Clearing

Clearing

1. CLEARING SUNIL KUMAR.P St.john’s Medical College Bangalore 10/13/2018 1 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

2. INTRODUCTION Clearing reagents acts as an intermediary between the dehydration and infiltration solutions. They should be Misible with both solutions. Most clearants are hydrocarbons with refractive indicies similar to protein. When the dehydrating agent has been entirely replaced by most of these solvents the tissue has a translucent appearance ; hence the term “ clearing agent”. 10/13/2018 2 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

3. DEFINITION The term “Clearing “ relates to the clear and translucent appearance of the tissue after treatment with the clearing agent. 10/13/2018 3 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

4. Criteria for choosing a suitable clearing agent are 1. Rapid penetration of tissue 2.Rapid removal of dehydrating agent 3.Ease of removal by melted paraffin wax 4.Minimal tissue damage 5.Low flammability 6.Low toxicity 7.Low cost 10/13/2018 4 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

5. Clearing agents suitable for routine use (TYPES) 1.Xylene 2.Toluene 3.Chloroform 4.Benzene 5.Cedar wood oil 6.Paraffin oil 7 .Citrus fruit oils 10/13/2018 5 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

6. XYLENE Xylene is a flammable Color less liquid Petroleum or aromatic odour Misible with most organic solvents and paraffin wax. It is suitable for clearing blocks less than 5 mm in thickness and rapidly replace the alcohol from the tissue. Over exposure to Xylene causes hardening of tissues. 10/13/2018 6 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

7. TOLUENE This has similar properties of Xylene but causes less brittleness. It is more flammable and volatile than xylene 10/13/2018 7 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

8. CHLOROFORM Chloroform is slower in action than xylene but causes less brittleness. Thicker tissue blocks can be processed, greater than 1 mm in thickness. It is non-flammable but highly toxic Most commonly used for CNS 10/13/2018 8 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

9. Infiltration or Impregnation After clearing the tissue is transferred into a chamber containing molten paraffin wax. During this stage…………. the clearing agent is eliminated from the tissue by diffusion into the molten wax and the wax in turn diffuses into the tissue. The tissue should pass at least two changes of wax to achieve proper impregnation by wax. 10/13/2018 9 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

10. Infiltrating agents 1.paraffin wax 10/13/2018 10 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

11. Other alternative infiltrating and embedding agents 1.Resin 2.Agar 3.Gelatin 4.Cellodin 10/13/2018 11 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

12. PARAFFIN WAX Paraffin wax is the most commonly used infiltrating and embedding medium in histopathology. Paraffin wax is a mixture of long chained hydrocarbons produced in the cracking of mineral oil. The waxes commonly used have a melting point ranging between 50 – 60oC. The temp. of the wax can be maintained using a thermostatically controlled oven. 10/13/2018 12 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

13. The average time taken for complete impregnation of a piece of tissue 5 mm thick is about 3 hours. But dense and thick tissues require longer time. 10/13/2018 13 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

14. Tissue processing Methods : Manual Automated Method by (Automatic tissue processor) 10/13/2018 14 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

15. Tissue processing by Using Automatic Tissue Processor Requirements 1. Alcohol solutions of various con% such as a.70 % b.95% c.100% ( absolute Alcohol) 2. Xylene or Acetone 3.Chloroform 4.Parafffin 5.Automatic tissue processor 6.Tissue 10/13/2018 15 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

16. Procedure 1. Place the solutions and paraffin in respective beakers of the equipment 2.The timing level is set at Zero and the machine is started (overnight Processing) 10/13/2018 16 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

17. conti……. 3.The basket with the cassettes automatically changes position and takes a bath in different reagents kept in the beakers in order to accomplish - Dehydration - Clearing - Infiltration - The final dip in the warm paraffin 4.Cassettes are opened next day morning for embedding. 10/13/2018 17 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

18. Automatic Tissue Processing Schedule 10/13/2018 18 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

19. Advantages of Automatic Tissue processing 1.Custom programs specific to tissues being processed, addition of vaccum, agitation or heat at any stage. 2.Rapid schedules. 3.Fluid and fume containment 4.Environmentally friendly reagents 5.Time delay for start of processing schedules 6.Reagent management 10/13/2018 19 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

20. Maintenance & care Of Automated Tissue Processor 1.Any spillage or overflow should be cleaned immediately. 2.Accumulation of wax on any surface should be removed. 3.The temp. of the paraffin wax bath should be set to 3oC above the melting point of wax. 4.Timings should be checked when placing the cassettes in the processor 10/13/2018 20 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

21. Quality control ….. For Automatic tissue processor Temp. of all paraffin wax dispensers, flotation water baths and automated processors are carefully monitored. 10/13/2018 21 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE