RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE,
KARNATAKA
TOPIC
“A CYTOMORPHOLOGICAL STUDY OF TOLUIDINE BLUE RAPID STAINING
METHOD IN FINE NEEDLE ASPIRATION CYTOLOGY REPORTING”
Dr. VIVEK T G
POSTGRADUATE
DEPARTMENT OF PATHOLOGY
K.V.G. MEDICAL COLLEGE & HOSPITAL
SULLIA (D.K)
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE-II
1.
NAME OF THE CANDIDATE AND
Dr. VIVEK T G
ADDRESS (IN BLOCK LETTERS)
S/O Dr T T GURURAJA
#004,
BLOSSOM
BLOCK,
VIVEK
MARVEL APARTMENTS, SURABHI
LAYOUT,
SHIVANAHALLI,
YELAHANKA, BANGALORE – 560064
KARNATAKA.
2.
NAME OF THE INSTITUTION
K.V.G. MEDICAL COLLEGE AND
HOSPITAL,
KURUNJIBAG,SULLIA
D.K. -574327
KARNATAKA.
3.
COURSE OF STUDY AND SUBJECT
M.D. PATHOLOGY
4.
DATE OF ADMISSION TO COURSE
31.08.2013
5.
TITLE OF TOPIC
“CYTOMORPHOLOGICAL STUDY OF
TOLUIDINE BLUE RAPID STAINING
METHOD IN FINE NEEDLE
ASPIRATION CYTOLOGY
REPORTING”
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need
for the Study
Fine needle aspiration cytology (FNAC) has been gaining importance as a result
of its cheap cost, easy performance, safety, along with fair specificity and specificity
especially in terms of sorting out malignant cases.1
Earlier rejection and scepticism by pathologist and clinicians of fine needle
aspiration cytology (FNAC) has gradually diminished and this technique is practiced
worldwide. 2
Routine use of FNAC and on-site assessment of the adequacy of cytopathologic
specimens should dramatically reduce the rate of failures and, thus, the cost of a true
diagnosis.3
FNAC can be used in place of open biopsy in most patients to facilitate
preoperative decision making and/or to avoid unnecessary invasive procedure for patients
with primary or metastatic lesions.2
There is an ever-increasing demand for rapid diagnosis in cytology. Quick–staining
techniques such as Diff-Quick or toluidine blue are routinely used. Papanicolaou (PAP) or
Hematoxylin and eosin (H&E) stains may also be used in a rapid stain setting, but these
take a little longer to prepare. 2
The onsite processing and interpretation of cytological specimens by
cytotechnologist has a significant clinical impact by increasing the diagnostic yield of
endoscopic ultrasound fine needle aspiration cytology.4
6.2 Review of Literature
Ling and Di in their study stained the smears with 0.06% toluidine blue ethanol
solution for rapid preliminary evaluation, then fixed in 95% ethanol again to de-stain the
toluidine, and finally stained with H&E.5
Camel et al concluded that the toluidine blue method is a reproducible and reliable
way of demonstrating ganglion cells in frozen rectal biopsies. This method provides faster
and easier identification than with H&E staining, which can ultimately be used to exclude
a diagnosis of Hirschsprung’s disease.6
Lambaht at al concluded the positive and the negative predictive value of the test
using toluidine blue and a consultant pathologist were very high which suggests that the
technique (imprint cytology stained with toluidine blue) could be applied as a diagnostic
tool with the reassurance that a positive result can be wholly relied upon.7
The accuracy of toluidine blue/eosin stained wet mount study is assessed by
confirming the rapid diagnoses with final diagnosis by conventional FNAC using H&E,
histopathological examination and clinical follow up. The reliability of this wet mount
study was also compared with works done by various authors and is proved to be accurate.
