Yob - Laboratory and Cryostat Techniques

11/11/2009 

THE CRYOSTAT AND 

CRYOSTAT TECHNIQUE: 

OBTAINING QUALITY MOHS 

SLIDES 

November, 2009 

Edward H. Yob, D.O. 

Dermatology Associates of Tulsa 

Clinical Associate Professor 

The University of Oklahoma - Tulsa 

Handout 2009 


DISCLOSURE 

I am a partner in DermFirst EHR, a 

consulting group which works 

exclusively with ENCITE - EMR 

The preparation of quality slides is 

a labor and skill intensive process 

requiring close coordination 

between the technician and Mohs 

surgeon. The necessary 

ingredients for success are, and 

will remain, attention to detail, 

practice and experience. 



ATTENTION TO DETAIL 

• Harvesting the tissue 

• Precise orientation 

• Preparing the specimen 

• Chroma-coding (inking the edges) 

• Temperature control - freezing the specimen 

• Cutting the specimen 

• Placement of the specimen on the slide 

CRYOSTAT 

• Basis of the fresh tissue technique 

• High tech refrigeration unit housing a 

microtome 

• Environmentally safe refrigerants 

• Microprocessors-maintain temperature 

• User friendly – fatigue factor 

• Routine Maintenance - Essential 



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11/11/2009 


Object (Chuck) Holder 

Blade 


Freeze Bar 

Handout 2009 Handout 2009 

PREPARING THE SPECIMEN 

Preparing the Mohs Map 

• Precise orientation – hatch marks 

• Relaxing incisions 

• Preparing the Mohs map 

• Dividing the specimen 

• Chroma-coding the specimen 

• Freezing the specimen 



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11/11/2009 

Dividing the Specimen 

Chroma-coding the Specimen 



Specimen Ready to Freeze 


CHROMA-CODING the specimen 


FREEZING AND MOUNTING 

• Fixed vs. adjustable object holder 

• Fixed vs. movable blade 

• Temperature control –20 to –25 

degrees C. 

epidermis / dermis – slightly warmer 

fat – slightly colder 

• Type of tissue >> technique used 

Adjustable Object Holder 

Fixed Blade 



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11/11/2009 

DIRECT METHOD 

Adjusting the chuck in the OBJECT HOLDER 


• Chuck is placed on the freeze bar 

and coated with OCT 

• Specimen is turned over and placed 

on the semi-solid OCT 

• Specimen is manually flattened and 

the lateral edges are teased up 

• Additional OCT is applied 

• Heat extractor may be used 


OCT 

Chuck 

Freeze Bar 

Placing the specimen on the OCT 



Teasing the edges 


Flattening the specimen 


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11/11/2009 

FREEZE BAR TECHNIQUE - 1 

• Specimen is placed directly on the 

clean quick freeze bar 

• Edges are gently manipulated to 

assure good contact 

• Specimen is coated with OCT and 

frozen 

• Chuck is prepared with OCT to a semisolid 

consistency 

Adding more OCT 



FREEZE BAR TECHNIQUE - 2 

• Frozen specimen is “popped” off the 

quick freeze bar 

• Specimen is inverted and frozen to the 

prepared chuck 


SPECIMEN IS PLACED 

ON THE FREEZE BAR 

DEEP MARGIN DOWN 

THE EDGES ARE 

GENTLY PUSHED 

DOWN 



OCT IS ADDED TO 

THE SPECIMEN 

THE OCT BEGINS 

TO SET 



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11/11/2009 

AS THE OCT BEGINS 

TO SET UP ON THE 

SPECIMEN… OCT IS 

ADDED TO THE 

CHUCK 

OCT BEGINS TO 

SET-UP ON THE 

CHUCK 



SPECIMEN IS 

‘POPPED OFF’ 

