Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue.

Author(s): Pollard K, Lunny D, Holgate CS, Jackson P, Bird CC

Publication: J Histochem Cytochem, 1987, Vol. 35, Page 1329-38

PubMed ID: 3309048 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to identify effects of fixative type, temperature, and pH as well as the use of 12 different additives and tissue processing reagents and conditions associated with paraffin embedding on the stability of CD3, CD4, and CD8 surface membrane antigens. Post-fixation variables were also addressed including the type of fixative (imidazole, tannic acid, osmium tetroxide), and a running tapwater wash to remove excess fixative. The size of tonsil specimens ranged from frozen sections to whole specimens (up to 20 x 15 x 5 mm). Stage-dependent effects were assessed using frozen tissue sections when necessary.

   Summary of Findings:

   While antigen-specific results were observed, fixing frozen sections in PLPD, PLP, acetone, or formol dichromate produced strong immunostaining for all three membrane surface antigens investigated when conducted at 4 degrees C at a pH of 7.5. For paraffin sections, superior immunostaining was obtained after fixation in PLPD, pH 7.5 for 24-36 h at 4 degrees C. For 10% formalin, optimal immunostaining was observed with a Tris buffer, as opposed to PBS, and fixation for 24 h or less. PEG 400 was the only additive investigated that reduced background staining and improved cell morphology preservation in frozen and paraffin specimens fixed in PLPD, PLP, or 10% formalin. Immunostaining was not significantly altered following post-fixation in PLPD, PLP, or 10% formalin, although immunostaining of formalin-fixed specimens incubated in Tris-saline buffer for 7 d enhanced CD3 immunoreactivity and reduced background staining. A tapwater wash for up to 6 h after post-fixation did not impact immunostaining, although a modest reduction in intensity was observed after a 24 h wash.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Formalin
   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Glycoprotein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Acetone Bouin's fixative Carbodiimide Dichromate periodate lysine paraformaldehyde Formaldehyde Formalin (buffered) Formol sublimate Frozen Glutaraldehyde Formol dichromate
   Biospecimen Preservation	Time in fixative	30 min -53 h (frozen sections) 18-102 h (whole tissue) 6 h- 7d (post-fixation)
   Biospecimen Preservation	Temperature of fixation/preservation	4 degrees C 22 degrees C
   Biospecimen Preservation	Fixative additive/buffer	Calcium chloride Cetyl pyridinium chloride Disodium phosphate buffer Imidazole Lidocaine Magnesium chloride Malachite green Osmium tetroxide Polyethylene glycol Potassium ferrocyanide Potassium oxalate Saturated ammonium sulfate Tannic acid Tris (hydroxymethyl) methylamine buffer
   Immunohistochemistry Specific	Targeted peptide/protein	CD3 CD4 CD8

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2. Study Purpose

   The purpose of this study was to identify the effects of a delay (at 4 degrees or room temperature) prior to specimen preservation via fixation or freezing on the stability of CD3, CD4, and CD8 surface membrane antigens.

   Summary of Findings:

   Although data was not shown the authors reported antigen-specific stability and superior immunostaining with frozen and paraffin sections stored for 2 h or less, and in specimens stored at 4 degrees C compared to those stored at room temperature.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Glycoprotein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0-72 h
   Storage	Storage temperature	4 degrees C 22 degrees C
   Immunohistochemistry Specific	Targeted peptide/protein	CD4 CD3 CD8

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3. Study Purpose

   CD3, CD4, and CD8 immunoreactivity was examined in frozen and paraffin embedded tissue sections following fixation in PLPD with PEG 400 at 4 degrees C for 30 min and 24 h, respectively, and dehydration and clearing with different reagents, temperatures, and durations.

   Summary of Findings:

   For frozen sections, dehydration in glycerol, PEG 400, or isopropanol and clearing with xylene, chloroform, carbon tetrachlrodie, toluene, or histoclear at -20 degrees C resulted in more intense immunostaining; although specific durations were not specified by the authors they note that shorter durations produced better results. Optimal staining among paraffin sections was observed after dehydration in several changes of isopropanol over a 10 h period at 4 degrees C, and clearing in xylene or chloroform at 4 or -20 degrees C for 15 min.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Other Preservative
   • Frozen
   • Ethanol

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Glycoprotein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Dichromate periodate lysine paraformaldehyde Frozen
   Biospecimen Preservation	Type of fixation/preservation	Dichromate periodate lysine paraformaldehyde Frozen
   Biospecimen Preservation	Dehydration reagent	Acetone Butanone Diethylene glycol Ethanol Ethoxyethanol Ethylene glycol Glycerol Isopropanol Methanol n-Propanol Propylene glycol Tertiary butanol
   Biospecimen Preservation	Dehydration duration/condition	15 min- 24 h -20 degrees C 4 degrees C 22 degrees C
   Biospecimen Preservation	Clearing agent	Benzene Carbon tetrachloride Chloroform Histoclear Methyl salicylate Toluene Trichlorethylene Xylene
   Biospecimen Preservation	Clearing duration/condition	15 min- 24 h -20 degrees C 4 degrees C 22 degrees C
   Immunohistochemistry Specific	Targeted peptide/protein	CD3 CD4 CD8

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4. Study Purpose

   The purpose of this study was to assess the potential effects of employing paraffin waxes with different melting points and drying mechanisms for tissue section/ slide adhesion on CD3, CD4, and CD8 antigenicity.

   Summary of Findings:

   Immunostaining was adversely and progressive impacted with the temperature of paraffin impregnation in frozen tissue sections. While impregnation with higher melting point paraffin resulted in superior preservation of cell morphology among whole tissue specimens, the intensity of immunostaining was superior with low melting point paraffin. A minimum of 1 h of paraffin impregnation was required, although differences in immunostaining were not observed with longer durations or vacuum extraction. Optimal immunostaining was observed when paraffin sections were dried at room temperature for 24 h compared to heating on a hot plate. Longer drying durations or the use of poly-L-lysine was required for adequate slide/tissue section adhesion when the PEG 400 additive was employed during fixation.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Glycoprotein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Dichromate periodate lysine paraformaldehyde Frozen
   Biospecimen Preservation	Dehydration reagent	Acetone Ethanol Ethoxyethanol Glycerol Isopropanol
   Biospecimen Preservation	Dehydration duration/condition	10-18 h 4 degrees C 22 degrees C
   Biospecimen Preservation	Clearing agent	Carbon tetrachloride Chloroform Histoclear Toluene Xylene
   Biospecimen Preservation	Clearing duration/condition	15 min- 6 h -20 degress C 4 degrees C 22 degrees C
   Biospecimen Preservation	Embedding medium	Paraffin with a 45 degree C melting point Paraffin with a 65 degree C melting point
   Biospecimen Preservation	Embedding duration/condition	1-2 h With vacuum extraction Without vacuum extraction
   Biospecimen Aliquots and Components	Type of slide	Dried for 20 min on a hot plate Dried for 1-7 d at 22 degrees C
   Immunohistochemistry Specific	Targeted peptide/protein	CD3 CD4 CD8

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