[Histonet] Unstained slides precut for IHC

[Tony Henwood (SCHN)]

Hi Carrie,
Over-heating of sections (and tissue blocks) for IPX is probably one of the most significant and unappreciated  pre-analytical factors that can affect immunolocalisation. We closely scrutinise slides closely for overheating, especially those sent to us for immunostaining. We strongly prefer air-dried sections that we place on our immunostaniners (we have both a Bond and Ventana Benchmark). The following might be useful:

Controlled Section Baking for Immunohistochemistry
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 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

________________________________________
From: Carrie Disbrow via Histonet <histonet at lists.utsouthwestern.edu>
Sent: Monday, 14 June 2021 12:04
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Unstained slides precut for IHC

Hi! Current lab is cutting extra slides for IHC and putting in 60 degree oven for 45 mins to dry.  If IHC is ordered a precut control and the already baked slides are again put in the 60 degree oven for 45 mins. Sometimes the control is cut and added to the dry slide or a separate slide.  So, the questions  have been should the   slides be air dried first using a fan before baking and slides not baked until the pathologist places an order? Should the tissue and the control all have the same amount of time in the oven to ensure consistency? Also, is it better to rack slides standing or on edge for IHC? Additionally, when sending slides for IHC to other labs is it preferred to send dry slides or baked slides? Thanks for your input!
Carrie Disbrow, HTL

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