Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry

Sheila Criswell; Kristina Zwerg; Cynthia Lazar; Caitlin Richmond; Chelsea Bridges

doi:doi:10.11648/j.ab.20221002.14

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Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry

Sheila Criswell, Kristina Zwerg, Cynthia Lazar, Caitlin Richmond, Chelsea Bridges

Published in Advances in Biochemistry (Volume 10, Issue 2)

Received: 6 May 2022 Accepted: 23 May 2022 Published: 31 May 2022

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Abstract

Glyoxal solutions are safer fixatives as compared to formalin in the anatomic pathology laboratory. While long-term effects of tissue stored in formalin are well-documented, studies of tissues fixed long-term in glyoxal are few. Using human autopsy tissues, 4 glyoxal solutions, and neutral buffered formalin, the effects of storage of tissues in fixatives for 8 hours, 1 month, and 4 months were compared. Special stain methods to include hematoxylin and eosin, melanin bleaching using either 10% hydrogen peroxide or potassium permanganate followed by oxalic acid, Verhoeff van Gieson, reticulin, Fontana Masson, mucicarmine, colloidal iron, alcian blue, trichrome, and Wright stain, as well as immunohistochemistry labeling of 18 antibodies were conducted and compared. Tissues fixed in glyoxal solutions exhibited nearly identical staining properties when compared with each other for all lengths of fixation. When fixed for 8 hours, they also produced the same results as tissues fixed in formalin. Although glyoxal-fixed tissues stored for 1 – 4 months in fixatives mimicked staining in formalin-fixed tissues for most special stain techniques, they retained adequate antigenic labeling for only 78% of the antibodies evaluated. Consequently, for special stain applications, tissues fixed in glyoxal for both short and long term yielded results comparable to those fixed in formalin. However, careful validation of specific antibody clones should be undertaken when conducting immunohistochemistry evaluations on tissues stored long-term in glyoxal fixatives.

Published in	Advances in Biochemistry (Volume 10, Issue 2)
DOI	10.11648/j.ab.20221002.14
Page(s)	59-70
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2022. Published by Science Publishing Group

Keywords

Fixation, Fixative, Formalin, Glyoxal, Immunohistochemistry, Long-Term, Special Stains

