A New Medium (HistoCold) for Surgical Specimens Preserving to Improve the Preanalytic Issues in Histopathological Samples Handling: Morphologic and Antigenic Analysis.
Author(s): Mandarano M, Pelliccia C, Tomasello L, Caselli E, Floridi C, Loreti E, Barberini F, Rulli A, Gili A, Potenza R, Puma F, Rosati E, Donini A, Petrina A, Baccari P, Del Sordo R, Colella R, Bellezza G, Sidoni A
Publication: Biopreserv Biobank, 2023, Vol. 21, Page 610-623
PubMed ID: 37192479 PubMed Review Paper? No
Purpose of Paper
This paper compared mitotic counts, histopathological grading scores, and immunohistochemical staining among surgically resected breast, lung, and colorectal carcinoma specimens that experienced a delay to formalin fixation (24-72 h) in the proprietary preservative HistoCold with case-matched formalin-fixed, paraffin-embedded (FFPE) specimens that were routinely processed.
Conclusion of Paper
When routinely processed FFPE carcinoma specimens were compared to case-match specimens that experienced a delay to fixation in HistoCold at 4°C prior to formalin fixation and paraffin embedding, mean mitotic counts were consistently higher in routinely processed FFPE specimens than HistoCold-treated specimens, although mitotic counts were still moderately to strongly correlated between the two specimen types. The agreement in histopathological grading scores between routinely processed FFPE specimens and those that experienced a delay to formalin fixation of 24-72 h in HistoCold ranged between fair and excellent, depending on the tissue and tumor type. The concordance of immunohistochemical staining scores between routinely processed FFPE specimens and those stored in HistoCold prior to formalin fixation ranged from absent to excellent depending on tissue/tumor type, antigen, and the duration of the delay. The authors note that longer delays in HistoCold were associated with an “underestimation” of the parameters examined, particularly with longer delays to fixation.
Studies
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Study Purpose
This study compared mitotic counts and histopathological grading scores of surgically resected carcinoma specimens that were formalin-fixed, paraffin-embedded routinely (no further details provided) with case-matched specimens that experienced a delay to formalin fixation (24-72 h) in the proprietary preservative HistoCold before formalin fixation(4% neutral-buffered formaldehyde) and paraffin-embedding. In total, 20 breast carcinoma and 25 non-small cell lung cancer (NSCLC) surgically resected tumor specimens that had a minimum tumor diameter of 10 mm and colorectal carcinoma (CRC) specimens that demonstrated an invasion of the muscularis propria layer were divided into three samples that were placed in HistoCold in a 1:10 ration of tissue to preservative and stored for 24, 48, or 72 h at 4°C. All samples were then fixed in 4% neutral-buffered formaldehyde in a 1:10 ratio for 24 h. Slide-mounted sections of FFPE blocks were stained with hematoxylin and eosin to determine mitotic count and histopathologic grading. Each HistoCold-treated, formaldehyde-fixed specimen was then compared to the case-matched routinely processed specimen that was used to determine the official diagnosis.
Summary of Findings:
The mean mitotic count in breast cancer specimens was consistently higher in routinely processed FFPE specimens (19.85±16.75) than case-matched specimens treated in HistoCold at 4°C during a 24 (13.90±14.86; ρ=0.799, p< 0.003), 48 (12.25±15.13; ρ=0.863, p< 0.0001), and 72 h (8.9±8.05; ρ=0.702, p< 0.003) delay to formalin fixation. Despite a progressive decline, mitotic counts in HistoCold-treated breast cancer specimens were still strongly (24 h, ρ=0.799, p< 0.003; 48 h, ρ=0.863, p< 0.0001) or moderately (72 h, ρ=0.702, p< 0.003) correlated with those of routinely processed case-matched specimens. Similarly, mean mitotic counts in NSCLC specimens were consistently higher in routinely processed adenocarcinoma (ADC) (6.00±7.46) and squamous cell carcinoma (SQCC) (12.30±5.81) specimens than in case-matched ADC (24 h= 4.53±5.82, 48 h=3.20±4.83, 72 h=3.27±4.25) and SQCC (24 h=9.60±7.06, 48 h=7.60±6.38, 72 h=6.70±5.98) specimens treated with HistoCold during a 24-72 h delay to formalin fixation. Despite these differences, mitotic counts in ADC NSCLC specimens treated with HistoCold for 24 (ρ=0.831, p<0.001), 48 (Rho, ρ=0.789, p=0.002), and 72 h (ρ=0.815, p=0.001) were still strongly correlated with those of routinely processed case-matched ADC specimens. In contrast, significant correlations in mitotic counts of routinely processed SQCC NSCLC specimens with those of SQCC specimens that were treated in HistoCold was only observed for the 24 h timepoint (ρ=0.818, p=0.022). Mitotic counts in colorectal cancer specimens that were routinely processed (24.00±8.92) only displayed a significant correlation to those in specimens that were treated with HistoCold for 48 h (23.90±9.60; ρ=0.840, p=0.014). Histopathological grading scores of routinely processed breast cancer specimens were fairly concordant with those from specimens treated in HistoCold for 24 (κ=0.481), 48 (κ=0.438), and 72 h (κ=0.460). Conversely, grading scores of ADC NSCLC specimens that were routinely processed demonstrated excellent agreement with those of HistoCold-treated specimens for all timepoints (κ=0.872), while agreement in grading scores of SQCC specimens were good or excellent among routinely processed specimens and HistoCold specimens treated for 24 and 48 h (κ=0.697 for both) and 72 h (κ=1), respectively. Among CRC specimens, routinely processed specimens had excellent agreement in grading scores with those treated with HistoCold when all time points were considered together (κ=1).