The study results yielded good diagnostic accuracy of 97.4% by combining rapid stain as a
supplementary procedure for conventional H&E.8
The results of Jing X et al proved the benefit of performing on-site assessment of
specimen adequacy in FNA diagnosis of thyroid nodules in terms of a decrease in non
diagnostic rate and an increase in diagnostic yield, regardless of the implementation of the
standard criteria for assessing specimen adequacy and diagnostic terminology.9
Eedes and Wang demonstrated that immediate assessment of specimen adequacy
increases (by approximately 20%) but does not guarantee the diagnostic yield of thyroid
FNA biopsies.10
Toluidine blue was found to be a reproducible and reliable way of demonstrating
the adequacy of material in FNACs of thyroid, breast lump and lymph node. This method
provides faster and easier identification of cells, which can be used to give a provisional
diagnosis in the majority of cases. The technique provided valuable information for the
pathologist and it was the comment that, it is easy enough to incorporate in the routine
investigation of general laboratories without extensive modification of their work style.2
Ammanagi et al used a simple, rapid toluidine blue method to stain smears for the
evaluation of the adequacy of the aspirate. They compared toluidine blue stained smears
with the same smears PAP stained later after decolorizing.2
Sumathy et al concluded that the supravital stained rapid wet mount FNAC (using
toluidine blue rapid staining) is useful as, a simple reliable, cost effective rapid staining
method. It would help to obtain sufficient cellularity in less cellular fibrotic lesions. It can
also used to assess adequacy of sample especially for deep seated lesions and minimize
false negative results. They observed that cytomorphology can be well appreciated in wet
mount study. It can be used for intra operative cytodiagnosis as an adjunct to frozen
section diagnosis. It can improve the diagnostic accuracy of conventional FNAC’s stained
with H&E. It can be routinely undertaken as a supplementary procedure for conventional
H&E.8
Joy et al they concluded that toluidine blue stain is a way of marking tissue for
microscopic examination. It stains nucleic acids blue and also increases the sharpness of
cytological smears.They observed that toluidine blue staining is not only a reliable method
for rapid staining and diagnosis, but also permits preservation of cytological material by
de-staining and re-staining with permanent stains.11
6.3. Objectives of the study:
a. To assess the adequacy of the sample of fine needle aspiration cytology.
b. To compare the conventional hematoxylin and eosin staining with toluidine blue.
c. To assess the efficacy in differentiating the benign and malignant lesion by using
toluidine blue stain.
7.
Materials and Methods
7.1 Source of Data
Analysis of fine needle aspiration cytology specimen of 150 patients
who are referred to the Department of Pathology, K V G Medical College and Hospital,
Sullia (D.K).
Study period will be for 15 months from November 2013 to April 2015.
Sample Size: Intended to study 150 cases.
7.2 Method of Data Collection
The FNAC will be performed using 22/21 gauze needles. Slides will be
fixed with fixatives, stained with toluidine blue and then examined and will be reviewed.
Patient will be explained about the procedure and consent will be taken.

Preparation of toluidine blue stain2,6:
Materials required
1. Toluidine blue-2.8gm
2. Urea-4.0 gm
3. Ethyl alcohol-280 ml
4. Distilled water- 12ml
Urea will be dissolved in water and will be slowly mixed with alcohol. Toluidne blue dye
will be added, mixed well and filtered.

Procedure of staining2,6:
1. Slides will be wet fixed for at least 15 seconds.
2. Slides will be stained in toluidine blue solution- 10 dips.
3. Slides will be rinse in tap water; water mounting with a coverslip will be done.
4. The undersurface of the slide will be wiped with tissue paper.
5. Evaluation under the microscope for the technical adequacy and preliminary diagnostic
impression, particularly concerning the presence of atypia in the cells and possible
neoplasia will be done. FNAC will be repeated if the material is inadequate.
6. Conventional H & E stain will be performed on the same smears after destaining with
95% ethyl alcohol for evaluation.
Inclusion Criteria
All the FNAC performed in KVG Medical College and Hospital, Sullia will
be included in the study.
Exclusion Criteria

Patients unwilling to undergo the FNAC will be excluded from the study.

Inadequate smears even after repeated aspirations will be excluded from the study
Follow up: Not required.
Follow up period: Not applicable.
Statistical analysis:
Analysis of Data will be done using appropriate statistical methods.
7.3 Does the study require any investigation\intervention to be conducted on patients\
humans\ animals? If so, please describe briefly:
Yes, As a routine patients referred from various departments are subjected to Fine Needle
Aspiration and stained as described above for evaluation.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes. Copy of Ethical Committee Clearance attached.
