Do Tissues Fixed in a Non-cross-linking Fixative Require a Dedicated Formalin-free Processor?

2021 ◽  
pp. 002215542110178
Author(s):  
Sonia G. Frasquilho ◽  
Ignacio Sanchez ◽  
Changyoung Yoo ◽  
Laurent Antunes ◽  
Camille Bellora ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Dna Integrity ◽  
Rt Pcr ◽  
Ki 67 ◽  
Fixed Tissue ◽  
Free System ◽  
Rna Integrity ◽  
Formalin Fixed Tissue ◽  
Rna Integrity Number ◽  
Formalin Fixed

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF−ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem:

scholarly journals Optimisation of DNA and RNA extraction from archival formalin-fixed tissue

1999 ◽  
Vol 27 (16) ◽  
pp. i-iii ◽  
Author(s):  
N. J. Coombs ◽  
A. C. Gough ◽  
J. N. Primrose
Keyword(s):  
Rna Extraction ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Dna And Rna ◽  
Formalin Fixed

Fatal Hantavirus Pulmonary Syndrome in an Adolescent

PEDIATRICS ◽  
1995 ◽  
Vol 95 (2) ◽  
pp. 276-280 ◽  
Author(s):  
Ali S. Khan ◽  
Thomas G. Ksiazek ◽  
Sherif R. Zaki ◽  
Stuart T. Nichol ◽  
Pierre E. Rollin ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Immunoglobulin M ◽  
Hantavirus Pulmonary Syndrome ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sin Nombre Virus ◽  
Rt Pcr ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Polymerase Chain ◽  
Formalin Fixed

Hantavirus pulmonary syndrome (HPS) is a recently recognized viral zoonosis characterized by a febrile prodrome progressing to severe noncardiogenic pulmonary edema.1-4 This syndrome is caused by at least three newly described hantaviruses: the first, Sin Nombre virus, is the Southwestern hantavirus that caused an outbreak of respiratory failure during the summer of 1993; the second, Black Creek Canal virus, caused a case of HPS in Florida; and the third hantavirus was identified in lung tissue from a patient in Louisiana. Diagnosis is by enzyme-linked immunosorbent assay (ELISA) serology with elevated immunoglobulin M (IgM) titers against heterologous and homologous hantaviral antigens, positive immunohistochemistry on formalin-fixed tissue, or reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of hantaviral nucleotide sequence from frozen tissue.

scholarly journals Use of proteinase K for RT-PCR of cytokine mRNA in formalin fixed tissue

1996 ◽  
Vol 49 (6) ◽  
pp. M364-M367 ◽  
Author(s):  
G. N Davies ◽  
I. S Bevan ◽  
J. B Lundemose ◽  
H. Smith ◽  
C. Sweet
Keyword(s):  
Proteinase K ◽  
Rt Pcr ◽  
Cytokine Mrna ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed

Sensitivity of Acid-Fast Staining forMycobacterium tuberculosisin Formalin-fixed Tissue

2002 ◽  
Vol 166 (7) ◽  
pp. 994-997 ◽  
Author(s):  
Hajime Fukunaga ◽  
Tomoyuki Murakami ◽  
Toshikazu Gondo ◽  
Kazuo Sugi ◽  
Tokuhiro Ishihara
Keyword(s):  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed ◽  
Acid Fast Staining

Generation of anti-DC-SIGN monoclonal antibodies capable of blocking HIV-1 gp120 binding and reactive on formalin-fixed tissue

2011 ◽  
Vol 135 (1-2) ◽  
pp. 165-172 ◽  
Author(s):  
Bertrand Canard ◽  
Hortense Vachon ◽  
Thomas Fontaine ◽  
Jean-Jacques Pin ◽  
Stéphane Paul ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Gp120 Binding ◽  
Hiv 1 ◽  
Formalin Fixed

scholarly journals A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.

1994 ◽  
Vol 42 (8) ◽  
pp. 1127-1134 ◽  
Author(s):  
J H Beckstead
Keyword(s):  
Simple Technique ◽  
Frozen Section ◽  
Lymphoid Tissues ◽  
Frozen Sections ◽  
Morphological Examination ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Important Approach ◽  
Reduced Toxicity ◽  
Formalin Fixed

Immunohistochemistry is a powerful tool for tissue diagnosis and research. Although the frozen section has remained the gold standard for this important approach to evaluation of antigens in tissues, there is widespread acknowledgment of many limitations. Routine paraffin-embedded sections ware widely used for morphological examination of tissues but are not optimal for antigen preservation. In this study, paraffin-embedded tissues fixed with a simple buffer containing zinc as the primary fixative were compared with tissues fixed with routine formalin, zinc-formalin, paraformaldehyde, ethanol, a variety of commercial (non-formalin-containing) fixatives that have been recommended for reduced toxicity and improved antigen survival, and frozen sections. Human lymphoid tissues and a group of antibodies to antigens (CD1, CD4, CD7, CD8, CD19) usually preserved only in frozen tissue were used as a model system. Fixation in a simple solution of zinc acetate and zinc chloride in a Tris-Ca acetate buffer resulted in antigen preservation comparable to that in frozen sections with antibodies to these cell surface markers. Morphological preservation was comparable to formalin-fixed sections. The work presents a new method that represents the closest approach yet to a technique that combines optimal antigenic survival with the convenience and morphological preservation of traditional formalin-fixed tissue embedded in paraffin.

scholarly journals Sequencing of Dna Isolated From Colorectal Sample Fixed in Liquid Formalin : Obstacles and Required Modifications

2020 ◽  
Vol 29 (2) ◽  
pp. 165-174
Author(s):  
Nahid Parvez ◽  
Mustak Ibn Ayub
Keyword(s):  
Dna Isolation ◽  
Low Cost ◽  
General Purpose ◽  
Proteinase K ◽  
Fixed Tissue ◽  
Target Region ◽  
Formalin Fixed Tissue ◽  
Successful Amplification ◽  
Cancer Tissues ◽  
Formalin Fixed

The necessary modifications in the protocol of general purpose DNA isolation kit to isolate and amplify a target region of genome from colorectal cancer tissues fixed in liquid formalin were made. It is shown that a one hour digestion with proteinase K yields enough DNA from formalin fixed colorectal tissue for subsequent PCR and sequencing. Moreover, using 100% ethanol instead of standard 50% during DNA binding step in the column improves the yield. As DNA fragmentation is unavoidable in formalin fixed tissue, PCR protocol was modified by increasing polymerase concentration to get successful amplification. Following these modifications, two regions of KRAS and BRAF genes were amplified and successfully sequenced from three different patients. These modifications provide a low cost option for Sanger sequencing of DNA isolated from formalin fixed tissue. Dhaka Univ. J. Biol. Sci. 29(2): 165-174, 2020 (July)

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