Biospecimens
Preservative Types
- Other Preservative
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Pre-preservation condition HistoCold at 4°C
No treatment
Biospecimen Acquisition Cold ischemia time 24 h at 4°C
48 h at 4°C
72 h at 4°C
-
Study Purpose
This study compared immunohistochemical staining of surgically resected carcinoma specimens that were formalin-fixed, paraffin-embedded routinely (no further details provided) with case-matched specimens that experienced a delay to formalin fixation (24-72 h) in the proprietary preservative HistoCold before formalin fixation(4% neutral-buffered formaldehyde) and paraffin-embedding. In total, 20 breast carcinoma and 25 non-small cell lung cancer (NSCLC) surgically resected tumor specimens that had a minimum tumor diameter of 10 mm and colorectal carcinoma (CRC) specimens that demonstrated an invasion of the muscularis propria layer were divided into three samples that were placed in HistoCold in a 1:10 ration of tissue to preservative and stored for 24, 48, or 72 h at 4°C. All samples were then fixed in 4% neutral-buffered formaldehyde in a 1:10 ratio for 24 h. Slide-mounted sections of FFPE blocks were immunohistochemically stained (breast: ER, PR, Ki-67, HER2; NSCLC: TTF-1, p63, PD-L1; colorectal: CDX2, MLH1, MSH2, MSH6, PMS2) and evaluated for the percentage of positively-stained tumor cells and the intensity of positive staining (mild, moderate, strong). Each HistoCold-treated, formaldehyde-fixed specimen was then compared to the case-matched routinely processed specimen that was used to determine the official diagnosis.
Summary of Findings:
In breast cancer specimens, in the agreement of immunohistochemical staining scores between routinely processed specimens and those treated with HistoCold were excellent for ER (κ=1 for all timepoints) and excellent or good for PR (24 h, κ=0.875; 48 h, κ=0.761; 72 h, κ=1.00). The percentage of tumor cells that were immunopositive for Ki-67 were similar and strongly and significantly correlated between routinely processed specimens (29.75±17.43) and those treated in HistoCold for 24 (32.40±15.35; ρ=0.890, p<0.001) or 48 h (29.90±16.08; ρ=0.750, p<0.001). However, the agreement between HER-2 scores ranged from good to fair with progressive delays to fixation in HistoCold-treated specimens relative to routinely processed breast cancer specimens (κ=0.712, 0.578, 0.485 for 24, 48, and 72 h, respectively). Antigenicity results were dependent on tumor type and the biomarker evaluated in NSCLC specimens. TTF-1 staining was absent (or lost)) in ADC specimens treated with HistoCold for 24, 48, and 72 h compared to immunopositive staining in routinely processed specimens, but p63 immunostaining was 100% concordant between routinely processed specimens and SQCC specimens that were treated in HistoCold for 24, 48, or 72 h (κ=1). The agreement between PD-L1 immunohistochemical staining of routinely processed specimens and those treated in HistoCold for 24, 48, and 72 h ranged from good to excellent in ADC specimens (κ=0.886 for 24 h and 0.783 for 48 and 72 h) but was only fair in SQCC specimens (κ=0.600 for 24 and 48 h and 0.455 for 72 h). In CRC specimens, the agreement in CDX2 immunostaining was excellent between routinely processed specimens and those treated in HistoCold for 24 or 72 h (κ=1.000 for both) and was 90% for the 48 h timepoint. When compared to routinely processed CRC specimens, concordance in MLH1 immunostaining was good and excellent for specimens treated with HistoCold for 24 (κ=0.737) and 48 h (κ=1.000), respectively, but displayed a wider range for MSH2 immunostaining (24 h, κ=1.000; 48 h, κ=0.615).
Biospecimens
Preservative Types
- Other Preservative
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 24 h at 4°C
48 h at 4°C
72 h at 4°C
Biospecimen Acquisition Pre-preservation condition HistoCold at 4°C
No treatment