A rapid manual processing technique for resource‑limited small laboratories
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Transcript of A rapid manual processing technique for resource‑limited small laboratories
GOOD MORNING
WORLD’S FASTEST HUMAN BEING!
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
ThankQ?