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Haemolytic diseases of new born

1. Cryostat

2. History: Frozen Section Resulted From Need for Rapid Intra operative Diagnosis.
Mayo Clinic Chief of Pathology Louis B. Wilson pioneered the frozen section at the Rochester, Minnesota clinic in 1905.

3. INDICATIONS OF FROZEN SECTION urgent diagnosis during surgery benign or malignant involvement of resection margins by malignancy example basal cell carcinoma. Ganglion cell in hirschprung disease Enzyme histochemistry (ATPase, NADPH, in muscle biopsy samples Non enzyme histo chemistry (lipids, carbohydrate) Identification of type of tissue Studying margins of tumors

4. INTRODUCTION Cryostat is used in medicine to cut histological sections. They are usually used in a process called frozen section histology. The cryostat is essentially an ultrafine delislicer called a refrigerated microtome The cryostat is usually a stationary upright freezer with an external wheel for rotating the microtome. The temperature can be varied depending on the tissue being cut usually from-20 C to-30C.

5. TYPES OF CRYOSTAT Single compressor (chamber cooling only) Double compressor(chamber and object cooling only) manual sectioning Motorized sectioning

6. The freezer is either powered by electricity or by a refrigerent like liquid nitrogen. Small portable cryostats are available and can run off generators or vehicle inverters.
To minimize unnecessary warming all necessary mechanical movements of the microtome can be achieved by hand via a wheel mounted outside the chamber.
Newer microtomes have electric push button advancement of the tissue.the tissue are sectioned as thin as 1 micrometer.

7. Handling of specimen Tissue must reach histopathology laboratory immediately To avoid tissue being dried it should be kept in saline The size of the tissue should be so thin so that good smooth sections can be obtained and freezing is good.
Thickness of the tissue should be about 3mm to 4 mm The tissue can directly be taken to cryostat or can be fixed with 10% formalin or formal alcohol.

8. Completion of freezing is observed by the change of colour of tissue which turns glossy white.
Freezing should be done fast. This will prevent ice crystal formation . The morphology is better preserved and artifacts are less.
Different freezing substances are used depending upon the availability and feasibility. Liquid nitrogen andCarbon dioxide gas are commonly used with freezing microtome. This gives good results.

9. MICROTOME The rotatory microtome controlled by external hand wheel that is mounted inside the cryostat cabinet has been specially manufactured and lubricated to work at low temperatures
it provide a mechanism for advancing a specimen towards a fixed knife with a section thickness ranging from 1-100micrometer or higher. To the side of the microtome there is area known as freezing shelf where the frozen samples are store prior to sectioning.
Newer instruments also incorporate peltier freezing stage with freezing shelf.

10. This stage is a thermoelectric device that when activated increases the diffusion of heat away from the sample to the cold stage resulting in higher cooling rate and thus faster freezing of sample. KNIFE HOLDER It is located in front of microtome or fixed either to the microtome base or cabinet
There are two types of knife holders that provides a means of clamping either a disposable blade or reusable steel blade. Disposable blades commercially available is either high or low profile are suspended in ridge on the rear pressure plate of knife holder and clamped firmly into place by pressure plate

11. It is important that clamping pressure be maintained e venly across the entire length of the disposable blade.
Damage to the either the front or rear pressure plate or section debris trapped between the pressure plates will significantly effect the clamping pressure and there by sectioning efficiently and quality.

12. For low profile disposable blade s the blade angle is in between 3 to 5 degrees and for high profile blade s the angle is in between 5-7 degrees .
Both types of knife holders are fitted with antiroll guides which as the name suggests this helps in prevent rolling or curling of sections .
The antiroll guide consists of a glassplate supported in a metal frame. There is gap between the underside of glass and knife surface allowing the section to slide under.
Gap sizes are available as 50, 100, 150 micrameter depending on thickness required.

13. An alternative to antiroll guide is cooling brush techniqu e to collect and gather sections.
Specimen holders or chucks for cryostat are available in various shapes and sizes.
The presence of stem or other specially designed projection on the bottom or side of chunk provides a hold to clamp the chuck and is often specific.

14. CRYOSTAT OPERATION Cryostat must be located in humidity controlled area with clearance of 30cm on all sides to ensure that there is unrestricted air movements around the Instruments failure of which leads to poor cooling performance.
All accessories, knife holders, anti roll guide, brush trays should be placed into chamber prior to instrument is turned on.
A start up the chamber temperature must be set a recommendation temp- 20c.allow at least 24hr for a cryostat to come down to selected operation temperature.

