Archival fixed histologic and cytologic specimens including stained and unstained materials are amenable to RT-PCR.

Author(s): Liu H, Huang X, Zhang Y, Ye H, El Hamidi A, Kocjan G, Dogan A, Isaacson PG, Du MQ

Publication: Diagn Mol Pathol, 2002, Vol. 11, Page 222-7

PubMed ID: 12459638 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of processing, storage, and amplicon length on successful amplification of G6PD from histological and cytological specimens. Specimens included archived formalin-fixed paraffin-embedded histological specimens stored as blocks, or H&E, immunostained or unstained slides, alcohol fixed cervical smears, acetone:alcohol fixed fine-needle aspirates, cell suspensions, or unstained or H&E stained bone marrow aspirates.

   Summary of Findings:

   All 5 fragment lengths of G6PD were successfully amplified in fresh specimens. Amplification of a 67 bp and 151 bp fragment was possible in most histological and cytological specimens, but amplification of a 242 bp fragment was only successful in a few specimens, and amplification of 379 bp product was possible in a few cytological and no histological specimens. The 453 bp fragments could not be amplified from either histological or cytological specimens. Storing the tissue as blocks, H&E stained sections, immunostained sections, or unstained sections did not result in any apparent effects, but in the 8 decalcified bone marrow core sections, no fragments larger than 67 bp were amplified. Amplification success rates for the 242 bp fragment were higher for fine needle aspirates and bone marrow aspirates than other types of cytological specimens, but the difference was not significant. For PAP-stained specimens there was an 88% success rate for the 242 bp fragment, but in MGG-stained specimens there was only a 12% success rate. There was no effect of storage on amplification of the 67 bp product, but amplification success of the 151 bp product was decreased for specimens stored over 5 y.

   Biospecimens

   • Tissue - Bone Marrow
   • Tissue - Tonsil
   • Tissue - Lymph Node
   • Cell - Cervix
   • Cell - Breast
   • Cell - Lymph Node
   • Cell - Thyroid Gland
   • Cell - Ovary
   • Cell - Bone Marrow
   • Cell - Other
   • Bodily Fluid - Urine
   • Bodily Fluid - Pleural Fluid
   • Bodily Fluid - Bronchial Lavage
   • Tissue - Other

   Preservative Types

   • Formalin
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	<5 years >5 years
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh) Alcohol Acetone:alcohol
   Analyte Extraction and Purification	Decalcification solution/ duration	None 5% formic acid
   RT-PCR Specific	Length of gene fragment	67 bp 151 bp 242 bp 379 bp 453 bp
   RT-PCR Specific	Targeted nucleic acid	Glucose-6-phosphate dehydrogenase
   RT-PCR Specific	Type of tissue stain	H&E stained PAP stained Unstained Immunostained MGG stained
   Biospecimen Acquisition	Method of cell acquisition	Fine needle aspiration Cervical smear
   Biospecimen Acquisition	Method of tissue acquisition	Surgical resection Aspirate

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