[Histonet] IHC questions

Tue Jul 25 19:22:50 CDT 2017

Hi Vicki,

We recently did a study that we published in our local journal (Histograph June 2017) that migh be of use:

Controlled Section Baking for Immunohistochemistry
Tony Henwood, Principal Scientist, Histopathology, The Children's Hospital at Westmead

One source of poor immunostaining is overheating of tissue and sections. Several authors have reported that heated slide drying adversely affects sensitivity in immunohistochemistry (1). Therefore, some have advocated the use of lower temperature drying using adhesive-coated slides to improve the sensitivity of the test (2-5).

In one study (1), half the antigens were adversely affected by section drying at 80oC including 5D3, CMV, S100, HMB45 and CEA. Oates (4) used antisera to epithelial membrane antigen from three different companies and found that for slides dried at 58"C, staining was often paler than slides dried at room temperature or at 37°C.

Low heat attachment of sections to slides can cause several issues including inadequate attachment of the tissue sections so that tissue sections may be lost during antigen recovery and/or immunostaining, the inability of some paraffins to melt well at 58oC, and the requirement of more than 1 hr before an immunohistochemical procedure may be started. It has been recommended that the most efficient protocol for mounting tissue sections to microscopic slides would be to attach the tissues overnight before applying the immunohistochemical procedure at a temperature at which all tissue mounting paraffins should melt (e.g., 65oC) (6). It should be remembered that a significant dewaxing of sections occurs when slides are heated a few degrees above the melting point of the wax.
Laboratory ovens seem to be variable in their ability to maintain a constant temperature with the implication that it is possible to either over-cook sections thus adversely affecting antigens or under-heat them, possibly compromising subsequent de-waxing. There is also the human element. How often are slides removed from the oven at the required time?

The modern automatic immunostainers have excellent on-board slide heating in order to achieve reproducible, accurate antigen retrieval. This feature also allows controlled "baking" of sections and being able to programme a set time, removes the possibility of human error. At the Children's Hospital, the Bond 3 is used for automated immunohistochemistry. A study was designed to assess the usefulness of on-board baking in routine immunohistochemistry.

Control sections were immunostained for several antigens (see table) using the Bond 3 on-board baking and dewax facility. Freshly cut sections were dried at 37oC for 5 minutes to remove excess water. Slides were then loaded onto the Bond and the baking procedure used was 35 minutes at 63oC. Stained controls were compared with control slides stained prior to the instigation of the on-board bake procedure. The historic procedure involved heating sections at 63-65oC for 35 minutes in a large fan-forced dry-air oven (7).

BCL-2

Mum-1

CD31

BCL-6

Calretinin

SATB2

BOB-1

S100

CyclinD1

CD20

ALK-1

MPO

CD21

Ki67

INI-1

CD3

Synaptophsin

BRG-1

HMB-45

Chromogranin

Inhibin

Melan A

Myogenin

Desmin

The results showed that there was no difference between controls stained with the historic compared to the on-board baking procedure except for BCL-6 which the new procedure gave stronger staining. (see figure).

In conclusion, we expect that on-board baking of sections should allow laboratories to have better control over the pre-analytical variables that can adversely affect the immunohistochemistry staining.

References

1.       Henwood, A. F. (2005). Effect of slide drying at 80oC on immunohistochemistry. Journal of Histotechnology, 28(1), 45-46.

2.       Wakins, J., Kellock, D., Gillet, C., Egan, M., Pontin, J. E., Millis, R. R., & Levinson, D. A. (1990). Enhancement of immunostaining. Histopathology, 17(2), 185-185.

3.       Dodson, A., Davies, E., & Waring, J. (1991). APTES, a section adhesive for immunocytochemistry; and experiences of slide drying at room temperature. Histopathology, 19(5), 484-485.

4.       Oates J. (1993) The effect of temperature on immunostaining. Br J Biomed Sci 50: 157-158,

5.       Williams, J. H., Mepham, B. L., & Wright, D. H. (1997). Tissue preparation for immunocytochemistry. Journal of clinical pathology, 50(5), 422-428.

6.       Jones, W. T., Stockard, C. R., & Grizzle, W. E. (2001). Effects of time and temperature during attachment of sections to microscope slides on immunohistochemical detection of antigens. Biotechnic & Histochemistry, 76(2), 55-58.

7.       Henwood, A. F. (2012). The application of heated detergent dewaxing and rehydration to immunohistochemistry. Biotechnic & Histochemistry, 87(1), 46-50.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)

Principal Scientist, the Children's Hospital at Westmead

Adjunct Fellow, School of Medicine, University of Western Sydney

Tel: 612 9845 3306

Fax: 612 9845 3318

Pathology Department

the children's hospital at westmead

Cnr Hawkesbury Road and Hainsworth Street, Westmead

Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-----Original Message-----
From: Gauch, Vicki via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Sent: Tuesday, 25 July 2017 11:25 PM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] IHC questions

Hi all,

I have been asked to post several questions regarding IHC stain issues and am hoping someone has some ideas...  We are having issues with IHC cases floating (particularly breast IHC) lately and are wondering....

1)      How are people storing IHC control slides?  Are they being precut and stored or cut at the time of the stain request?

2)      If storing controls- are you doing anything special ?

3)      How long are the IHC slides being air dried before placing in the oven ?  How long are they in the oven prior to staining?

This has not been such an issue in the past but recently we are experiencing it more frequently. We are looking at various things  but if anyone has any suggestions, they would be greatly appreciated !!!

Thanks,

Vicki Gauch

AMCH

Albany, NY

-----------------------------------------

CONFIDENTIALITY NOTICE: This email and any attachments may contain confidential information that is protected by law and is for the sole use of the individuals or entities to which it is addressed. If you are not the intended recipient, please notify the sender by replying to this email and destroying all copies of the communication and attachments. Further use, disclosure, copying, distribution of, or reliance upon the contents of this email and attachments is strictly prohibited. To contact Albany Medical Center, or for a copy of our privacy practices, please visit us on the Internet at www.amc.edu<http://www.amc.edu>.

_______________________________________________

Histonet mailing list

Histonet at lists.utsouthwestern.edu<mailto:Histonet at lists.utsouthwestern.edu>

http://lists.utsouthwestern.edu/mailman/listinfo/histonet

This message is intended for the addressee named and may contain confidential information. If you are not the intended recipient, please delete it and notify the sender.

Views expressed in this message are those of the individual sender, and are not necessarily the views of NSW Health or any of its entities.