Factors determining whether diffuse large B-cell lymphoma samples are detected by flow cytometry.

INTRODUCTION: Flow cytometry (FCM) is widely used in the diagnosis of mature B-cell neoplasms (MBN), and FCM data are usually consistent with morphological findings. However, diffuse large B-cell lymphoma (DLBCL), a common MBN, is sometimes not detected by FCM. This study aimed to explore factors that increase the likelihood of failure to detect DLBCL by FCM. METHODS: Cases with a final diagnosis of DLBCL that were analysed by eight-colour FCM were retrospectively collated. Clinical, FCM, histopathological and genetic data were compared between cases detected and cases not detected by FCM. RESULTS: DLBCL cases from 135 different patients were analysed, of which 22 (16%) were not detected by FCM. In samples not detected by flow cytometry, lymphocytes were a lower percentage of total events (p = 0.02), and T cells were a higher percentage of total lymphocytes (p = 0.01). Cases with high MYC protein expression on immunohistochemistry were less likely to be missed by FCM (p = 0.011). Detection of DLBCL was not different between germinal centre B-cell (GCB) and non-GCB subtypes, not significantly affected by the presence of necrosis or fibrosis, and not significantly different between biopsy specimens compared to fine-needle aspirates, or between samples from nodal compared to extranodal tissue. CONCLUSION: The study identifies several factors which affect the likelihood of DLBCL being missed by FCM. Even with eight-colour analysis, FCM fails to detect numerous cases of DLBCL.

Comparison of flow cytometry with other modalities in the diagnosis of myelodysplastic syndrome.

INTRODUCTION: The myelodysplastic syndromes (MDSs) are heterogeneous myeloid malignancies, conventionally diagnosed by cytomorphology and cytogenetics, with an emerging role for flow cytometry. This study compared the performance of a 4-parameter flow cytometry scoring system, the Ogata Score, with other modalities in the diagnosis of MDS. METHODS: Bone marrow aspirate and trephine biopsies from 238 patients performed to assess for possible MDS were analysed, and the flow cytometry score was retrospectively applied. The sensitivity and specificity of the flow cytometry score, the aspirate microscopy, the trephine microscopy with immunohistochemistry, and cytogenetic and molecular results were determined relative to the final diagnosis. RESULTS: The medical records of the 238 patients were reviewed to determine the final clinical diagnosis made at the time of the bone marrow examination. This final diagnosis of MDS, possible MDS or not MDS, was based on clinical features and laboratory tests, including all parameters of the bone marrow investigation, except for the flow cytometry score, which was only determined for this study. The flow cytometry score was 67.4% sensitive and 93.8% specific. Aspirate microscopy had higher sensitivity (83.7%) and similar specificity (92.0%), whereas trephine microscopy had similar sensitivity (66.3%) and specificity (89.4%) to flow cytometry. Although the flow cytometry score had a lower sensitivity than aspirate microscopy, in 18 patients (7.6% of the total) the flow cytometry score was positive for MDS, whereas aspirate microscopy was negative or inconclusive. CONCLUSION: The flow cytometry score and trephine microscopy exhibited reasonable sensitivity and high specificity, and complement aspirate microscopy in the assessment of MDS.

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

BACKGROUND: There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. METHODS: A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. RESULTS: FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. CONCLUSIONS: FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society.

Absence of simian virus 40 (SV40) DNA in lymphoma samples from Tasmania, Australia.

AIMS: It has been postulated that the recent world-wide increase in the incidence of non-Hodgkin's lymphoma (NHL) may have been caused by human infection with simian virus 40 (SV40) (a lymphotropic monkey virus that was introduced to man from contaminated poliovirus vaccines between 1955 and 1963); therefore, we set out to determine the incidence of SV40 DNA positivity in lymphoma samples from patients in Tasmania, Australia. METHODS: One hundred lymph node samples, 50 from patients with lymphomas and 50 from controls, were tested using PCR amplification of three SV40-specific primer pairs followed by dot-blot hybridisation. RESULTS: All of the samples tested contained amplifiable DNA, but none contained amplifiable SV40 sequences with any of the primer sets used. CONCLUSIONS: Our results demonstrate absence of SV40 in the lymphoid tissues of our study population in Tasmania, Australia. SV40 does not explain the increasing incidence of NHL in our population.

One in four patients with follicular thyroid cytology (THY3) has a thyroid carcinoma.

BACKGROUND: Fine-needle aspiration (FNA) biopsy is the cornerstone of assessment of thyroid nodules. Cytological criteria for benign (THY2) and malignant (THY5) aspirates are well established and reliable. When cytology suggests a follicular neoplasm (THY3), only formal histological assessment can differentiate between benign and malignant lesions. The objective of this study was to determine the factors predictive of malignancy in thyroid nodules when cytological assessment is restricted to euthyroid patients living in an area without endemic goiter who undergo routine diagnostic lobectomy once the FNA raises the suspicion of a follicular neoplasm. METHOD: Retrospective review of histological and clinical data in a cohort of patients with a palpable thyroid nodule and THY3 cytology. RESULTS: Between January 2000 and December 2007, 1981 patients (346 males and 1635 females) underwent 2809 thyroid FNAs. There were 201 THY3 reports (9%). Histology demonstrated thyroid carcinomas in 57 patients (31 follicular carcinomas, 11 Hurthle cell carcinomas, 11 papillary carcinomas, 1 medullary thyroid carcinoma, 1 poorly differentiated thyroid cancer, 1 lymphoma, and 1 metastatic renal carcinoma). Benign tumors were found in 144 patients with follicular adenomas (n = 76), Hurthle cell adenomas (n = 33), multinodular goiter (n = 13), adenomatoid nodules (n = 15), colloid nodules (n = 4), and thyroiditis (n = 3). THY3 cytology was more predictive of malignancy in men (13/34 male symbol vs. 44/167 female symbol, p < 0.001, chi(2) test). The risk for malignancy was 1:4 for the entire group and 1:3 for patients under 30 years and over 60 years. About 17/46 nodules over 40 mm in diameter were carcinomas, compared with only 35/140 in nodules under 40 mm (p < 0.01, chi2 test). CONCLUSION: One in four patients with cytological features of a follicular neoplasm has a thyroid carcinoma. A large nodule (>4 cm) with THY3 cytology has a high likelihood of being a cancer, and arguably such patients could be offered total thyroidectomy rather than diagnostic lobectomy.