COMPARATIVE STUDY OF COMPARATIVE STUDY OF ER/PR/Cerbb2 USING FAA ER/PR/Cerbb2 USING FAA FIXATIVE AND 10% BUFFER FIXATIVE AND 10% BUFFER FORMALINFORMALIN

Mr. G. P. TiwariDepartment of Pathology

Tata Memorial Hospital

IntroductionIntroduction

ER/PR/Cerb2 are the most important prognostic and predictive factors in breast carcinomas.

IHC demonstration of these biomarkers remains the main stay of assessment.

However it is affected by many technical factors especially fixation.

Introduction ctd Introduction ctd

FAA fixative or AFA is an alcoholic based fixative with a combination of 3 chemicals formaldehyde, alcohol, acetic acid. It was first described by William Davidson and William Hartmann in the year 1953.

Most European countries use alcoholic formalin to fix breast cancer tissues for better preservation of ER receptor.

QuestionsQuestions

Is FAA cost effective?Should FAA be advocated for all

round use?- as many labs may not have the resources.

AIMS & OBJECTIVESAIMS & OBJECTIVES

To evaluate if FAA helps in improving the results of ER/PR/cerb2 in breast cancer.

To compare the results with buffered formalin.

INGREDIENTS OF ORIGIONAL INGREDIENTS OF ORIGIONAL FAAFAA

ETHANOL(95-100%): 85.0 ML

FORMALIN(37-40%): 10.0 ML

GLACIAL ACETIC ACID: 5.0 ML

If the concentration of the acetic acid is 5%, it brings swelling in the tissue and Slides becomes hazy and patchy staining with IHC was observed but 0.5% acetic acid concentration is sufficient to over come this problem.

MODIFIED FAA SOLUTIONMODIFIED FAA SOLUTION

ETHANOL (95 – 100 %) :- 85.0 ML

BUFFERED FORMALIN(37-40%) :-10 ml

GLACIAL ACETIC ACID :- 0.5 ML

The amount of acetic acid or formalin may be varied.

Methanol or isopropyl alcohol may be used in stead of ethanol

Acetone, methanol, ethanol, isopropanol

Displace water molecules from pertinacious materials and extract lipids from the tissue

CHEMISTRY OF FAA FIXATIVECHEMISTRY OF FAA FIXATIVE

Acetic acid

Coagulates nucleic acid and enhances the rate of penetration of formalin in the

tissue

The tissue is fixed rapidly as compared to 10% buffered formalin

CHEMISTRY OF FAA FIXATIVECHEMISTRY OF FAA FIXATIVE

ADVANTAGES OF FAAADVANTAGES OF FAA

Inhibits formation of formalin pigment Rapid fixative(5mm in 4 hrs.) LESS-toxic Less expensive Easy to prepare Osmotic pressure is well maintained It permits all most all type of staining and

immunohistochemistry stains Turn around time is improved for 2 days

TECHNICAL CONSIDERATIONSTECHNICAL CONSIDERATIONS

The mixture can be kept indefinitely but its chemical composition changes considerably with storage..

The concentration of ethanol and formaldehyde decline, so the fixative property also declines, hence must be prepared fresh everyday.

ROUTINE PROCESSING SCHEDULE ROUTINE PROCESSING SCHEDULE FOR BREAST TISSUESFOR BREAST TISSUES

Fixation- 48 hours Dehydration- 06 hours Clearing- 06 hours Impregnation- 06 hours Total Processing Time-22-23 hours. Turn Around Time-4 days for slide

submission, 6 days after ER,PR

Material and MethodMaterial and Method

25 cases of primary operable breast cancer tissues who had not received prior chemotherapy were evaluated.

Two sections from tumour as similar as possible were taken. One was fixed in buffered formalin and the other in FAA.

The tissue sections were coded blindly unknown to the pathologist for identification as to what was the nature of fixation.

RESULTSRESULTS

H & E – Cellular swelling but less shrinkage

Out of 25 samples 10 cases are negative 13 cases are positive 2 cases are without tumor

Marker FAA BF

ER+ PR + 8++ 8

ER+ PR - 2 2

ER- PR + 1 0

ER- PR - 9 10

Cerb2 + 0 1*

Cerb2 - 20 19

Two cases were without tumor

*On review false reported as positive due to cytoplasmic staining

++ Score improved by score + 1 in three cases

Assessment in 23 casesAssessment in 23 cases

Improved H & E

NBF FAA

COST/BENEFITCOST/BENEFIT

NBF COSTLY LESS

PENETRATION TURN AROUND

IS MORE

FAA CHEAPER RAPID

PENATRATION TURN AROUND

TIME IS LESS

CONCLUSIONCONCLUSION

Though number of cases is small – a marginal benefit was observed

Results are promising and we are processing more cases

Similar study for molecular preservation in FAA Fixed tissues should be done

REFERENCESREFERENCES

1. .JOHN D. BANCROFT AND ALAN

2. STEVEN THEORY AND PRACTICE OF HISTOLOGICAL TECHNIQUES 2nd EDITION 1982

3. CARLETON H. M HISTOGICAL TECHNIQUE LONDON4. C.F.A CULLING R.T. ALLISON, W.T. BARR CELLULAR

PATHOLOGICAL TECHNIQUES 4th EDITION 1985

5. HISTOLOGICAL AND HISTOCHEMICAL METHOD BY KIERNAN 1985

6. Biotech Histochem. 1993 Mar;68(2):75-82.