Re: [Histonet] validation

[Tony Henwood (SCHN) via Histonet]

Immunohistochemistry validation is much more than simply 20 positives and 20 
negative cases, though this will allow you to meet most accreditation 
requirements.

Validation is a multi step process:
1.  Optimisation: following a thorough literature review (what should and 
should not stain, clones used (any difference), recommended assay conditions 
(eg antigen retrieval type), choose an appropriate control to confirm staining 
conditions and perform an optimum titre.
2.  Validation: based on the literature review, choose appropriate known 
(or expected) positives and negative cases that the pathologists would 
entertain in the differential diagnosis that should be negative.

Take Prostate Specific Antigen as an example, include known prostatic 
carcinomas with a mix of well, moderately and poorly differentiated prostatic 
carcinomas and include negative tumours that should not express PSA (eg 
melanomas, lymphomas, colonic and lung carcinomas).

This is a scientific approach that inspires confidence in the usefulness of the 
test.

3.  Another task that we find extremely useful is on-going Verification, 
where we compare staining results with final diagnosis of cases immunostained 
for the marker.

Food for thought?

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: Nancy Schmitt via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 25 October 2017 4:16 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] validation

Good Day!
When performing validation on IHC, we use 20 positives and 20 negative cases.

When testing for negative, can the normal tissue surrounding the tumor be 
counted as negative?

Does the negative tissue need to be tumor that is negative for the AB or just 
normal tissue?

Thank you for your consideration,

Nancy
Dubuque, IA
Ph. 563-589-9810



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[Histonet] validation inquiry regarding negative tissue

[Eddie Martin via Histonet]

Hi Nancy,

The use of negative internal control tissue on your antibody testing for 
negative cases would have to be determined by your medical director/chief 
pathologist as acceptable. 

You may also use Lipoma tissue as a negative control tissue. It works as a 
negative control for just about every antibody I can think about. There may 
also be a few antibodies that would have false positive staining with a lipoma, 
but a pathologist could read through that stuff. 

Hope this helps!

Kind regards,
Eddie M.

Edward M. Martin, HTL, HT(ASCP), QIHC(ASCP)
Lead Hematopathology Medical Technologist
The National Institutes of Health
and
IHC Histotechnologist III
The Joint Pathology Center
Walter Reed NMMC
Bethesda, MD


> On Oct 24, 2017, at 1:16 PM, Nancy Schmitt  wrote:
> 
> Good Day!
> When performing validation on IHC, we use 20 positives and 20 negative cases.
> 
> When testing for negative, can the normal tissue surrounding the tumor be 
> counted as negative?
> 
> Does the negative tissue need to be tumor that is negative for the AB or just 
> normal tissue?
> 
> Thank you for your consideration,
> 
> Nancy
> Dubuque, IA
> Ph. 563-589-9810
> 
> 
> 
> NOTICE: This email may contain legally privileged information. The information
> is for the use of only the intended recipient(s) even if addressed
> incorrectly. If you are not the intended recipient, please notify the sender
> that you have received it in error and then delete it along with any
> attachments. Thank you.
> 
> 
> 
> 
> NOTICE: This email may contain legally privileged information. The information
> is for the use of only the intended recipient(s) even if addressed
> incorrectly. If you are not the intended recipient, please notify the sender
> that you have received it in error and then delete it along with any
> attachments. Thank you.
> 
> 
> 

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Re: [Histonet] H LFB

[Patrick Dooley via Histonet]

Here's my lab's SOP for an LHE. 7uM sections. Hope this helps!

1.Deparaffinize and hydrate to absolute ethanol.

2.Luxol Fast Blue 60° C oven 1hr.

3.Cool to room temperature.

4.Hydrate to water.

5.2 - 3 dips reducer. The fresher the reducer the less number of dips
you need. You'll need to look under the microscope to determine an adequate
differentiation; the best is when you can still clearly see the axonal
processes extending into the neocortex but with not much staining in the
grey matter at all.

6.Rinse in tap water until water is clear.

7.Harris Hematoxlyn 10 min.

8.  Rinse in tap water until water is clear.

9.  Differentiate in acid alcohol.  This usually takes 1-2 dips. Again,
look under the microscope to detemine a sufficient stopping point to
differentiation.

10.  Rinse in tap water until water is clear.
11.  Eosin for 5 - 7 minutes.
12. Rinse in tap water until water is clear.
13. Dehydrate to absolute ethanol. This step adequately reduces the
background eosin in our lab, but you may need to leave/dip more in 95%
ethanol as that will differentiate it the best.
14. 10 dips in two separate changes of xylene.
15. Coverslip.

Solutions are as follows:

Luxol Fast Blue10 g Luxol Fast Blue MBS

1 liter 95% ethanol

0.5 ml glacial acetic acid



Reducer 10 g hydroquinone

50 g Na2SO4

1 liter distilled water



Acid Alcohol 10 ml conc. HOAc

1 liter 70% ethanol



Eosin  0.5% aqueous Eosin Y

when first used, add 1 ml glacial HOAc
/ 400 ml eosin

Patrick Dooley, HTL(ASCP)

Research Tech I / Tissue Bank Coordinator

MGH Alzheimer’s Disease Resource Center

CNY 114 Rm. 2650

617-643-6994(office)
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[Histonet] Cryostat question

[Charles Riley via Histonet]

Does anyone out there use the Avantik QS12 cryostats? We have a machine that is 
only a year and a half old and have had to get the Peltier system (the cryobar) 
replaced 4 times. Is anyone else having this problem consistently? 

Sent from my iPhone
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