title: One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens.
abstract: BACKGROUND
  There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.
  
  
  RESULTS
  Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.
  
  
  CONCLUSIONS
  In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.
authors.affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, United States of America. cmuelle1@gmu.edu
authors.forename: Claudius
authors.initials: C
authors.lastname: Mueller
authors.forename: Kirsten H
authors.initials: KH
authors.lastname: Edmiston
authors.forename: Calvin
authors.initials: C
authors.lastname: Carpenter
authors.forename: Eoin
authors.initials: E
authors.lastname: Gaffney
authors.forename: Ciara
authors.initials: C
authors.lastname: Ryan
authors.forename: Ronan
authors.initials: R
authors.lastname: Ward
authors.forename: Susan
authors.initials: S
authors.lastname: White
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Memeo
authors.forename: Cristina
authors.initials: C
authors.lastname: Colarossi
authors.forename: Emanuel F
authors.initials: EF
authors.lastname: Petricoin
authors.forename: Lance A
authors.initials: LA
authors.lastname: Liotta
authors.forename: Virginia
authors.initials: V
authors.lastname: Espina
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D035843:Biomedical Research; D002415:Cats; D005260:Female; D005629:Frozen Sections / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008813:Mice, Inbred ICR; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008875:Middle Aged; D016612:Paraffin Embedding / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis*; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D010750:Phosphoproteins