Qualitative Comparison of Cryostat- versus Snap-Frozen Neurosurgical Intraoperative Consultations.
Author(s): Priemer DS, Wysozan T, Zahedi F, Alrabadi N, Mesa H, Vortmeyer AO
Publication: Int J Surg Pathol, 2023, Vol. 31, Page 949-956
PubMed ID: 35971290 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to semi-quantitatively and quantitatively assess the effect of freezing method on the histologic quality of brain tumor biopsies that were embedded in OCT and frozen in a cryostat (-18 to -21°C) or snap-frozen by submersion in isopentane pre-cooled with dry ice (-78°C). Histologic quality was assessed by the Histologic Preservation Score (HPS), which quantifies distortions/differences relative to a case-matched formalin-fixed, paraffin-embedded specimen and the Ice Crystal Vacuolization Score (ICVS), the mean size (µm) of the largest five vacuoles observed.
Conclusion of Paper
OCT-embedded sections of brain tumor biopsies that were preserved by snap-freezing in isopentane pre-cooled with dry ice (-78°C) were of higher histological quality than specimens that were frozen in a cryostat (-18 to -21°C) as reflected by lower mean HPS (2.28 versus 2.84, p=0.0175) and ICVS (7.47 versus 14.56, p<0.001). While not statistically significant, small vacuoles (<10 µm) were more common in snap-frozen specimens than cryostat-frozen specimens (72.7% versus 40.3%) while larger vacuoles (>20 µm) were more prominent in cryostat-frozen specimens than snap-frozen ones (22.6% versus 0%). HPS and ICVS also differed significantly between neuroglial and mesenchymal tissue, but only in specimens frozen in a cryostat (HPS: 3.23 versus 2.33, p<0.001; ICVS: 16.86 versus 10.26, p<0.001). HPS and ICVS were strongly and significantly correlated (r2=0.62, p<0.0001). Regarding clinical impact, most of the 15 specimens with an HPS score indicative of severe distortion that affected diagnosis (score of 4 or 5) were frozen in a cryostat (86%, 13/15). There were five specimens for which the reviewing neuropathologists could not make a specific diagnosis, all five specimens were frozen in a cryostat (-18 to -21°C).
Studies
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Study Purpose
The purpose of this study was to semi-quantitatively and quantitatively assess the effect of freezing method on the histologic quality of brain tumor biopsies that were embedded in OCT and frozen in a cryostat (-18 to -21°C) or snap-frozen by submersion in isopentane pre-cooled with dry ice (-78°C). Histologic quality was assessed by the Histologic Preservation Score (HPS), which quantifies distortions/differences relative to a case-matched formalin-fixed, paraffin-embedded specimen and the Ice Crystal Vacuolization Score (ICVS), the mean size (µm) of the largest five vacuoles observed. In total, biopsies of central nervous system tumors (44 neuroglial, 26 intracranial mesenchymal lesions, 8 metastases of head and neck malignant tumors, and 8 miscellaneous lesions) were collected (details not provided) and bisected; one-half of the specimen was fixed in 10% neutral buffered formalin and paraffin embedded and the other half was coated in OCT and frozen in a cryostat (frozen, -18 to -21°C) or immersed in isopentane that was pre-cooled with dry ice (-78°C) and covered with an aluminum heat extractor (snap-frozen). Cryostat-generated sections (3 µm thick) of frozen and snap-frozen specimens were mounted on glass slides, fixed in 95% ethanol, hematoxylin and eosin (H&E) stained. Two neuropathologists performed a blind review of slides from FFPE, cryostat-frozen, and snap-frozen specimens to determine the histological preservation score (HPS), which is based on differences and distortions present relative to the case-matched FFPE specimen (1=little to no difference, 2=slight differences that do not affect interpretation, 3=moderate distortion that does not compromise interpretation, 4= severe distortion that compromises interpretation, 5= severe distortion that prevents interpretation). To determine the ice crystal vacuolization score (ICVS), H&E-stained slides of cryostat-frozen and snap-frozen biopsy specimens were scanned and the mean diameter of the five largest vacuoles located in a 0.05 mm2 area with the “best-preserved histology” were determined; the ICVS represents the mean of those vacuole diameters.
Summary of Findings:
OCT-embedded sections of brain tumor biopsies that were preserved by snap-freezing in isopentane pre-cooled with dry ice (-78°C) were of higher histological quality than specimens that were frozen in a cryostat (-18 to -21°C) as reflected by lower mean HPS (2.28 versus 2.84, p=0.0175) and ICVS (7.47 versus 14.56, p<0.001). While not statistically significant, small vacuoles (<10 µm) were more common in snap-frozen specimens than cryostat-frozen specimens (72.7% versus 40.3%) while larger vacuoles (>20 µm) were more prominent in cryostat-frozen specimens than snap-frozen ones (22.6% versus 0%). HPS and ICVS values also differed significantly between neuroglial and mesenchymal tissue, but only in specimens frozen in a cryostat (HPS: 3.23 versus 2.33, p<0.001; ICVS: 16.86 versus 10.26, p<0.001). HPS and ICVS were strongly and significantly correlated (r2=0.62, p<0.0001). Regarding clinical impact, most of the 15 specimens with an HPS score indicative of severe distortion that affected diagnosis (score of 4 or 5) were frozen in a cryostat (86%, 13/15). There were five specimens for which the reviewing neuropathologists could not make a specific diagnosis, all five specimens were frozen in a cryostat (-18 to -21°C).
Biospecimens
Preservative Types
- OCT
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Lymphoma
- Neoplastic - Melanoma
- Neoplastic - Germ Cell
- Neoplastic - Mixed type
- Neoplastic - Other
Platform:
Analyte Technology Platform Morphology H-and-E microscopy Morphology Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Cooling or freezing method/ rate Cryostat (-20 or -30 degrees C)
Pre-cooled isopentane
Biospecimen Preservation Type of fixation/preservation Frozen
Snap frozen
Formalin (buffered)