15. DEFROST All commercial cryostat use a regular once every 24hr automated defrost cycle to ensure that chamber, microtome and accessories are kept free of frost. During the de frost phase the compressor goes into a hot gas phase and for pre set period of time gas circulates warm air into chamber. Any frost or ice present on chamber surfaces is melted and fluid is drained into sealed container. For majority of cryostats the defrost time is set to midnight this ensure that the defrost procedure is completed and instrument is cooled down to set operating temperature outside of normal working hours.

16. FREEZING OF FRESH UNFIXED TISSUE Tissue for freezing should be fresh, the specimen must be fixed as soon as possible without creating freeze artifacts. Techniques for suitable feezing includes: liquefied nitrogen -190 isopentane cooled by liquid nitrogen
dry ice
carbon dioxide gas
aerosol sprays

17. LIQUID NITROGEN AND ISOPENTANE The best frozen sections are obtained when the tissue is frozen very quickly. The method of choic e is isopentane and liquid nitrogen.
The problem in using liquid nitrogen alone is formation of nitrogen vapour bubbles around the tissue acting as an insulator and so inhibiting rapid even cooling of tissue.
This can produce freeze artifact s in the tissue which may make diagnostic interpretation difficult especially in muscle biopsy.

18. This problem can be overcome by snap freezing the tissue in an agent with high thermal conductivity which has been cooled to -160 c by immersion of liquid nitrogen in isopentane .
A beaker of isopentane is suspended in flask of liquid nitrogen
When the temperature of isopentane is reached to-160C the tissue is submerged in isopentane .
Insufficient time in freezing medium can lead to freezing artifact while prolong freezing can cause crack the block compromising the sample and causing sectioning problems
The tissue may be rolled in talc prior to snap freezing to reduce artifacts.

19. Dry ice Solid carbon dioxide may be used for freezing tissue blocks. Two pieces of dry ice are held in gloved hands against the cryostat block holder containing the tissue which has been oriented in a cryo embedding medium such as OCT. As the tissue freezes a white line will be seen passing through the tissue, the dry ice is then to be removed to avoid over freezing of the tissue.
This method is not usually recommended to as it is not economical since dry ice will change to gaseous state upon storage.
The need for regular deliveries and wastage of large amount of

20. Carbon dioxide gas Carbon dioxide from the co2 cylinder has been successfully used in the past.
Tissue blocks are frozen by adopting a conventional freezing microtome with a gas supply or by using special adopter for CO2 tank that holds the tissue chuck. Aerosol spray s have gained popularity as means of freezing small tissue blocks. These sprays are readily available and easily stored.
A major problem is environmental issues of aerosol emission and safety problem of inhalating aerosol emissions.

21. Tissue can be successfully frozen directly in the cryostat using the freeze bar and the heat extractor.
The tissue is frozen simultaneously from the block face and the block holder.
Freeze artifacts may be reduced if all objects are kept cold and ready for use.
This method is very quick and is often used for intra operative frozen section.

22. FIXED TISSUES AND CRYOSTAT Although cryostat is used for sectioning unfixed tissue certain methods require post fixation on cold formal calcium . However the effect of freezing unfixed tissues is the diffusion of labile substances.
This may not cause problem for diagnosis but it can effect the accurate localisation of some abundant enzymes such as acid and alkaline phosphatases.

23. To accurately localise these hydrolysing enzymes and other enzymes and other antigens it is useful to fix the tissue prior to sectioning in the cryostat . The tissue must be absolutely fresh and placed in formal calcium at 4c for 18 hours

24. Frozen specimen are mounted on specimen holder with cryo compound and
placed on freezing shelf and allowed to stabilize at chamber temperature. Fresh specimen can be directly frozen onto a specimen holder that is placed
on a freezing shelf. A heat extractor can be used to aid freezing and ensure good adherence and flat front block surface.
A heat extractor is a large piece of stainless steel that is supported on a hinged arm that is usually mounted above freezing stage.

