Impact of formalin fixation on mismatch repair protein evaluation by immunohistochemistry.
Author(s): Grillo F, Ali M, Paudice M, Pigozzi S, Anselmi G, Scabini S, Sciallero S, Piol N, Mastracci L
Publication: Virchows Arch, 2023, Vol. , Page
PubMed ID: 37773452 PubMed Review Paper? No
Purpose of Paper
This paper compared immunohistochemical staining intensity, patchiness, and occurrence of central artefacts of four mismatch repair proteins among matched formalin-fixed paraffin embedded (FFPE) colorectal carcinoma specimens that were fixed for <20 h (hypofixed), 24-48 h (standard fixed), and >90 h (hyperfixed) at room temperature; in specimens fixed for 24-48 h at 4°C (cold-fixed), and among standard fixed specimens that differed in size (0.5 x 0.5 cm versus 2 x 1 cm). The incidence of inadequate staining, patchiness, and central artefacts was also compared among the four mismatch repair antibodies.
Conclusion of Paper
Compared to standard fixed specimens, hypofixed and hyperfixed specimens both had reduced immunostaining intensity, hypofixed specimens tended to have more patchiness and central artefact staining, and hyperfixed specimens had less patchiness and central artefact staining. Cold fixation (4°C) resulted in stronger staining intensity and less patchiness compared to standard fixed specimens, but the significance of differences in intensity was dependent on the cut-off values applied. Using a smaller-sized specimen led to reduced patchiness and less pronounced central artefacts but did not improve staining intensity. Based on their findings, the authors recommend fixation at 4°C for 24 h. MLH1, followed by PMS2, staining was responsible for the majority of specimens with inadequate staining, patchiness, and central artefact staining.
Studies
-
Study Purpose
This study compared the intensity, patchiness, and occurrence of central artefacts after immunohistochemistry (IHC) analysis of case-matched FFPE colorectal carcinoma specimens that were fixed for <20 h (hypofixed), 24-48 h (standard fixed), and >90 h (hyperfixed) at room temperature; in specimens fixed for 24-48 b at 4°C (cold-fixed); and in standard fixed specimens that differed in size (0.5 x 0.5 cm versus 2 x 1 cm). Leftover colorectal carcinoma specimens from 30 patients (nine female and twenty-one male) were cut into five pieces (four pieces were 2 x 1 cm and one piece was 0.5 x 0.5 cm) and placed in formalin within 30 min of resection. Matched 2 x 1 cm specimens were fixed for <20 h (hypofixation, mean 17.27 h), 24-48 h (standard fixation, mean 31.1 h), and >90 h (hyperfixation, mean 115.73 h) at room temperature and for 24-48 h (mean 26.9 h) at 4°C; the 0.5 x 0.5 cm specimen was fixed at room temperature for 24-48 h. Following fixation, all specimens were dehydrated in 95% ethanol at 4°C before processing using a Leica ASP6025S Processor. Details of block storage and sectioning were not provided. Levels of MLH1, PMS2, MSH2, and MSH6 were quantified by IHC using a BenchMark ULTRA Immunostainer and evaluated based on nuclear staining intensity (scoring system: 1-3), patchiness (percentage of immunostaining), and presence or absence of a central artefact (rim of adequate staining).
Summary of Findings:
Inadequate staining (intensity score of 0) was reported for 46 immunoreactions. Of these 46 specimens with inadequate staining, 47.8% (22) were hypofixed specimens (<20 h), 28.3% (13) were hyperfixed specimens (>90 h), 13.1% (6) were standard fixed specimens (24-48 h), 6.5% (3) were small specimens (0.5 x 0.5 cm), and 4.3% (2) were cold fixed (4°C) specimens. Patchiness (staining in 10-69% of the specimen) was observed in 281 specimens and of these, 29.1% (74) were hypofixed, 24.8% (63) were standard fixed, 18.1% (46) were hyperfixed, 15% (38) were cold-fixed, and the remaining 13% (33) were small specimens. Central artefact staining that was moderate (half of the outermost portion of the section was adequately stained and half of innermost portion was inadequately stained) or marked (staining only occurred in the outer rim with only weak or absent staining in the interior of the specimen) was found in 157 specimens. Of these 157 specimens, 31.8% (48) were hypofixed, 25.9% (39) were standard fixed, 11.9% (28) were hyperfixed specimens, 11.9% (18) were cold-fixed, and 11.9% (18) were small specimens. When IHC staining was evaluated by antibody, MLH1 and PMS2 were responsible for the majority of inadequate staining (45.7% and 34.8% of specimens, respectively), patchiness (33.1% and 31.5% of specimens, respectively), and central artefact staining (34.4% and 27.8% of specimens, respectively). Hypofixed specimens had lower immunostaining intensity (P=0.0013) and a trend to more patchy immunostaining and central artefact staining than standard fixed specimens. Similarly, hyperfixed specimens were found to have reduced staining intensity (P<0.00001), but a reduced incidence of patchy immunostaining and fewer central artefacts in comparison to standard fixed specimens (P=0.046 and P=0.045, respectively). Cold fixation resulted in a stronger staining intensity and less patchiness compared to standard fixed specimens, but the significance of differences in intensity was dependent on the cut-off values applied. Using a smaller size specimen led to reduced patchiness and less pronounced central artefacts (P=0.0008 and P=0.0014, respectively) but did not improve staining intensity.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative <20 h (hypofixed)
24-48 h (standard fixed)
>90 h (hyperfixed)
Immunohistochemistry Specific Targeted peptide/protein MLH1
MSH2
MSH6
PMS2
Biospecimen Aliquots and Components Aliquot size/volume 2 x 1 cm
0.5 x 0.5 cm
Biospecimen Preservation Temperature of fixation/preservation 4°C
Room temperature