I Clin Pathol 1996;49:749-754 749 Immunoglobulin light chain mRNA detected by in situ hybridisation in diagnostic fine needle J Clin Pathol: first published as 10.1136/jcp.49.9.749 on 1 September 1996. Downloaded from aspiration cytology specimens C J R Stewart, M A Farquharson, T Kerr, J McCorriston Abstract erative lesions is generally more Aims-To demonstrate expression of im- problematic.' Whereas malignant lymphoma munoglobulin light chain mRNA in diag- may be diagnosed on the basis of routine cyto- nostic fine needle aspiration (FNA) cytol- logical preparations,2 3 in some cases it is not ogy specimens using an in situ possible to distinguish reactive and malignant hybridisation (ISH) technique; and to lymphoid lesions on morphology alone.' 4 As in evaluate ISH in a series of reactive histopathology, special techniques often offer a lymphoid proliferations and malignant useful aid to the assessment of lymphoid lymphomas. proliferations and together with the cytomor- Methods-Forty diagnostic FNA speci- phology permit an accurate diagnosis in most mens showing a lymphoid cell population specimens.57 were examined for immunoglobulin light Immunocytochemistry has been used in chain mRNA expression using ISH. Aspi- cytology specimens mainly to demonstrate T rates were obtained from lymph node (n = and B cell phenotypes, aberrant expression of 34), salivary gland (n = 3), subcutaneous antigens in T cell lymphomas and immu- tissue, thyroid and breast (n = 1 each). noglobulin light chain restriction in B cell The cases included 20 B cell lymphomas, lymphomas.8"- More recently, material ob- five cases of Hodgkin's disease and 15 tained by FNA has been used for genotypic reactive lymphoid proliferations. Com- analysis including Southern blotting and parison with light chain immunoreactivity polymerase chain reaction (PCR) for demon- was made in 36 cases and histological cor- stration of T cell antigen receptor and relation from biopsy material was avail- immunoglobulin gene rearrangements.'3- The able in 24. latter are valuable techniques but are limited Results-Immunoglobulin light chain re- by loss of correlative cell morphology and the striction was demonstrated in 14 of 20 B requirement for relatively high cell yields. http://jcp.bmj.com/ cell lymphomas using ISH and in six of 17 Monoclonal B cell proliferation may also be B cell lymphomas using immunocyto- demonstrated using in situ hybridisation (ISH) chemistry. A polytypic pattern of light for detection of restricted immunoglobulin chain expression was observed in four of light chain mRNA.'6 This technique offers the five cases of Hodgkin's disease with both specificity of genotypic analysis but with techniques, and in 12 of 15 and 11 of 14 preservation of cell morphology. ISH detection reactive lymphoid proliferations using is also unaffected by serum immunoglobulin on September 28, 2021 by guest. Protected copyright. ISH and immunocytochemistry, respec- which produces background staining and diffi- tively. culties in interpretation with some immunocy- Conclusions-The assessment of immu- tochemistry preparations. In this study we have noglobulin light chain expression is a use- examined light chain mRNA expression by ful adjunct to morphology in the diagnosis ISH and immunocytochemistry in a series of of reactive and malignant lymphoid pro- reactive and malignant lymphoid proliferations liferations in FNA specimens. Light chain obtained by FNA. restriction can be shown using either immunocytochemistry or ISH, but the latter is more sensitive in the diagnosis of Methods B cell lymphoma. DIAGNOSTIC CASES (7 Clin Pathol 1996;49:749-754) FNA specimens were obtained from 40 patients attending Glasgow Royal Infirmary Keywords: B cell lymphoma, in situ hybridisation, with clinically palpable lesions. Two or three immunocytochemistry, fine needle aspiration. aspirates were performed by Cytology staff Departments of using 23 or 25 gauge needles and 10 or 20 ml Cytology and syringes. The aspirates were obtained from Pathology, Royal Fine needle aspiration (FNA) cytology offers lymph node (34 cases), salivary gland (three Infirmary, Glasgow major advantages in the initial assessment of cases), subcutaneous tissue, thyroid and breast Correspondence to: patients with lymphadenopathy. The technique (one case each). Direct smears were prepared Dr C J R Stewart, is simple, inexpensive, safe, and is applicable to for routine staining by the Diff-Quik, Department of Pathology, May- Glasgow Royal Infirmary, both inpatient and outpatient settings. A diag- Grunwald-Giemsa or Papanicolaou methods Glasgow G4 OSF. nosis of metastatic carcinoma or melanoma is and needles thereafter rinsed in normal saline. Accepted for publication usually straightforward in lymph node FNA Cytospin preparations from the needle rinses 27 June 1996 specimens but the assessment of lymphoprolif- were used for immunocytochemistry and ISH. 750 Stewart, Farquharson, Kerr, McCorriston The optimum dilution for cytospin prepara- Table 1 Light chain expression