References

[1]	Werner M, Kaloutsi V, Buhr T, Bernhards J, Georgii A. 1992. Immunohistochemical examination of routinely processed bone marrow biopsies. Pathol. Res. Pract. 188 (6): 707-713.
[2]	Collins B, MacNichol E. 1978. Long term fixation for histological studies. Microsco Acta. 81 (2): 155-158.
[3]	Leong A, Gilham P. 1989. The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology. 21: 266-268.
[4]	Kim O. 2003. Effect of fixation time and freeze-thaw cycles on the molecular analysis of viral DNA. J. Vet. Sci. 4 (2): 203-204.
[5]	Dapson RW, Feldman AT, Wolfe D. 2006. Glyoxal Fixation and Its Relationship to Immunohistochemistry. J Histotechnol. 29 (2): 65-76.
[6]	Arber D. 2002. Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast markers. Appl Immunohistochem. Mol. 10 (2): 183-186.
[7]	Clark N. 2002. Fluorochrome Labeled Bone (Part I): Long-term Fixation Results, J. Histotechnol. 25 (2): 101-103.
[8]	Merrell G, Troiano N, Coady C, Kacena M. 2009. Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate. Biotech Histochem. 80: 3-4, 139-146.
[9]	Mastracci L, Gambella A, Bragoni A, Pigozzi S, Pastorino L, Vanni I, Tosi I, Campora M, Fiocca R, Grillo F. 2019. Coping with formalin banning in pathology: under vacuum long-term tissue storage with no added formalin. Histochem. Cell Biol. 151: 501-511.
[10]	Liu X, Schumann C. 2014. Optimization of electron microscopy for human brains with long-term fixation and fixed-frozen sections. Acta Neuropathol. Commun. 2: 42.
[11]	Pikkarainen M, Martikainen P, Alafuzoff I. 2010. The effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers. J Neurophathol Exp Neural. 69 (1): 40-52.
[12]	Nagai M, Minegishi K, Komada M, Tsuchiya M, Kameda T, Yamada S. 2016. Extraction of DNA from human embryos after long-term preservation in formalin and Bouin’s solutions. Congenit. Anom. 56: 112-118.
[13]	Hopwood D. 1969. A comparison of the crosslinking abilities of glutaraldehyde, formaldehyde and α-hydroxyadipaldehyde with bovine serum albumin and casein. Histochemie. 17: 151-161.
[14]	Dapson R. 2007. Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval. Biotech Histochem. 82 (3): 161-166.
[15]	Criswell S, Altman S, Peeler C, Drake T, Lazar C, Douglas S, DeJarnatt V. 2021. Glyoxal fixation: an immunohistochemistry assay evaluation. J Histotechnol, DOI: 10.1080/01478885.2021.1996125.
[16]	Lassalle S. Hofman V, Ilie M, Gavric-Tanga V, Breast P, Havet K, Butori C, Selva E, Santini J, Mograbi B, et al. 2009. Assessment of morphology, antigenicity, and nucleic acid integrity for diagnostic thyroid pathology using formalin substitute fixatives. Thyroid. 19 (11): 1239-1248.
[17]	Gatta L, Cadei M, Balzarini P, Castriciano S, Paroni R, Verzeletti A, Cortellini V, De Ferrari F, Grigolato P. 2012. Application of alternative fixatives to formalin in diagnostic pathology. Eur J Histochem. 56: e12.
[18]	DeJarnatt V, Criswell S. 2020. Glyoxal: a proposed substitute for formalin in H&E and special stains, J. Histotechnology, 44 (1): 37-45.
[19]	Umlas J, Tulecke M. 2004. The effects of glyoxal fixation on the histological evaluation of breast specimens. Hum Pathol. 35 (9): 1058-1062.
[20]	Paavilainen L, Edvinsson A, Asplund A, Hober S, Kampf C, Ponten F, Wester K. 2010. The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells. J Histochem Cytochem. 58 (3): 237-246.
[21]	Richter K, Revelo N, Seitz K, Helm M, Sarkar D, Saleeb R, D’Este E, Eberly J, Wagner E, Vogl C, et al. 2017. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. The EMBO J. 37: e95709.
[22]	Celikkan F, Mungan C, Sucu M, Uysal F, Hayme S, Hayme S, Kuscu N, Ozkavukcu S, Celik-Ozenci C, Can A. 2020. PFA is superior to glyoxal in preserving oocyte, embryo, and stem cell proteins evidenced by super-resolution microscopical surveys of epitopes. J Assist Reprod Gen. 37: 369-384.
[23]	Cook H. Manual of histological demonstration techniques. London: Butterworths; 1974.

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APA Style

Sheila Criswell, Kristina Zwerg, Cynthia Lazar, Caitlin Richmond, Chelsea Bridges. (2022). Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry. Advances in Biochemistry, 10(2), 59-70. https://doi.org/10.11648/j.ab.20221002.14

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ACS Style

Sheila Criswell; Kristina Zwerg; Cynthia Lazar; Caitlin Richmond; Chelsea Bridges. Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry. Adv. Biochem. 2022, 10(2), 59-70. doi: 10.11648/j.ab.20221002.14

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AMA Style

Sheila Criswell, Kristina Zwerg, Cynthia Lazar, Caitlin Richmond, Chelsea Bridges. Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry. Adv Biochem. 2022;10(2):59-70. doi: 10.11648/j.ab.20221002.14

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@article{10.11648/j.ab.20221002.14,
  author = {Sheila Criswell and Kristina Zwerg and Cynthia Lazar and Caitlin Richmond and Chelsea Bridges},
  title = {Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry},
  journal = {Advances in Biochemistry},
  volume = {10},
  number = {2},
  pages = {59-70},
  doi = {10.11648/j.ab.20221002.14},
  url = {https://doi.org/10.11648/j.ab.20221002.14},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ab.20221002.14},
  abstract = {Glyoxal solutions are safer fixatives as compared to formalin in the anatomic pathology laboratory. While long-term effects of tissue stored in formalin are well-documented, studies of tissues fixed long-term in glyoxal are few. Using human autopsy tissues, 4 glyoxal solutions, and neutral buffered formalin, the effects of storage of tissues in fixatives for 8 hours, 1 month, and 4 months were compared. Special stain methods to include hematoxylin and eosin, melanin bleaching using either 10% hydrogen peroxide or potassium permanganate followed by oxalic acid, Verhoeff van Gieson, reticulin, Fontana Masson, mucicarmine, colloidal iron, alcian blue, trichrome, and Wright stain, as well as immunohistochemistry labeling of 18 antibodies were conducted and compared. Tissues fixed in glyoxal solutions exhibited nearly identical staining properties when compared with each other for all lengths of fixation. When fixed for 8 hours, they also produced the same results as tissues fixed in formalin. Although glyoxal-fixed tissues stored for 1 – 4 months in fixatives mimicked staining in formalin-fixed tissues for most special stain techniques, they retained adequate antigenic labeling for only 78% of the antibodies evaluated. Consequently, for special stain applications, tissues fixed in glyoxal for both short and long term yielded results comparable to those fixed in formalin. However, careful validation of specific antibody clones should be undertaken when conducting immunohistochemistry evaluations on tissues stored long-term in glyoxal fixatives.},
 year = {2022}
}