THE FREEZE BAR 

SPECIMEN IS INVERTED 

AND PLACED ON THE 

CHUCK 



ADDITIONAL OCT 

IS PLACED ON THE 

SPECIMEN 

A HEAT SINK CAN 

BE USED TO SPEED 

UP THE FREEZING 



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11/11/2009 


GLASS SLIDE TECHNIQUE - 1 

• Specimen is placed cut side down on a 

glass slide and inked 

• Lateral margins are gently pushed down to 

assure good contact 

• Slide is placed on the quick freeze bar and 

the specimen is coated with OCT 

• Chuck is prepared with OCT to a semi-solid 

consistency 



GLASS SLIDE TECHNIQUE - 2 

• Slide and specimen are inverted and 

frozen to the prepared chuck 

• Bottom of the slide is warmed with a 

gloved finger and the slide is slipped 

off 


SPECIMEN BROUGHT INTO 

THE LAB ON A GLASS SLIDE 

CUT SIDE DOWN 



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OCT BEING APPLIED 

MARGINS BEING CHROMACODED 

(INKED) 



OCT SETTING UP 

APPLYING OCT TO 

THE CHUCK 



INVERTING THE SLIDE 

ON TO THE CHUCK 

SLIDE WITH SPECIMEN 

ADHERING TO THE 

CHUCK 



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11/11/2009 

WARMING THE SLIDE 

THE SLIDE SLIPPING OFF 



APPLYING ADDITIONAL 

OCT TO THE SPECIMEN 

SPEEDING UP THE 

PROCESS WITH THE 

HEAT SINK 




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11/11/2009 

CRYOMOLD TECHNIQUE 

• Small amount of OCT is placed in a room 

temperature plastic cryomold 

• Specimen is placed face down in the 

cryomold 

• Cryomold is placed on the quick freeze bar 

and filled with OCT 

• When freezing begins a chuck is placed in 

the cryomold 

• When frozen the cryomold is peeled away 

OCT Added 

Specimen Placed in a 

Room Temp Cryomold 



Cryomold 

Placed In Cryostat 

To Freeze 

Cryomold Removed 

From The Specimen 

Chuck Added 



The BEST technique is one that 

produces consistent results 

efficiently 

Techniques evolve based on 

the experience of the 

technician and the 

preferences of the surgeon 


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11/11/2009 

CUTTING THE TISSUE 

• Chuck is placed in the object holder 

• Excess OCT is rough cut away 

• When specimen becomes visible final 

alignment is accomplished 

• Micrometer is set to proper thickness 

• Anti-roll mechanism is moved into place 

• Specimen is cut and placed on the slide 

SPECIMEN READY TO 

BE ROUGH CUT 



FACING THE BLOCK 

Rough cutting away 

the OCT 

TISSUE BEGINS TO 

APPEAR 



BLOCK BEING ADJUSTED 

TO THE PROPER ANGLE 

WITH THE MICROMETER ADJUSTED TO THE 

PROPER THICKNESS THE TISSUE IS CUT 



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USING A BRUSH TO MINIMIZE CURLING 

PICKING UP THE SPECIMEN WITH A SLIDE 





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11/11/2009 

MICROSCOPE SLIDES 

• Types of slides 

– Frosted – color coded 

– Electrostatically charged 

• Labeling slides 

– Accession number? (M09-102) 

– Name, date, stage, piece and slide # 

• John Doe, 11/5/09 I 1 A 


TECHNIQUE TIPS - 1 

• Blades must always be sharp 

• Microtome must be cleaned and lubricated 

• Microtome must be securely fastened in the 

unit 

• Blades should have blade guards in place 

• Align epidermis 90° to the blade 

• A smooth cutting motion is essential 

TECHNIQUE TIPS - 2 

• Brushes and other implements should be 

kept cold in the cryostat 

• Consistency is key when placing the 

specimen on the slide 

• Always pre-label l slides before cutting any 

tissue 

• Fresh tissue and all the associated debris is 

bio-hazardous – universal precautions are 

as essential in the lab as in the operating 

room 



BLADE GUARDS IN PLACE 


KEEP ALL BRUSHES ETC. IN THE CRYOSTAT 


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11/11/2009 

The ASMS Clinical Meeting 

Monterey CA May 27-30, 2010 


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Yob - Laboratory and Cryostat Techniques

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