using in situ hybridisation was x (ISH) and immunocytochemistry (ICC) tions 150-200 10' cells/ml. J Clin Pathol: first published as 10.1136/jcp.49.9.749 on 1 September 1996. Downloaded from The cytology diagnoses, which were based Monotypic Polytypic both on morphology and the results of special techniques, fell into three main groups: Cytology diagnosis ISH ICC ISH ICC B cell lymphoma 14/17 6/14 0/17 0/14 A: B (n = 20) Suspicious of BCL 0/3 0/3 0/3 0/3 Group cell lymphoma Hodgkin's disease 0/5 0/5 4/5 4/5 Seventeen cases were considered diagnostic of Reactive lymphoid 0/15 0/14 12/15 11/14 B cell lymphoma on the FNA specimen. In proliferations three aspirates the appearances were consid- BCL = B cell lymphoma. ered highly suggestive but not conclusive of B cell lymphoma. Further subclassification of the In addition to light chain analysis, most cases lymphomas was not generally attempted on the were also examined with a panel of T and B basis of the FNA findings. cell antisera for routine diagnostic purposes. The diagnosis of B cell lymphoma was con- These included CD3 (SAPU), CD43, CD20, firmed on subsequent excision biopsy in 14 of CD79, and CD45 (all from Dako). Selected the 20 cases (1 1 of those considered diagnostic cases were also examined for immunoreactivity and all three considered suspicious on FNA). to CD 15 (SAPU) and CD30 (Dako). Four cases considered diagnostic of B cell lymphoma on FNA represented recurrent IN SITU HYBRIDISATION tumour after treatment and did not undergo The ISH technique was similar to that used for re-biopsy subsequently. The remaining two paraffin wax embedded tissue sections. Initial patients were unfit for biopsy for medical studies using aspirates from excised human reasons; however, the diagnosis of lymphoma tonsil had shown that optimum fixation was was consistent with the clinical and radiologi- achieved using neutral buffered formalin, and cal findings. that light chain mRNA could be detected up to The biopsy findings in those 18 patients with seven days after sampling in both fixed and histological correlation were of follicle centre unfixed material. cell lymphoma (seven cases), large B cell Cytospin preparations were prepared on lymphoma (five cases), low grade mucosa asso- silane treated slides and fixed for 24 hours in ciated lymphoid tissue (MALT) lymphoma neutral buffered formalin. The slides were (three cases), and lymphocytic lymphoma, my- washed twice in water and then digested with eloma and monocytoid B cell lymphoma (one 20 gg/ml proteinase K (Sigma, Poole, Dorset, case each). UK) for 30 minutes at 370C. Proteinase activ- Light chain expression was also examined by ity was stopped by washing in 0.2% glycine. immunocytochemistry in 17 cases. Cells were post-fixed with 0.4% paraformalde- hyde in phosphate buffered saline for 20 minutes at 4°C. Prior to applying the probes, http://jcp.bmj.com/ Group B: Hodgkin 's disease (n = 5) slides were washed twice in water followed by Biopsy confirmation of Hodgkin's disease was one wash in industrial alcohol, then air dried. obtained subsequently in four patients, show- All solutions used before hybridisation were ing nodular sclerosis subtype in three and treated with diethylpyrocarbonate. nodular lymphocyte predominance (LP) sub- The probes used were fluorescein isothiocy- type in one. The remaining patient who anate (FITC) labelled deoxyribonucleotide presented with a subarachnoid haemorrhage cocktails to K and X mRNA (Dako). Probes on September 28, 2021 by guest. Protected copyright. was considered medically unfit for biopsy. were diluted 1 in 1 with 30% hybridisation Light chain expression was also examined by buffer (30% formamide BDH, 10% dextran immunocytochemistry in all five cases. sulphate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 5 mM EDTA, 50 mM Tris HCl, pH Group C: reactive lymphoid proliferation (n = 7.5); 20 p1 of diluted probe was applied to the 15) slides, which were then covered with a Biopsy in two patients with persistent and coverslip and incubated overnight at 37°C. clinically suspicious lymphadenopathy con- Coverslips were removed in 2 x SSC (0.3 M firmed the presence of a reactive lymph node. NaCl, 0.03 M sodium citrate) and sequentially The remaining 13 patients had no evidence of washed in 0.1% Triton X/TBS (0.05 M Tris lymphoma on clinical follow up of two to 24 HCI, pH 7.6, 0.15 M NaCl) for 10 minutes (average 14) months. and TBS for five minutes. Thereafter, slides Light chain expression was also examined by were incubated with alkaline phosphatase con- immunocytochemistry in 14 cases. jugated rabbit anti-FITC (Dako) for 30 minutes at room temperature. Detection was IMMUNOCYTOCHEMISTRY achieved using NBT/BCIP (Sigma) as sub- Cytospin preparations for light chain analysis strate and cells were counterstained with 1% were air dried and then fixed in acetone for 10 light green. minutes. The primary antibodies were polyclo- Reactive tonsillar tissue was used as a nal anti-K and anti-X (Dako, High Wycombe, positive control.