STANDARD OPERATING PROCEDURE (SOP) FOR PAPANICOLAOU STAINING

1.0 Purpose

To establish a consistent procedure for Papanicolaou staining of FNA Fluids and conventional smear LBCs received in the Cytopathology section.

2.0 Scope

This procedure is applicable to all types of smears after initial fixation.

3.0 Introduction

Papanicolaou staining is utilized to differentiate cells in smear preparations of various bodily secretions, including gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells. It is a reliable technique commonly employed for cervical cancer screening in gynecology, collectively known as Pap smear.

4.0 Principle

The classic Pap stain involves three dyes in separate solutions:

Haematoxylin is used as a nuclear stain, imparting a blue-black color to cell nuclei.

Orange G-6 is employed as a counterstain, primarily staining keratinized cells.

Eosin Azure, comprising Eosin Y and Light Green SF yellowish, stains superficial epithelial squamous cells, cytoplasm of other cells, nucleoli, cilia, and red blood cells.

5.0 Analytical Measuring Range and Limit of Detection

Not Applicable

6.0 Responsibility

Technical staff and pathologists are responsible for the execution and supervision of the procedure.

7.0 Abbreviations

Nil

8.0 Requirements

Kit Reagents:

a) Alcohol with varying concentrations (70%, 90%, absolute).

b) Harris's Hematoxylin

c) 0.5% aqueous HCL

d) 0.1% Ammonia aqueous

e) OG6-(Ready to Use)

f) EA50-(Ready to Use)

g) DPX

h) Light gree

Instrumentation and Software:

a) Centrifuge

b) Slides

c) Cover slip

d) Diamond pencil

Disposables:

a) Gloves

Specimen Collection and Handling:

a) Specimen Collection: Room Temperature, slides must be fixed immediately after smear preparation.

b) Specimen Transport: Room Temperature

c) Specimen Storage: Room Temperature

d) Specimen and Control Preparation: Nil

9.0 Precautions

All specimens should be handled with gloves.

Instruments should be handled carefully to prevent injury.

10.0 Limitations & Interferences

Not Applicable

11.0 Documentation

All documents are maintained as follows:

a) Cytology entry register

b) Cytology stain adequacy form

c) Cytology reagent change form

12.0 Instructions

12.1 Pre Analytical Steps: Including sample preparation and reagent preparation.

12.2 Analytical Steps:

Smears washed with running tap water for 2 to 5 minutes.

Hematoxylin application for 3 minutes.

Wash with running tap water for 2 to 5 minutes.

Differentiation with 0.5% aqueous Acid Alcohol for 5 to 10 seconds, followed by washing with tap water for 2 to 5 minutes.

Blueing in 0.1% aqueous ammonia solution for 5 to 10 seconds, then washing with running tap water for 2 to 5 minutes.

OG6 application for 4 minutes or LBC for 5 minutes, with each change using 95% alcohol for 10 seconds.

EA50 application for 2 minutes or LBC for 1 minute and 30 seconds, with each change using 95% alcohol for 10 seconds.

Clearing with Xylene for 2 to 3 minutes.

Mounting with DPX and labeling the slides.

Submitting slides to the Pathologist along with the requisition form.

12.3 Quality Control and Assurance

Evaluation of a suitable slide of each type for staining adequacy before each batch.

Calibration and Calibration verification: Not Applicable

12.4 Storage of Samples

Cytology smears are retained for 10 years at room temperature. Trained staff are available for backup in case of staff shortage.

13.0 Reference Range

Not Applicable

14.0 Critical Value

Not Applicable

15.0 Reporting of Results and Interpretation

Results are reported following established Bethesda Protocols in approved report formats.

16.0 Contingency Plan

Suitable trained Staff are available for backup in case of staff shortage or other contingencies.

17.0 References

Bancroft Theory and Practice of Histological Techniques by John D Bancroft and Marilyn Gamble; 2007 (6th ed).

END OF THE DOCUMENT

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