STANDARD OPERATING PROCEDURE (SOP) FOR CUTTING PARAFFIN WAX BLOCKS

Standard Operating Procedure (SOP) for Cutting Paraffin Wax Blocks

1.0 Purpose

To establish a procedure for cutting all paraffin blocks in the Surgical Pathology Department.

2.0 Scope

This SOP is applicable to all biopsy specimens received and outlines the procedures for all technical staff in the section.

3.0 Introduction

Good quality cutting of sections and staining are prerequisites for satisfactory morphological examination of tissue. Poor quality sections increase the risk of missed findings or erroneous reporting by the pathologist.

4.0 Principle

Paraffin wax acts as a solid medium to support the tissue, enabling thin sections to be cut without damaging the knife or tissues.

5.0 Analytical Measuring Range and Limit of Detection

Not applicable.

6.0 Responsibility

- Technical Staff

- Pathologist

7.0 Abbreviations

None.

8.0 Requirements

Instrumentation and Software

- Rotary Microtome

- Water Bath

- Slide Warmer

- Brush

9.0 Precautions

- Handle knives with care to prevent injury.

- Blot the surface of the water in the flotation bath after each block to prevent floaters.

10.0 Limitations & Interferences

Not applicable.

11.0 Documentation

All documents are maintained in the following:

- Block entry register

- Temperature log file

12.0 Instructions

12.1 Pre-Analytical Steps

12.1.1 Preparation of Egg Albumin

1. Take the white of 1 egg into a measuring cylinder.

2. Add equal parts of glycerine.

3. Mix well and add a pinch of thymol powder.

4. Keep at room temperature.

5. Discard when turbid.

12.1.2 Preparation of Polylysine Slides

1. Take 5 ml of 0.1% commercial polylysine solution.

2. Add 45 ml of distilled water (to achieve a 1 in 10 dilution).

3. Dip the clean, grease-free slides in the solution for 10-15 minutes at room temperature.

4. Drain the slides and allow them to dry.

5. Store at room temperature. Use the stored slides within 1 week to 10 days.

12.2 Analytical Steps

A. Prerequisites for Section Cutting

1. Clean, fresh, grease-free glass slides are coated with section adhesive (egg albumin/polylysine).

2. Number the coated slides with a diamond pencil, corresponding to the block to be cut.

3. Fill the flotation bath (Leica Hi 1210) with distilled water and switch it on. Set the temperature below the melting point of the wax.

4. Switch on the slide warmer (Leica HI1210) and adjust the temperature to 60-65°C.

B. Trimming of Blocks

1. Fix the trimming knife to the knife holder at a 3-6 degree clearance angle.

2. Place the cutting surface of the block parallel to the edge of the knife.

3. Adjust the thickness indicator of the microtome to 10-30 microns.

4. Trim the block until the entire surface of the tissue to be cut is exposed.

5. Remove the block and place it on ice, with the cutting surface downwards for cooling.

C. Section Cutting

1. Adjust the microtome thickness to the desired microns.

2. Replace the trimming knife with the cutting knife in the knife holder, maintaining a 3-6 degree clearance angle.

3. Remove the block from the ice and fix it to the block holder of the microtome.

4. Cut ribbons of sections, usually 4-6 microns thick for routine tissues and 2-3 microns for renal biopsy, liver biopsy, and bone marrow biopsies.

5. Use a fine needle or brush to hold the free end of the ribbon with fine forceps and ease the last section from the knife edge.

6. Float the ribbons on a flat dish with 60% alcohol for stretching, then transfer them to the pre-warmed flotation bath.

7. Place the tail end of the ribbon in contact with the water in the bath, with the smooth surface facing down.

8. Allow 30 seconds to 1 minute for the sections to float.

9. Draw the flattened sections onto adhesive-coated, pre-numbered slides.

10. Place the slides with the sections on the pre-warmed slide warmer for drying for 10-15 minutes.

11. The sections are now ready for further staining.

12. Maintain a daily temperature log of the tissue flotation water bath.

12.3 Quality Control and Assurance

1. At the beginning of each daily run, review one tissue section for quality of processing, embedding, cutting, and staining. Proceed with staining the rest of the sections after results are found acceptable or after performing required corrective actions.

2. Calibration and calibration verification: Not applicable.

12.4 Storage of Samples

Retain paraffin blocks and slides for 10 years at room temperature.

13.0 Reference Range

Not applicable.

14.0 Critical Value

Not applicable.

15.0 Reporting of Results and Interpretation

Not applicable.

16.0 Contingency Plan

1. In case of temporary breakdown of one microtome or water bath, the technical staff will complete their work using the second one.

17.0 References

- Theory and Practice of Histological Techniques. John D. Bancroft, Fourth Edition

END OF THE DOCUMENT

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