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A rapid manual processing technique for resource limitedsmall laboratories

Journal of Oral and Maxillofacial Pathology Vol. 19 Issue 3 Sep - Dec 2015

Dr. RAM KUMAR TIRANDAS,III YR PG IN OMFP

CONTENTS

•INTRODUCTION•OBJECTIVES•MATERIALS AND METHODS•STATISTICAL ANALYSIS•RESULTS•CONCLUSION

BEFORE

NOW

SOURCE OF INFORMATION

BEFORE

NOW

LIBRARy

IMPORTANCE OF TIME

•INTRODUCTION

•Rapid diagnosis

•Management planning- prognosis

•Efficient pathology service

•PROCESSING TECHNIQUES

•Surgical pathology

•Small laboratories- lack -------Power supply, High cost of automation.

•FFPE tissue sections- Treasures of any pathology department.

a b

(Blocks)

manual

•16-48 hr rapid processing (2days).

•Test processing schedule is 8-9 hrs a day?

•Quality of the tissue processing.

STEPSOF TISSUE

PROCESSING

•FIXATION

•DEHYDRATION

•CLEARING

•IMPREGNATION

•EMBEDDING

•SECTIONING

•STAINING

Types

Depending on the

availability of the

reagents, type of

tissue and

standardization

protocols, tissue

processing

depends!

(Why is fixation not considered as a step in tissue processing?)

•OBJECTIVES

To evaluate the effectiveness of a new rapid processing schedule and compare it with two existing rapid processing schedules.

A B C D

Godkar’s Bancroft’s Test schedule Automatic TP

AUTOMATIC TISSUE PROCESSOR

Bancroft’s

(2 working days)

Godkar’s

(11 hrs)

CONTROLHigh cost

Power supply

Test schedule

?

17 hrs (long cycle)

•MATERIALS AND METHODS

•Comparison among processing schedules

•Ethical clearance

•Animal housed medical college animal house

•Uniform firm tissue- tongue (8mmx8mmx6mm)

22 tongues were included to harvest a total of 80 tissue samples of the

desired size. The specimens were fixed in freshly prepared 10% neutral buffered formalin for 24 hrs. (1 day here?)

Study design and sample source

400-600 g, 2-2 1/2 yrs, chloroform given.

Figure 1: The schematic representation of grossing of rat tongue

•GROSSING, TRIMMING AND FIXATION

Cylindrical sections

Which tissues are to be washed in water after fixation?

Pre processing preparation and measurements

Begg’s wire

Linear measurement in mm (length)

3 chip CCD camera on Trinocular stereo microscope(Olympus SZX7, Japan) calibrated for millimetre under × 10 magnification

Morphometric analysis

[8 mm × 8 mm × 6 mm]

a two dimension measurement of surfacearea in square millimetres was done

Tissue processing

The chemicals were chosen based on a combination of performance, availability, safety and cost.

Table 1

Isopropyle alcohol, xylene, acetone ( in test schedule )

Paraffin tissue blocks were made

Quality of tissue blocks by means of adequate ribbon formation assessed.

Mounting and staining

Figure 4: Adequate ribbon formation Figure 5: Inadequate ribbon formation

Diagnostic adequacy was examined by comparing preprocessing, post-processing and post staining by using a stereomicroscope.

Fragmentation, epithelial stripping, irregular voids and shrinkage were examined by single inter-observer.

Figure 7: Measurement for shrinkage in cross-sectional surface area

after staining

stereomicroscope

After the tissue processing, there will definatly be the shrinkage of the tissues

1 and 4 schedules showed slight higher shrinkage compared to other schedules.

TISSUE PROCESSING SCHEDULE

11 hr

2 days

8 hrs

17 hrs

6hrsD

81/2hrsD

4 hrsC

4hrsD

3 hr C

81/2hrsD

4hrsC4hrs P

2hrsC4 hrsP

2hrP

P

D- dehydration, C- clearing, P- paraffin imprignation

STATISTICAL ANALYSIS•One‑way ANOVA was used for multiple

group.

•Tukey’s test for group‑wise comparisons.

•Chi‑square test was used for analyzing categorical data.

•Interobserver reliability was tested by Kappa measure of agreement.

•P ≤ 0.05 was considered statistically significant.

Figure 3: Measurement for linear shrinkage after processing

Figure 2: Measurement for linear shrinkage

Figure 6: Small folds restricted to epithelium and lamina propria

(Unstained, x10 under stereomicroscope)

Figure 8: Steromicroscopic image showing fragmentation of tissuesection (H&E stain, x10 under stereomicroscope)

Figure 9: Steromicroscopic image showing stripping of epithelium.(H&E stain, x10 under stereomicroscope)

Figure 10: Steromicroscopic image showing irregular voids seen inthe epithelial sections (H&E stain, x10 under stereomicroscope).

RESULTSEffect of processing schedules on shrinkage

Table3: 3 and 4 schedules showed slight higher shrinkage compared to other schedules.

Correlate with the article, statistically insignificant.

Test Schedule III has slight higher shrinkage compared to other schedules.

•Summary of the mean percentages [Table 5] calculated from overall shrinkage values demonstrated a range of 14.4–19% shrinkage of specimens by the Schedules and showed schedule II to have the least overall shrinkage followed by schedule III, schedule I and schedule IV.

Effect of processing schedules on ease of sectioning and quality of sections

The overall performance of the schedules also found them in a narrow range of performance with the best schedule being schedule with 48.3% total score in terms of shortcomings, followed by schedule II at 50%, schedule I at 51.7% and schedule IV at 56.7%.

Effect of processing on staining

Sections from all schedules were of acceptable diagnostic quality.

BEST

II followed by III, I and IV.

•Fixation ( 24hrs) - 1st day

•Overnight dehydration 15 hrs – 2nd day- 1 STATION

70% alcohol - 1 station

90% alcohol- 1 station

100% alcohol- 1 station

Acetone + xylene- 1 station

Xylene- 2 station

Paraffin- 2 station

8hrs

TEST SCHEDULE

REFERENCE

34

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