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Validation of a novel one-step tissue fixation chemistry that preserves phosphoproteins and histomorphology
Virginia A. Espina1, Claudius Mueller1, Svetlana Rassulova2, Holly S. Gallimore2, Elisa Baldelli1,3, Svetlana Senina1,
Joel Pachter4, Kirsten H. Edmiston2, Lance A. Liotta1
1 George
Mason University, Manassas, VA, 2Inova Fairfax Hospital, Department of Surgery, Falls Church, VA, 3S.Maria della Misericordia
Hospital, Perugia, Italy, 4University of Connecticut, Farmington, CT
Abstract
Unmet need: A significant and underappreciated issue is the fact that excised tissue is alive
and reacting to ex vivo stress [1]. During this “cold ischemia time” cells within the tissue react
and adapt to the absence of vascular perfusion, ischemia, hypoxia, acidosis, and
accumulation of cellular waste. Challenged by the realization that phosphoprotein signaling
pathways were reactive and fluctuating immediately following procurement, we developed a
non-formalin fixative chemistry for the preservation of biomarker molecules and
histomorphology in one step, in epithelial and calcified tissues, using standard clinical
pathology processing protocols [2] (US Patent #8,460,859).
Technology and potential advantages: A novel, non-formalin, one-step phosphoprotein
preservation chemistry was created, characterized in a wide variety of human and animal
tissues, independently validated by pathologists, published, patented, licensed, and samples
were distributed to the scientific community. Our preservative stabilizes phosphoproteins,
immunohistochemical antigens, glycoproteins, nucleic acids, decalcifies bone, and renders
exquisite diagnostic cellular histomorphology superior, or equivalent, to formalin. This fixative
reduces biospecimen pre-analytical variability in research or clinical molecular profiling. Our
novel preservative makes it possible to ask, and answer, research questions, and conduct
clinical trial studies that were never before possible. For example, we can now assess
molecular targets in calcified tissue, and reliably monitor phosphorylated signal transduction
proteins - the substrates of kinase drug targets.
Progress to date: Completed independent performance validation of the fixative in a wide
variety of tissues and labile cancer related antigens. The histomorphology of our fixative was
equal or superior to formalin under the following histomorphology rankings: overall color and
fidelity, cell size, preservation of nuclear membrane, preservation of nucleoli and nuclear
chromatin, preservation of overall cell structure, and nuclear:cytoplasmic ratio maintained.
RNA quality was equal to frozen brain tissue prior to paraffin embedding, and is compatible
with laser capture microdissection and qRT-PCR. In the case of calcified tissues, our fixative
aided a simplified processing of bony tissues (18.5 hour reduction in processing time) while
supporting immunohistology, histomorphology, and FISH, superior to or equivalent to
formalin.
Methods
Preservation of clonal heterogeneity in tumor tissue. TheraLin fixed tissue has been
used to conduct Immuno-Laser Capture Microdissection for full genome sequencing of
specific cell populations.
Simultaneous preservation and decalcification of human bone metastasis for
molecular analysis. TheraLin obviates the need for a separate decalcification step and
preserves histomophology and IHC superior to formalin for bone, as judged by independent
pathologist analysis. TheraLin preserves and decalcifies entire mouse embryos in one step.
TheraLin fixation replaces specimen freezing and eliminates the requirement for dry
ice shipping. Comparison of signal pathway phosphoprotein networks in vivo, in core
biopsies taken before/after administration of molecular targeted therapy, is now possible.
TheraLin has been validated in clinical research trials.
Nuclear Chromatin: Improved nuclear histomorphology of eye, testes, and brain
tissue. TheraLin preserves nuclear chromatin detail, without chromatin smudge artifacts,
that is superior to formalin.
Carbohydrate chemistry in tissue: Preservation of mucin and glycoproteins. Superior
PAS staining of mucin, enabling visualization of glycocalyx gradients.
Biodefense research: Inactivation of viral biohazard select agents by TheraLin in
tissue specimens. Rapid inactivation of select agents in tissue by TheraLin, while
preserving biomarkers, improves safety of tissue handling and eliminates the one-month
quarantine time for formalin fixed tissue.
RNA Preservation. A modified protocol based on the PAXgene Tissue miRNA Kit
(PreAnalytix) was optimal for RNA extraction from TheraLin fixed cells. RNA quality and
quantity in cells fixed in TheraLin was verified to be maintained at high quality (RIN 8.2) for
72 hours. This time exceeds the recommended maximum length of time of fixation, prior to
paraffin embedding, for Anatomical Pathology Clinical Practice Guidelines. TheraLin has
made possible the RNA analysis of microdissected brain vessels.
Centro di Riferimento Oncologico, Aviano, Italy
University of Brescia, Italy
Istituto Nazionale Tumori, Milano, Italy
Istituto Regina Elena, Rome, Italy
Istituto Oncologico del Mediterraneo, Catania,
Italy
• Istituto Ortopedico Rizzoli, Bologna, Italy
• St. James’ Hospital, Dublin, Ireland
Gall bladder (5)
Liver (10)
Nerve (2)
Testis (3)
Thyroid (18)
Tonsil (5)
Skin (4)
Spleen (2)
Stomach (7)
8A
Figure 2. Bayesian unsupervised clustering of signal transduction proteins for microdissected
matched FFPE, TheraLin fixed, or frozen lung tissues. The matched frozen and TheraLin samples
clustered together, indicating similar molecular characteristics. None of the FFPE samples clustered with the
matched frozen or TheraLin-fixed samples. (BHP=TheraLin fixed tissue, FFPE=formalin fixed paraffin embedded)
Figure 5. TheraLin preserves mucins and
glycoproteins. A) Human colon stained with Periodic
Acid Schiff (PAS) stain. B) Thyroid tissue stained with
Masson’s Trichrome stain demonstrating porosity
gradients within the colloid.
