HOPE-BAL: improved molecular diagnostics by application of a novel technique for fixation and paraffin embedding.

Author(s): Marwitz S, Abdullah M, Vock C, Fine JS, Visvanathan S, Gaede KI, Hauber HP, Zabel P, Goldmann T

Publication: J Histochem Cytochem, 2011, Vol. 59, Page 601-14

PubMed ID: 21430262 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 preservation method on immunohistochemical staining in bronchoalveolar lavage cells. Specimens fixed in 4% neutral buffered formalin were embedded in normal paraffin while HOPE-fixed specimens were embedded in low-melting point paraffin. The authors used both whole sections and tissue microarrays for immunostaining analysis.

   Summary of Findings:

   HOPE-fixation yielded good morphological preservation and CD8, CD68, Ki67, and pan-cytokeratin immunostaining was similar between HOPE-fixed and formalin-fixed specimens. However, HOPE-fixed specimens showed clear CD4 immunostaining while formalin-fixed specimens did not. Further, leukocyte common antigen (LCA) staining was superior in HOPE-fixed specimens compared to formalin-fixed specimens. Two-dimensional gel electrophoresis was possible with protein extracted from HOPE-fixed specimens.

   Biospecimens

   • Cell - Bronchial Lavage

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Asthma
   • Chronic Obstructive Pulmonary Disease (COPD)
   • Emphysema
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Protein	Tissue microarray
   Protein	Colorimetric assay
   Protein	1D/2D gels
   Cell count/volume	Light microscopy
   Morphology	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) HOPE
   Preaquisition	Diagnosis/ patient condition	Tuberculosis Sarcoidosis Fibrosis Chronic cough
   Immunohistochemistry Specific	Targeted peptide/protein	CD4 CD8 CD68 LCA Ki67 Cytokeratins

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2. Study Purpose

   The purpose of this study was to determine the effects of preservation method (freezing at -80 degrees C, HOPE-fixation, or 4% neutral buffered formalin) on RNA yields and integrity in bronchoalveolar lavage cells.

   Summary of Findings:

   RNA with lower integrity was obtained from HOPE-fixed specimens compared to RNA from fresh-frozen specimens, and RNA integrity was unable to be determined for formalin-fixed specimens. RNA yields (4 specimens averaged) were 157 ng/uL, 138 ng/uL, and 3.2 ng/uL for frozen, HOPE-fixed, and formalin-fixed specimens, respectively. In situ hybridization, real-time qRT-PCR, and transcription microarray analysis were successfully performed using HOPE-fixed specimens.

   Biospecimens

   • Cell - Bronchial Lavage

   Preservative Types

   • Frozen
   • Other Preservative
   • Formalin

   Diagnoses:

   • Asthma
   • Chronic Obstructive Pulmonary Disease (COPD)
   • Emphysema
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	In situ hybridization
   RNA	Tissue microarray
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Electrophoresis
   RNA	Real-time qRT-PCR
   Cell count/volume	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen HOPE
   Preaquisition	Diagnosis/ patient condition	Tuberculosis Sarcoidosis Fibrosis Chronic cough

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