25. Before mounting the specimen onto the microtome ensure that the hand wheel is in locked position.
In unlocked position the object head can move when insertioning the chuck placing the operator at risk.
The specimen holder is clamped firmly onto the object head mounted on the spindle of microtome.
The handwheel is unlocked and the specimen is lowered until it is at same height as the knife holder.
The knife holder can then be unlocked and moved manually towards the specimen until it is close to specimen surface

26. Adjust the micrometer setting of the microtome to trimming thickness of 50micrometer and begin to turn the microtome handwheel the specimen will advance by this set value and will make contact with the knife and surface of block will be sectioned.
This process is called trimming or facing the block and purpose is to achieve full face section of specimen
As soon as this is achieved stop sectioning and adjust the micrometer setting to desired section value.
Brush all trimmings from the blade or knife edge, carefully lower the antiroll plate in to place and continuous sectioning

27. Carefully lift the antiroll guide or move the sections on the pressure plate or knife surface
Take a glass slide and holding it at angle gently lower it on to the section Sections of fres frozen tissue will adher to the plane glass slides due to the presence of free protein and lipid
Sections of fixed frozen tissue will need to be mounted on coated slides

28. Sections of fresh frozen tissue should be fixed immediately unless they are going to be stored for future study
A standard histology fixer such as neutral buffered formalin is the most suitable fixiative for frozen sectio n
Sections of fresh frozen tissue will rapidly dry if exposed to warm air and this will result in cellular artifact.
If sections mounted on glass slides that are going to be placed immediately in to a plastic slide box that have been coiled and kept in a chamber and the entire box is wrapped in double layer of aluminium foil, laballed and moved immediately in to -80 c.

29. CABINET TEMPERATURE The temperature of microtome and cabinet temperature must be monitored Many cryostats have digital displays of the block temperature and the cabinet temperature
The temperature should be suitable for the tissue type and the type of preparation to be cut.
Most of the unfixed material will section well between-15 to -23 C Tissue containing large amount of water will section better at warmer temperature and harder tissues are those that contain fat require a colder temperature.

30. If sections are shattered with chatter lin es this is an indication that block is too cold. Most fixed tissues are will section best within range - 7 to 12C depending on hardness of tissue Small blocks of un decalcified cancellous bone can be sectioned but care must be taken to remove any cortical bone fragments prior to freezing.

31. ARTIFACTS When freezing tissue for frozen sections freezing artifacts are formed, the water in the tissue freezes and forms ice crystals and the crystal size and quantity of crystals is proportionate to speed which tissue is frozen.
The tissue is cut and placed on a room temperature slide at this point the tissue is thawed.
The thawing of ice crystals produces freezing artifacts that appears as holes or discontinuation of the tissue architecture when viewed microscopically.

32. Disinfection The wash tray underneath the microtome spindle should be removed.
And other shavings and specimen debris can be wiped up using either paper towel or guaze swab soaked in 70% alcohol.
Disinfectant systems are now available such as disinfectant spray
(MICROM) system
formaldehyde vapour (THERMO FISCHER)
A combination of disinfectant spray
UV light (LEICA)
ozone(SAKURA).

33. Common problems specimen identification
specimen orientation benign vs malignant
artifacts
cutting of the fat temperature regulation
infectious specimens

34. Quality assurance Never 2 cases in one grossing area
System of identification of blocks and slides has to be done Correlation has to be done in each case
The cryostat must be cleaned periodically
Record keeping.

35. Cryostat maintainance Daily….. Lock the microtome handle wheel
Remove all the section debries either by alcohol soaked paper towel or proper vacuum system
Remove all the specimens, blades/knives
Remove all used specimen holder and soak in warm op water to ensure all cryo compounds are removed.

36. Weekly … Empty fluid container (the fluid which is collected during the daily defrost cycles) As the fluid is considered to be biohazard and disposed accordingly. Monthly Spindle lubrication-ensue that hand wheel of microtome is locked and bring the microtome blade farward . Using a disposable pipette place the oil onto the rear upper surface of spindle. Retract spindle to its home position this will ensure that spindle is kept well lubricated and free of dust particles……… …..as spindle issues lead to poor sectioning.

37. Checklist….. Confirm the chamber temperature is set a recommended temp of -20C
Confirm that hand wheel is locked Check the knife holder clearance angle has set correctly
Insert the first blade and check the blade is clamped firmly
Confirm that desired section thickness has set
Unlock the hand wheel and start sectioning
When sectioning is completed lock the hand wheel
Remove the specimen from the object holding process for either low temperature storage or fixation or subsequent paraffin embedding.

38. Disadvantages 1. morphology is distorted
2. cellular details are not well seen
3. Staining is not very good
4. some special stains cannot be performed