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Published on: March 7, 2013
The use of archival bone marrow specimens in detecting B-cell non-Hodgkin's lymphomas using polymerase chain reaction
1Division of Pathology, Clinical Laboratory Nagoya University Hospital, Japan.
Insights
Archival bone marrow tissues can detect B-cell non-Hodgkin's lymphoma (B-NHL) using PCR. Formalin-fixed tissues and aspirate smears are valuable DNA sources, improving diagnostic sensitivity for B-NHL bone marrow involvement.
Area of Science:
- Hematology
- Molecular Diagnostics
- Oncology
Background:
- Bone marrow (BM) involvement is critical in staging B-cell non-Hodgkin's lymphoma (B-NHL).
- Detecting B-NHL in BM often utilizes polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH/JH) gene rearrangement.
- Fresh BM aspirates are standard, but the utility of archival BM specimens requires further evaluation.
Purpose of the Study:
- To assess the efficacy of archival bone marrow specimens for molecular detection of B-NHL.
- To compare different fixation methods (B5 vs. formalin) and specimen types (trephine biopsy, aspirate clots, aspirate smears) for DNA extraction and PCR analysis.
- To determine if archival tissues can enhance the overall sensitivity of B-NHL detection in bone marrow.
Main Methods:
- Studied bone marrow from 13 patients with nodal B-NHL.
- Categorized cases based on histology and fresh BM PCR results.
- Analyzed archival tissues including paraffin-embedded trephine biopsy (TB), aspirate clots (AC), and air-dried aspirate smears (AS) using PCR for IgH/JH gene rearrangement.
- Compared DNA yield and PCR results from B5-fixed and formalin-fixed tissues.
Main Results:
- B5-fixed tissues (TB and subset of AC) failed to yield analyzable DNA.
- Formalin-fixed AC provided sufficient DNA in 8/13 cases, with positive PCR results in 2/3 of group 1 and 3/3 of group 2.
- Microdissection of formalin-fixed AC identified additional positive cases.
- Air-dried aspirate smears consistently yielded sufficient DNA, identifying one additional positive case in group 1.
- Overall molecular detection rates were 100% for H(+)/M(+), 75% for H(+)/M(-), and 40% for H(equivocal)/M(-) groups using archival specimens.
Conclusions:
- Archival bone marrow specimens are a valuable source of DNA for molecular detection of B-NHL.
- Formalin fixation is superior to B5 fixation for preserving DNA integrity in archival BM tissues.
- Utilizing archival BM specimens, particularly formalin-fixed aspirate clots and aspirate smears, can improve the overall sensitivity for detecting bone marrow involvement in B-NHL.
Abstract:
The detection of B-cell non-Hodgkin's lymphoma (B-NHL) involving the bone marrow (BM) can be enhanced by assessing immunoglobulin heavy chain (IgH/JH) gene rearrangement using PCR. While the fresh BM aspirate has been the most commonly used specimen, the utility of archival BM tissues has not been extensively evaluated. We studied the BM from 13 patients with nodal B-NHL (7 low-grade and 6 intermediate grade), which were categorized into three groups based on the histologic finding of lymphoma (H) and the presence of a monoclonal IgH/JH band by PCR using fresh BM aspirates (M): (1) H(+)/M(+), 4 cases; (2) H(+)/M(-), 4 cases; and (3) H(equivocal)/M(-), 5 cases. Archival tissues available for study included paraffin-embedded trephine biopsy (TB)/aspirate clots (AC) and air-dried aspirate smears (AS). All TB (13/13) and a subset of AC (5/13) were B5-fixed, and all these tissues failed to yield analyzable DNA. In contrast, sufficient DNA was consistently obtained in AC that were formalin-fixed (8/13). Of these 8 cases, 2/3 of group 1, 3/3 of group 2, and 0/2 of group 3 had a monoclonal IgH band. Using DNA extracted from microdissected lymphoid aggregates morphologically evident in the AC sections, additional positive cases were identified: 1/3 of group 1 and 2/2 of group 3. In those 5 cases that did not have formalin-fixed TB/AC, sufficient DNA was extracted from AS in all cases; one additional positive case was identified in group 1. Overall, 4/4 (100%) of group 1, 3/4 (75%) of group 2, and 2/5 (40%) of group 3 showed molecular evidence of lymphoma. To conclude, archival BM specimens are a useful source of DNA for molecular detection of B-NHL involvement, and formalin appears to be a better fixative than B5. The use of these samples may improve the overall detection sensitivity.
