematoxylin counterstained, and mounted with histologic resin.Pathologists evaluated all assays with common microscopy; the immunohistochemical reaction controls were positive.
Pathologists carried out the final histologic evaluation under a common light microscope, based on the 2001 and 2008 tumor classification systems of the World Health Organization (WHO), as recommended in the literature (14) .

Flow cytometry immunophenotyping
FNAB samples were placed in a collecting medium (Vitrocell), and lymph node/mass samples were imbibed in a saline solution or a collecting medium (RPMI, Vitrocell).Cells were first counted in a Neubauer chamber.The slides were prepared in Cytospin and colored with a Rosenfeld dye for cytomorphology.The 7-AAD cell viability assay was used.After the morphologic analysis, the specimens were pipette in 12 x 75 mm tubes depending on the sample volume and the number of cells.The specimens were then PBS (phosphate buffer) washed before marking with monoclonal antibodies.These were obtained from several manufacturers: Beckman Coulter (BC), Becton Dickinson (BD), IQ Products (IQP), Immunotech (IM).
After marking with monoclonal antibodies, cells were incubated during 15 minutes at room temperature and away from light.A hemolytic buffer (ammonium chloride) was applied during 15 minutes at room temperature for lysis of red blood cells.
Specimens were washed three times with PBS and fetal bovine serum, and incubated during one hour in a water bath at 37 o C for surface marking into light and heavy chain immunoglobulins.The IntraPrep kit (Beckman Coulter) was used for intracytoplasmatic marking.Data gathering and analysis was done in an EPICS XL-MCL and FC-500 (Beckman Coulter) flow cytometer.Analyses were interpreted based on the resulting histograms together with cytomorphology of the specimens, according to the tumor classification system of the WHO( 2001 and 2008) or others recommended previously in the literature (14) .
The combined results of lymph node analysis by FNAB cytology and pathology/immunohistochemistry were compared with the results of flow cytometry immunophenotyping.Sensitivity and specificity were used as parameters for assessing the performance of flow cytometry immunophenotyping relative to pathology (the gold standard).
Sensitivity was calculated to assess the proportion of diseases subjects that tested positive, and specificity was calculated to assess the proportion of disease-free subjects that tested negative.The positive predictive value was calculated to assess the probability of a subject having the disease when tested positive, and the negative predictive values was calculated to assess the probability of a subject not having the disease when tested negative.

reSUltS
There were 157 specimens of 142 patients during the period from 1999 to 2009, of which 75 were male and 67 were female; the mean age was 55 years (ranging from 4 to 92 years).
The procedures for obtaining the specimens consisted of biopsies in 119 patients, FNAB in 16 patients, and FNAB followed by biopsy in 7 patients.
The sites for 145 lymph node specimens were the neck, inguinal, axillary, mediastinal, peripancreatic, paraaortic, and juxtacarotid regions; the sites of 12 tumor mass samples were the spleen, kidney, small intestine, lung, ischium, parotid, scalp, and nasopharynx.There were more than one specimens in 12 patients because of different procedures (for instance, FNAB specimens followed by biopsy specimens), different sites obtained at the same time, different years in a single patient, or duplication of specimens (Table 1).
To investigate the efficacy of associating flow cytometry immunophenotyping with pathology for accurate diagnoses, we assessed the agreement percentage between the two techniques for each disease group in the study (Figure 2).The 142 study patients were classified according to the diagnosis of the disease (Table 1).The bars show the percentage of agreement in diagnosis by each technique (Figure 2).

Pathological diagnosis number of patients
Interestingly, agreement was above 80% in 9 of 11 disease groups; it was above 70% in one group.Only the diagnosis of Hodgkin's lymphoma (LH) was mostly discordant, which has been predicted in the literature, as will be discussed below.
Figure 3 shows the diagnoses of patients according to pathology.

DiScUSSiOn
The traditional technique of choice for diagnosing lymph node diseases has been histopathology of paraffi n-included tissues.Immunohistochemistry is an important tool for analyzing biopsies of lymph nodes and other tissues; cell morphology and tissues architecture are preserved, and immunophenotypic analysis of histological sections are possible (23,24) .Detecting specifi c antigens in lymphoid cells is fundamental for classifying tumors, assessing the outcome, and identifying specifi c targeted therapy (13,14) .However, pathology of immunohistochemical results in routine laboratory work has its limits: the analysis may be subjective, reproducibility is limited, and the process is time-consuming.Inter-and intra-observer variability is high because so many factors may interfere with the processing of specimens and interpretation of results.Thus, lack of consensus in quantifying antigen expression and defi ning positive and negative results in poor reproducibility.Cytomorphological assays and fl ow cytometry immunophenotyping overcome some of these hurdles by providing faster diagnoses, quantitative and qualitative analysis of cell antigens, and multiparametric analyses (2,7,17,18) .However, fl ow cytometry also has its limits: variability in antigenic signature expression; loss of cells during the pre-analytical process; specimen preparation issues; work with fresh specimens; and availability of suffi cient neoplastic cells.Large cell lymphoma cells may be lost in the preparation process because of cell frailty.According to the literature, a negative result does not exclude malignancy (11,18,19,23) .
FNAB is a minimally invasive procedure for which cytomorphological analysis combined with fl ow cytometry immunophenotyping are important tools, which are able to rapidly differentiate lymphoproliferative diseases from reactional lymphoid hyperplasia in most cases of enlarged lymph nodes (20,23,25) .
In our study, the diagnosis by fl ow cytometry immunophenotyping and pathology differed in 27 cases; this occurred more often when diagnosing Hodgkin's lymphoma in 14 patients out of 15 patients with positive pathology for this disease (51.85% of the total number of discordant cases), in 17 specimens (56.7%).
Although fl ow cytometry is useful for diagnosing several hematopoetic neoplasms, and may often detect small cell populations (< 0.01% of leukocytes), it is a limited technique for the diagnosis of Hodgkin's lymphoma involving lymph nodes.Many studies on fl ow cytometry immunophenotyping in Hodgkin's lymphoma have shown changes in reactional lymphocytes, such as the CD4/CD8 ratio in T cells; however, this technique fails to detect Reed Sternberg cells, especially because of their large volume (26) .In 2009, Wood described a highly sensitive and specifi c technique based on fl ow cytometry using nine colors and three lasers for diagnosing classic Hodgkin's lymphoma (27) .
Flow cytometry failed to diagnose non-Hodgkin's B lymphoma in ten patients of our sample.In these cases, there was partial distribution of anomalous cells in lymphoid tissues in two patients, which affected neoplastic cell representation.In two cases, the fi nal diagnosis was large B cell diffuse lymphoma rich in T cells/histiocytes, where those few detected