Figure 7. Viral plaque assay demonstrates
viral inactivation during tissue fixation.
Liver samples from animals infected with the
BSL2 strain of Rift Valley Fever Virus showed
total inactivation of RVFV after 7 days fixation
in TheraLin.
Frozen BHP 3h BHP 24hNBF 3h NBF 24h
8B
651 bp
438 bp
170 bp
IHC Stains Better or
Equal to FFPE
1A4
AE1/AE3
AFP
BCC
Bcl-2
Brachyury
CA125
Calretinin
CD 10
CD 20
CD 21
CD 23
CD 3
CD 31
CD 34
CD 38
CD 45
CD 5
CD 56
CD 68
CDX2
CK 20
CK 7
CK 8/18
CK HMW
CrA
E-Cadherin
EGFR
EMA
ER α
HEPA
Her2
HMB45
HMB-45
Ki-67
MDM2
Osterix
Pankeratin
Pax 8
P-Glycoprotein
Phospho-AcetylCoA Carboxylase
(Ser79)
Phospho-Akt
(Ser473)
Phospho-Bcl-2
(Ser70)
Phospho-eIF4G
(Ser1108)
Phospho-ERK
(Thr202/Tyr204)
Phospho-GSK3 α/β
(Ser21/Ser9)
Phospho-p38 MAPK
(Thr180/Tyr182)
Figure 8. RNA is preserved during fixation with TheraLin. A) Human colon mucosa was snap frozen or fixed in
TheraLin or NBF for 3 or 24 hours prior to freezing. RNA was extracted using the Qiagen AllPrep DNA/RNA/Protein
Mini kit (frozen and TheraLin fixed samples) or the Qiagen FFPE miRNeasy kit (NBF samples). 200 ng of RNA were
reverse transcribed to cDNA and 0.5uL cDNA was used to amplify three different sized amplicons of Beta-Actin
mRNA. B) LCM/qRT-PCR. Microdissected (Arcturus PixCell IIe) mouse brain was fixed or frozen. RNA was directly
reverse transcribed without isolation. TheraLin fixed tissue showed lower Ct values compared to frozen tissue
indicating preservation of RNA. (BHP=TheraLin, NBF=neutral buffered formalin)
TheraLin fixative components
 Precipitating fixative – stabilize proteins
 Permeation enhancer – rapid penetration
 Phosphatase & Kinase inhibitors – inhibit
reactive cell signaling post excision
 Reversible cross-linkers – stabilize proteins,
facilitate extraction of biomolecules post fixation
Under evaluation
 Carboxylic acid – maintain nuclear morphology
Figure 4. Histomorphology of murine tissue and
whole embryos. 31 murine tissues, including whole
mouse embryos, show adequate preservation in
TheraLin with paraffin embedding.
RNA is Preserved for Downstream PCR and qRT-PCR
206 samples
27 tissue types
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18.5 day mouse embryo
Figure 6. TheraLin is a one-step fixation/decalcification
solution that is compatible with immunohistochemistry.
TheraLin preserves nuclear volume, does not require
additional decalcification, preserves phosphoprotein epitopes,
and is compatible with laser capture microdissection, and with
various Hematoxylin formulations. Top right panel shows bone
samples without additional decalcification.
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Bone Cancer (50)
Breast Cancer (20)
Lung Cancer (19)
Ovarian Cancer (11)
Prostate Cancer (15)
Renal Cancer (10)
Uterus/Ovary (13)
Pancreas (2)
Other Tissues (10)
5B
5A
Figure 1. Immuno-LCM for PCNA
positive cells in prostate tissue. TheraLin
fixed tissue is compatible with laser capture
microdissection and immuno-LCM, coupled
with downstream next gen sequencing (Ion
Torrent).
Seven Pathology Groups
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One-step Preservation and Decalcification
Performance Features of TheraLin Fixative
PR
RCC
S100
SMA
Sox9
Thyroglobulin
TTF1
Vimentin
WT1
Conclusions
1. This fixative formulation is now commercially available as TheraLin™ from Grace Bio-Labs (www.gracebio.com).
2. TheraLin retains cellular morphology and antigenicity equivalent to formalin fixed tissue and is compatible with
immunohistochemistry, PCR, qRT-PCR, reverse phase protein microarrays, and laser capture microdissection.
3. TheraLin has successfully undergone clinical pathology validation at multiple international sites.
Figure 3. Ki-67 antigenicity is preserved on cut tissue section slides. Formalin and TheraLin matched
tissue sections were cut and stored on slides then stained side-by-side. Robust Ki-67 reactivity by IHC is
evident up to 131 days after tissue sections were cut onto slides.
References: 1. Espina V. et al, A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Molecular & Cellular Proteomics (2008), 7: p19982018; DOI 10.1074/mcp.M700596-MCP200. 2. Mueller C. et al, One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and
research specimens. PLoS One, (2011) 6(8): p. e23780.
This work was funded by IMAT R21CA125698-01A1 and R33CA157403-01 to L. Liotta and V. Espina

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