8. REFERENCES
1. Rekhi B, Gorad BD, Kakade AC, Chinoy RF. Scope of FNAC in the diagnosis of soft
tissue tumors- A study from a tertiary cancer referral center in India, Cyto J 2007;20 (4).
2. Ammanagi AS,Dombale VD, Patil SS.On- site toluidine blue staining and screening
improves afficacy of Fine-Needle Aspration Cytology Reporting. Actacytologica
2012;56:347-351.
3. Borget I, Vielh P, Leboulleux S, Allyn M, Iacobelli S, Schlumberger M, et al.
Assessment of the Cost of Fine-Needle Aspiration Cytology as a Diagnostic Tool in
Patients With Thyroid Nodules. American J Clinical Pathology 2008;129:763-771.
4 Alsohaibani F, Girgis S, Sandha GS. Does onsite cytotechnology evaluation improve the
accuracy of endoscopic ultrasound-guided fine-needle aspiration biopsy?. Cancer J
Gastroenterology 2009;23(1):26-30.
5 Wong NL, Di F. Pseudosarcomatous Fasciitis and Myositis Diagnosis by Fine-Needle
Aspiration Cytology. American J Clinical Pathology 2009;132:857-865.
6. Canil M, Meir K, Jevon G, Sturby T, Moerike S, Gomez A. Toluidine Blue Staining Is
Superior to H&E Staining for the Identificationof Ganglion Cells in Frozen Rectal
Biopsies. Histologic 2007;40:1-3.
7. Lambah PA, McIntyre MA, Chetty U, Dixon JM. Imprint cytology of axillary lymph
nodes as an intraoperative diagnostic tool. European J Surgical Oncology 2003;29:224228.
8. Sumathy C, Suresh Durai J, Swaminathan K, Vallimanalan S, Munavarah SA.
Supravital Stained Rapid Wet Mount Preparation of Fine Needle Aspirates – A
Cytomorphological Study. Tirunelveli e J Medical Sciences 2012;3:62-66.
9. Jing X, Michael CW, Pu RT. The Clinical and Diagnostic Impact of Using Standard
Criteria of Adequacy Assessment and Diagnostic Terminology on Thyroid Nodule Fine
Needle Aspiration. Diagnostic Cytopathology 2008;36:161-166.
10. Eedes CR, Wang HH. Cost - Effectiveness of Immediate Specimen Adequacy
Assessment of Thyroid Fine-Needle Aspirations. American J Clinical Pathology
2004;121:64-69.
11 Joy MP, Iyer VK, Aron M, Kapila K, Verma K. Rapid staining using toluidine blue: a
reliable method for quick diagnosis in ultrasound guided aspiration cytology. Indian J
Pathology and Microbiology 2003 ;46(4):589-592.
9
SIGNATURE OF CANDIDATE
(Dr. VIVEK T G)
10 REMARKS OF THE GUIDE
11 11.1 Name and designation of Dr. K P KARUMBAIAH
Guide
ASSOCIATE PROFESSOR OF PATHOLOGY,
K.V.G
MEDICAL
COLLEGE
&
HOSPITAL,
SULLIA (D.K).
11.2 Signature
11.3 Head of department
Prof. T M KARIAPPA
PROFESSOR & H.O.D OF PATHOLOGY,
K.V.G
MEDICAL
SULLIA (D.K).
11.4 Signature
12 12.1 Remarks of the Principal
12.2 Principal signature
COLLEGE
&
HOSPITAL,
ETHICAL COMMITTEE CLEARANCE
1
TITLE OF DISSERTATION:
A
CYTOMORPHOLOGICAL
STUDY
OF
TOLUIDINE BLUE RAPID STAINING METHOD
IN FINE NEEDLE ASPIRATION CYTOLOGY
REPORTING.
2
NAME OF THE CANDIDATE:
Dr. VIVEK T G
3
NAME OF THE GUIDE:
Dr. K P KARUMBAIAH
ASSOCIATE PROFESSOR OF PATHOLOGY.
4
APPROVED/NOT APPROVED:
5
LAW EXPERT:
Mr. KRISHNA MURTHY
ADVOCATE
PRINCIPAL
K.V.G MEDICAL COLLEGE AND HOSPITAL, SULLIA (D.K).