a B
neoplastic cells are spread out in a rich background of T lymphocytes and histiocytes; furthermore, the cells are large and more fragile compared to other lymphocytes.Thus, they are not well represented, which may mask flow cytometry analysis.Meda et al. (28) and Verstovsek et al .
(1) also reported this finding.They are often wrongly characterized as a polyclonal population because of significant contamination by residual normal cells.Therefore, a flow cytometry immunophenotyping result showing no evidence of malignancy did not exclude a cancer; in these situations, a detailed cytomorphological exam is required, as demonstrated in the literature (9,29) .According to Meda et al. (28) and Zardawi et al., (18) if κ and λ light chains are not restricted, further evidence of clonal proliferation may be investigated, such as major antigen proliferation (CD19, CD20) in specific tissues (over 85%), CD10 ≥> 18% or CD20+CD5+ ≥> 35%.
Among other studies, Martins et al. (7) conducted a retrospective study of 627 lymph node FNAB specimens and underlined the importance of cytomorphological analysis for the diagnosis of large cell non-Hodgkin's lymphoma.
In three of our patients, the specimens consisted of necrotic material, which was frail or sparse for flow cytometry immunophenotyping; the specimen with sparse material was obtained in a bone biopsy, and was not representative of the neoplasm.Representativeness of malignancy was lost in three patients, and flow cytometry immunophenotyping diagnosis was not possible (in one, the PCR technique for B clonality was used to supplement the study); furthermore no distinct representative IgH loci monoclonal rearrangement band was found, which demonstrated the paucity of cells in the specimen.The presence of necrosis, accelerated tumor growth, and bone, affected pre-analytical processing, thereby interfering with cell viability for analysis in the cytometer; this again has been reported in the literature (19,23) .
In T cell lymphomas, cell clonality may only be characterized if antigenic expression of a T lineage marker is absent.A demonstration of T clonality may be done using PCR, the Southern Blot molecular biology technique, or flow cytometry for clonality detection in the Vβ family with simultaneous analysis of more than 20 monoclonal antibodies -a technique that is not available in Brazil.
In our study, flow cytometry was more specific than sensitive in diagnostic agreement, a result of poor agreement in patients with Hodgkin's lymphoma, (14,15) and in ten patients with large cell diffuse lymphoma, as described previously (10) .These factors also affected the global positive predictive value.
Immunohistochemistry is an important tool in biopsies of lymph nodes and other tissues; it is possible to analyze the immunophenotype of histological sections where identification of the tissue architecture is also possible (30) .For example, identifying CD5 antigen expression in some lymphomas, such as between the small cell lymphocytic lymphoma/chronic lymphocytic leukemia (LL/CLL) and the mantle cell lymphoma (ML), which are two types of lymphoproliferative diseases that progress differently, thereby requiring a correct definition.These cases require investigating and charactering the D1 cyclin marker, which is found in 70 to 80% of mantle cell lymphoma cases (15) -a technique that is done successfully only with immunohistochemistry. Detection is also possible of the typical t(11:14) of the mantle cell lymphoma in classic cytogenetics, fluorescent in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR).
In mature B cell lymphomas, the main differential diagnosis to be made among those that are positive for the CD10 marker is between the follicular lymphoma, the large cell diffuse lymphoma, and Burkitt's lymphoma.Demonstrating histologically the Bcl-2 marker helps identify neoplastic follicles in follicular lymphomas and differentiate them from reactional follicles in follicular lymphoid hyperplasia.Identifying Bcl-2 is difficult in flow cytometry, which however may differentiate follicular lymphomas from lymphoid hyperplasia by testing clonality in the κ and λ ratio (16,21,31) .
Diagnostic centers that currently provide these technologies as supplementary diagnostic tools reduce the limitations of each method, add speed, and further choices for staging and defining the most appropriate treatment.

cOnclUSiOn
We have been able to show that in several situations, for many hematologic diseases, flow cytometry associated with cytomorphology and immunohistochemistry made it possible to diagnose and differentiate reactional processes from neoplasms, and to subclassify lymphoproliferative diseases.
In our experience with the majority of suspected cases of lymphoproliferative diseases, flow cytometry data supplemented the findings of cytomorphology, immunohistochemistry (FNABs) and biopsy specimens.

Figure 1 .
Figure 1.Flow cytometry analysis of axillary lymphadenomegaly of patient with follicular lymphoma.

Figure 2 .
Figure 2. Percentage of diagnosis with agreement in pathological examination and flow cytometry immunophenotyping per studied group.

LFigure 3 .
Figure 3. Distribution of diagnosis agreement in percentage of patients per subtype of lymphoma.Source: Department of Pathology -Hospital Israelita Albert Einstein

table 1 .
List of patients per pathological diagnosis and types of specimen collection