Diagnostic concordance of pathological methods and reports of hematopathologists compared to local nonspecialized pathologists in the diagnosis of lymphoma in Mexico.

OBJECTIVES: To assess the concordance between lymphoma diagnoses made via tissue biopsy by local pathologists and also to assess the after review of these specimens by more specialized hematopathologists. METHODS: A prospective, non-interventional and multicenter study was conducted at seven sites in Mexico from January 2017 to October 2017. Eligible biopsies were sampled from patients with a previous diagnosis of lymphoma on lymph node biopsy or a diagnosis of extranodal lymphoma, with adequate amount and tissue preservation for the review analysis. The biopsy tissues reviewed by local pathologists were also reviewed by hematopathologists participating in the study. The concordance in diagnosis results was classified into three categories: diagnostic agreement, minor discrepancy and major discrepancy. RESULTS: Out of 111 samples received, 105 samples met the eligibility criteria and were included for full analysis. The median patient age (range) was 54 (16-94) years. A diagnostic agreement was observed in 23 (21.9%) biopsies, minor discrepancies were observed in 32 (30.5%) biopsies and major discrepancies were observed in 50 (47.6%) biopsies. Diagnostic concordance varied across the seven study sites; the rate of major discrepancies ranged from 0% to 100% and the rate of diagnostic agreement ranged from 0% to 81.8%. Out of the 105 reviewed biopsies, a total of 89 cases were diagnosed as lymphoma by hematopathologists. CONCLUSIONS: This study showed that major discrepancies were observed following the review by hematopathologists compared with that of the local pathologist's initial diagnosis in nearly one-half cases. In addition, there was a wide variation in the percentage of diagnostic agreements and discrepancies among different study sites.

Comparative expression of a set of genes to an internal housekeeping control in CDNA amplified and not amplified by PolyAPCR in non-Hodgkin's lymphoma samples obtained from fine-needle aspiration cytology.

AIM: We aimed to evaluate the amount and quality of the RNA obtained from lymph nodes of non-Hodgkin lymphomas (NHLs) patients using fine-needle aspiration cytology (FNAC), and to develop strategies to overcome eventual technical drawbacks. MATERIALS AND METHODS: Twenty-six patients with NHL and 10 tonsils from children submitted to tonsillectomy underwent FNAC. The aspirates were performed using both cytoaspirator (sample A) and syringe and needle (sample B). The RNA was extracted using Trizol reagent and transcribed with the Superscript kit (Invitrogen). The quality of RNA was verified through the amplification of a beta-actin 155-bp fragment. RESULTS: Fifty-two NHL and 20 tonsil samples were analyzed. The total amount of RNA in the tonsil samples varied from <1.0 to 6.2 microg with cytoaspirator (A) and from <1.0 to 4.7 microg with syringe and needle (B). The total amount of RNA obtained from NHL varied from <1.0 to 6.5 microg with cytoaspirator (A) and <1.0 to 5.5 microg with syringe and needle. In an attempt to increase the amounts of RNA in each sample, we standardized the polyAPCR technique, which increased by 10 times the amount of cDNA in most of the test and control samples. The efficiency of the reaction was verified through the amplification of beta-actin, in which 100% of the test and control samples were amplified. When polyAPCR cDNA and nonamplified cDNA samples were paired to be evaluated by real-time PCR, using glyceraldehyde-3-phosphate dehydrogenase as the constitutive gene and nuclear factor-kappa B and NFkappaBIA as target genes, there was equivalence in the amplifications of 100% of the 15 evaluated samples. CONCLUSIONS: Our results showed that FNAC, obtained either by cytoaspirator or syringe and needle, is a good source of small amounts of RNA. The polyAPCR technique significantly increased the amount of genomic material, which might be a cDNA source for future gene expression studies.

Evaluation of quantitative rt-PCR using nonamplified and amplified RNA.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.