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Effects of Decalcifying Agents of Variable Duration on PD-L1 Immunohistochemistry.

Author(s): Strickland AL, Blacketer S, Molberg K, Markantonis J, Lucas E

Publication: Am J Clin Pathol, 2020, Vol. 153, Page 258-265

PubMed ID: 31603186 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of four different decalcifying agents and treatment duration on PD-L1 expression status as determined by IHC in FFPE lung and placental tissue. Pieces from 10 placentas collected surgically and 10 non-neoplastic lungs obtained during autopsy were fixed in 10% neutral buffered formalin (NBF) for 24 to 48 h and then subjected to decalcification in one of four decalcifying solutions: EDTA, formic acid/MasterCal IM Plus (FA/MC), 12% HCl, and Decal STAT/23% HCl for 0, 1, 2, 6, or 24 h. Slides of 4 μm-thick FFPE tissue sections were prepared, deparaffinized, rehydrated, and target retrieval was performed by submersion in preheated (65°C) target retrieval solution at low pH (6.1) and incubation at 97°C for 20 min. PD-L1 staining performed using an autostainer and assessed by two pathologists that estimated the percentage of positive stained cells and scored the staining intensity on a scale from 0 to 3+ (absent to strong). Change in staining intensity compared to non-decalcified specimens was defined as minimal (1-5%), mild (5-20%), mild to moderate (20-40%), moderate (40-60%), significant (60-99%), and complete (99-100%). The H-score (range 0-300) was calculated for each case and rounded to the nearest 5 or 0 for easier visualization.

   Summary of Findings:

   In the placenta tissue, there was no change in proportion of cells staining positively for PD-L1 or staining intensity from non-decalcified specimens after decalcification with EDTA or FA/MC, regardless of treatment duration. There was no change in proportion of positively stained cells in placenta decalcified for 1, 2, or 6 h with 12% HCl but a 2% decrease in the percentage of positive cells relative to non-decalcified specimens was observed when decalcified for 24 h. PD-L1 staining intensity in 12% HCL decalcified specimens was reduced compared to non-decalcified specimens with a minimal decrease in staining intensity at 1 and 2 h, a moderate decrease at 6 h, and a significant decrease at 24 h. The proportion of PD-L1 positive cells was comparable after decalcification of placenta in Decal STAT for 1, 2, or 6 h to that in non-decalcified placenta but a 20% decrease in the proportion of PD-L1 positive cells was observed after decalcification for 24 h. Compared to non-decalcified placenta, Decal STAT decalcified placenta displayed  a mild to moderate decrease in staining intensity after 1 h, moderate decrease after 2 h, significant decrease after 6 h, and almost complete loss of staining after 24 h. In lung tissue, there was no change in the proportion of positively stained cells or staining intensity for EDTA-decalcified specimens regardless of duration or in lung decalcified with FA/MC for 1, 2, or 6 h but there was an average 15% decrease in positive cells and a mild to moderate change in staining intensity after decalcification of lung in FA/MC for 24 h. Lung specimens decalcified with 12% HCl had a 5% decrease in the proportion of positive cells after 1 or 2 h, 20% decrease after 6 h, and 50% decrease after 24 h. The PD-L1 staining intensity was with not affected by decalcification of lung tissue in 12% HCL for 1 or 2 h, but displayed a mild to moderate decrease after 6 h, and a moderate decrease after 24 h. The proportion of positive cells decreased in lung tissue after decalcification with Decal STAT with a 10% decrease in the proportion of positive cells after 1 h, 25% after 2 h, 40% after 6 h, and 70% after 24 h. Further, the intensity of PD-L1 staining decreased with Decal STAT decalcification with a mild to moderate decrease after 1 h, moderate decrease after 2 h, significant decrease after 6 h, and significant to complete loss of staining after 24 h. The average H-score remained at baseline (300) for all timepoints among the EDTA and FA/MC-treated placenta specimens but decreased to 280 after 1 or 2 h, 260 after 6 h, and 170 after 24 h of decalcification with 12% HCl - and decreased to 250 after 1 h, 235 after 2 h, 175 after 6 h, and 85 after 24 h decalcification with Decal STAT specimens. Similarly, the baseline average H-score in lung tissue remained at baseline (160) when decalcified for 1 to 6 h with EDTA and decreased to 155 after EDTA decalcification for 24 h. Among FA/MC-decalcified tissue, the average H-score remained at 160 after 1 h then decreased to 155 after 2 h, 150 after 6 h, and 125 after 24 h. The average H-score decreased to 140 after 1 and 2 h of treatment, 105 after 6 h, and 50 after 24 h in 12% HCl-treated tissue and decreased to 120 after 1 h, 100 after 2 h, 60 after 6 h, and 5 after 24 h in Decal STAT-treated specimens.

   Biospecimens

   • Tissue - Placenta
   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Autopsy
   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Decalcification solution/ duration	EDTA for 0, 1, 2, 6, or 24 h Formic acid/MasterCal IM Plus (FA/MC) for 0, 1, 2, 6, or 24 h 12% HCl for 0, 1, 2, 6, or 24 h Decal STAT/23% HCl for 0, 1, 2, 6, or 24 h
   Immunohistochemistry Specific	Targeted peptide/protein	PD-L1

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