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TY  - JOUR
T1  - Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry
AU  - Sheila Criswell
AU  - Kristina Zwerg
AU  - Cynthia Lazar
AU  - Caitlin Richmond
AU  - Chelsea Bridges
Y1  - 2022/05/31
PY  - 2022
N1  - https://doi.org/10.11648/j.ab.20221002.14
DO  - 10.11648/j.ab.20221002.14
T2  - Advances in Biochemistry
JF  - Advances in Biochemistry
JO  - Advances in Biochemistry
SP  - 59
EP  - 70
PB  - Science Publishing Group
SN  - 2329-0862
UR  - https://doi.org/10.11648/j.ab.20221002.14
AB  - Glyoxal solutions are safer fixatives as compared to formalin in the anatomic pathology laboratory. While long-term effects of tissue stored in formalin are well-documented, studies of tissues fixed long-term in glyoxal are few. Using human autopsy tissues, 4 glyoxal solutions, and neutral buffered formalin, the effects of storage of tissues in fixatives for 8 hours, 1 month, and 4 months were compared. Special stain methods to include hematoxylin and eosin, melanin bleaching using either 10% hydrogen peroxide or potassium permanganate followed by oxalic acid, Verhoeff van Gieson, reticulin, Fontana Masson, mucicarmine, colloidal iron, alcian blue, trichrome, and Wright stain, as well as immunohistochemistry labeling of 18 antibodies were conducted and compared. Tissues fixed in glyoxal solutions exhibited nearly identical staining properties when compared with each other for all lengths of fixation. When fixed for 8 hours, they also produced the same results as tissues fixed in formalin. Although glyoxal-fixed tissues stored for 1 – 4 months in fixatives mimicked staining in formalin-fixed tissues for most special stain techniques, they retained adequate antigenic labeling for only 78% of the antibodies evaluated. Consequently, for special stain applications, tissues fixed in glyoxal for both short and long term yielded results comparable to those fixed in formalin. However, careful validation of specific antibody clones should be undertaken when conducting immunohistochemistry evaluations on tissues stored long-term in glyoxal fixatives.
VL  - 10
IS  - 2
ER  -

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Author Information

Sheila Criswell

Department of Diagnostic and Health Sciences, University of Tennessee Health Science Center, Memphis, USA
Kristina Zwerg

Department of Pathology, Methodist University Hospital, Memphis, USA
Cynthia Lazar

Department of Pathology, Methodist University Hospital, Memphis, USA
Caitlin Richmond

Department of Diagnostic and Health Sciences, University of Tennessee Health Science Center, Memphis, USA
Chelsea Bridges

Department of Diagnostic and Health Sciences, University of Tennessee Health Science Center, Memphis, USA

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APA Style

Sheila Criswell, Kristina Zwerg, Cynthia Lazar, Caitlin Richmond, Chelsea Bridges. (2022). Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry. Advances in Biochemistry, 10(2), 59-70. https://doi.org/10.11648/j.ab.20221002.14

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ACS Style

Sheila Criswell; Kristina Zwerg; Cynthia Lazar; Caitlin Richmond; Chelsea Bridges. Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry. Adv. Biochem. 2022, 10(2), 59-70. doi: 10.11648/j.ab.20221002.14

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AMA Style

Sheila Criswell, Kristina Zwerg, Cynthia Lazar, Caitlin Richmond, Chelsea Bridges. Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry. Adv Biochem. 2022;10(2):59-70. doi: 10.11648/j.ab.20221002.14

