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Histologic and Quality Assessment of Genotype-Tissue Expression (GTEx) Research Samples: A Large Postmortem Tissue Collection.

Author(s): Sobin L, Barcus M, Branton PA, Engel KB, Keen J, Tabor D, Ardlie KG, Greytak SR, Roche N, Luke B, Vaught J, Guan P, Moore HM

Publication: Arch Pathol Lab Med, 2024, Vol. , Page

PubMed ID: 38797720 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper evaluated the potential effects of postmortem interval (PMI), rapidity of death (modified Hardy Score), and tissue type on the tissue and RNA quality markers, autolysis score and RNA integrity number (RIN), respectively, in specimens that appeared normal upon autopsy collected as part of the Genotype-Tissue Expression (GTEx) Project. Postmortem interval (the time between donor death and tissue preservation) and modified Hardy Score (a measure of the rapidity of death, 0=mechanical ventilation, 1= <10 min, 2= <1 h, 3= 1-24 h, 4= >24 h) were recorded for each donor. Two blocks of tissue were collected from up to 40 tissue types from each of 956 eligible GTEx Project donors (641 males, 315 females; age: 21-70 y; body mass index: 18.5-35). One block was PAXgene-fixed and paraffin-embedded (PFPE) and used for histologic assessment to determine autolysis score (0=none, 1=slight, 2=moderate, 3=severe) by project pathologists and the other was fixed in PAXgene fixative and stored at -80°C until RNA extraction using Qiagen’s PreAnalytix PAXgene Tissue RNA/miRNA Kit for RNA quantification by Nanodrop spectrophotometer and RNA integrity by RIN using a bioanalyzer. 

   Summary of Findings:

   Tissue-specific differences in autolysis score and RIN were observed among specimens from GTEx Project donors. Twenty-four of the 40 tissue types collected had a mean RIN ≥6, with the remaining 16 tissue types having a mean RIN that ranged between 4.34- 5.96.  Skeletal muscle (7.70), minor salivary gland (7.48), and esophagus mucosa (7.18) had the highest mean RINs while the lowest mean RINs were observed in kidney (4.34, cortex; 4.73, medulla), spleen (4.63), and pancreas (5.20). The severity of autolysis also varied among the tissue types examined, being more prominent in pancreas and gastrointestinal mucosa and less evident in skeletal muscle, nerve, and skin). RIN and autolysis score were strongly and negatively correlated in pancreas (r= - 0.70, p< 0.00001, n=768) but not skeletal muscle (r= -0.07, n=940).  The relationships between PMI and RIN or autolysis score were also tissue-dependent.  PMI was modestly and negatively correlated with RIN in pancreas (r= -0.68, p<0.00001, n=768) but only weakly and negatively correlated in skeletal muscle (r= -0.13, p=0.00011).  

   
   This relationship was also reflected in shifts in the percentage of specimens with a RIN >6, as the percentage of pancreas specimens with a RIN >6 was higher for specimens with a PMI <8 h than ≥8 h (83% versus 17%), and the percentage of tibial nerve specimens with a RIN >6 was higher for specimens with a PMI <4 h than ≥20 h (70% versus 58%). However, this relationship was not observed in skeletal muscle where the percentage of specimens with a RIN >6 remained stable across the PMI range examined (86-95%). Similarly, PMI and autolysis score were positively and strongly correlated in pancreas (r=0.72, p<0.0001, n=807) but not in skeletal muscle (r=0.094, n=944). In pancreas specimens, the mean autolysis score was higher with longer PMIs (AS= 0.96 autolysis score when PMI <4 h versus AS=2.63 when PMI ≥20 h) and there was an observable increase in the percentage of pancreas specimens with severe autolysis with PMI (1% when PMI <4 h versus 56% when PMI ≥20 h). Skeletal muscle and tibial nerve specimens displayed comparatively stable autolysis scores over the PMI range evaluated.  An examination of effects associated with the rapidity of death of the donor (measured by modified Hardy Score, HS) was confounded by a disproportionate representation of PMIs between HS categories. For example, while the mean RIN of pancreas specimens was higher for a HS of 0 than those with a HS of 1-4 (6.40 versus 3.53-3.92), donors that received mechanical ventilation prior to death (HS=0) tended to have short PMIs than those that did not (HS 1-4)(PMI < 8 h: 91% versus 3-13%). Thus, donors who received mechanical ventilation were excluded from HS comparisons of individual tissue types. Weak, negative correlations between HS and RIN were observed for heart (r= -0.32), testis (r= -0.29), adrenal gland (r= -0.25), liver (r= -0.24), skeletal muscle (r= -0.20), and pituitary (r= -0.20)(p<0.00025 for all). A correlation between HS and autolysis score was limited to stomach specimens (r= 0.27, p<0.00001).
    

   Biospecimens

   • Tissue - Pancreas
   • Tissue - Nerve
   • Tissue - Muscle (Skeletal)
   • Tissue - Adipose
   • Tissue - Adrenal Gland
   • Tissue - Aorta
   • Tissue - Bladder
   • Tissue - Brain
   • Tissue - Breast
   • Tissue - Cervix
   • Tissue - Colorectal
   • Tissue - Esophagus
   • Tissue - Fallopian Tube
   • Tissue - Heart
   • Tissue - Ovary
   • Tissue - Salivary Gland
   • Tissue - Lung
   • Tissue - Liver
   • Tissue - Kidney
   • Tissue - Skin
   • Tissue - Small Bowel
   • Tissue - Uterus
   • Tissue - Thyroid Gland
   • Tissue - Testis
   • Tissue - Stomach
   • Tissue - Spleen
   • Tissue - Vagina

   Preservative Types

   • PAXgene

   Diagnoses:

   • Normal
   • Autopsy

   Platform:

   Analyte	Technology Platform
   Morphology	H-and-E microscopy
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Postmortem interval	<1 - 27 h
   Preaquisition	Rapidity of death	Hardy Score = 0 (mechanical ventilation preceding death) Hardy Score = 1 Hardy Score = 2 Hardy Score = 3 Hardy Score = 4
   Biospecimen Acquisition	Biospecimen location	Adipose-Subcutaneous Adipose-Visceral Adrenal gland Artery-Aorta Artery-Tibial Bladder Brain-Cerebellum Brain- Cortex Breast Cervix-Ectocervix Cervix-Endocervix Colon-Sigmoid Colon-Transverse Esophagus-Gastroesophageal junction Esophagus- Mucosa Fallopian tube Heart-Atrial appendage Heart-Left ventricle Kidney-Cortex Kidney-Medulla Liver Lung Minor salivary gland Nerve-Tibial Ovary Skin-Not sun exposed Skin-Sun exposed Small intestine Spleen Stomach Testis Thyroid Uterus Vagina

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