Copy | Download

@article{10.11648/j.ab.20221002.14,
  author = {Sheila Criswell and Kristina Zwerg and Cynthia Lazar and Caitlin Richmond and Chelsea Bridges},
  title = {Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry},
  journal = {Advances in Biochemistry},
  volume = {10},
  number = {2},
  pages = {59-70},
  doi = {10.11648/j.ab.20221002.14},
  url = {https://doi.org/10.11648/j.ab.20221002.14},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ab.20221002.14},
  abstract = {Glyoxal solutions are safer fixatives as compared to formalin in the anatomic pathology laboratory. While long-term effects of tissue stored in formalin are well-documented, studies of tissues fixed long-term in glyoxal are few. Using human autopsy tissues, 4 glyoxal solutions, and neutral buffered formalin, the effects of storage of tissues in fixatives for 8 hours, 1 month, and 4 months were compared. Special stain methods to include hematoxylin and eosin, melanin bleaching using either 10% hydrogen peroxide or potassium permanganate followed by oxalic acid, Verhoeff van Gieson, reticulin, Fontana Masson, mucicarmine, colloidal iron, alcian blue, trichrome, and Wright stain, as well as immunohistochemistry labeling of 18 antibodies were conducted and compared. Tissues fixed in glyoxal solutions exhibited nearly identical staining properties when compared with each other for all lengths of fixation. When fixed for 8 hours, they also produced the same results as tissues fixed in formalin. Although glyoxal-fixed tissues stored for 1 – 4 months in fixatives mimicked staining in formalin-fixed tissues for most special stain techniques, they retained adequate antigenic labeling for only 78% of the antibodies evaluated. Consequently, for special stain applications, tissues fixed in glyoxal for both short and long term yielded results comparable to those fixed in formalin. However, careful validation of specific antibody clones should be undertaken when conducting immunohistochemistry evaluations on tissues stored long-term in glyoxal fixatives.},
 year = {2022}
}

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TY  - JOUR
T1  - Comparison of Long-Term Fixation Effects on Tissues Stored in Glyoxal and Formaldehyde as Evaluated by Special Stains and Immunohistochemistry
AU  - Sheila Criswell
AU  - Kristina Zwerg
AU  - Cynthia Lazar
AU  - Caitlin Richmond
AU  - Chelsea Bridges
Y1  - 2022/05/31
PY  - 2022
N1  - https://doi.org/10.11648/j.ab.20221002.14
DO  - 10.11648/j.ab.20221002.14
T2  - Advances in Biochemistry
JF  - Advances in Biochemistry
JO  - Advances in Biochemistry
SP  - 59
EP  - 70
PB  - Science Publishing Group
SN  - 2329-0862
UR  - https://doi.org/10.11648/j.ab.20221002.14
AB  - Glyoxal solutions are safer fixatives as compared to formalin in the anatomic pathology laboratory. While long-term effects of tissue stored in formalin are well-documented, studies of tissues fixed long-term in glyoxal are few. Using human autopsy tissues, 4 glyoxal solutions, and neutral buffered formalin, the effects of storage of tissues in fixatives for 8 hours, 1 month, and 4 months were compared. Special stain methods to include hematoxylin and eosin, melanin bleaching using either 10% hydrogen peroxide or potassium permanganate followed by oxalic acid, Verhoeff van Gieson, reticulin, Fontana Masson, mucicarmine, colloidal iron, alcian blue, trichrome, and Wright stain, as well as immunohistochemistry labeling of 18 antibodies were conducted and compared. Tissues fixed in glyoxal solutions exhibited nearly identical staining properties when compared with each other for all lengths of fixation. When fixed for 8 hours, they also produced the same results as tissues fixed in formalin. Although glyoxal-fixed tissues stored for 1 – 4 months in fixatives mimicked staining in formalin-fixed tissues for most special stain techniques, they retained adequate antigenic labeling for only 78% of the antibodies evaluated. Consequently, for special stain applications, tissues fixed in glyoxal for both short and long term yielded results comparable to those fixed in formalin. However, careful validation of specific antibody clones should be undertaken when conducting immunohistochemistry evaluations on tissues stored long-term in glyoxal fixatives.
VL  - 10
IS  - 2
ER  -

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