PURPOSE, PROCEDURES, QUALITY ASSURANCE, AND

SAFETY PROCEDURES IN THE SPECIAL STAINS LABORATORY

1. Purpose of the Special Stains Laboratory – The Special Stains Laboratory is a service-oriented laboratory where special stains and histochemical procedures are performed on surgical, autopsy, and other tissues for diagnostic and research purposes.  Special stains are also performed on material received from cytology.

2. Procedure for Requesting Special Stains – Special stain requests can be made via electronic ordering or by using a Histopathology Special Stain Request form.  Special stain requests on surgical and autopsy tissue sections must be countersigned by a senior staff member or by a senior resident.  The request form must be properly and completely filled out and should indicate the diagnosis which the special stain is expected to help confirm.

3. Procedure for Processing Special Stain Requests – The request form should be taken to the main Histopathology Laboratory.  The request form should be placed in the proper request container.  If electronically ordered the order will be retrieved periodically throughout the day.  Rush requests should be given directly to the laboratory supervisor or the technologist who is responsible for cutting special stains.

4. Procedure for Cutting Special Stain Requests – All special stain requests are cut in the main Histopathology Laboratory.  Surgical requests take priority over autopsy and research requests.  Sections for special stains are cut routinely on liver, kidney, bone marrow, and other biopsy tissues.  After the special stain sections are cut, they are placed in a 60o-65o C. drying oven for a minimum of one hour.  The cut off time for same day requests is 11:00 a.m.  If a request is received before 12 noon you must verbally indicate if it is a rush for that day or can be microwaved dry.

5. Procedure for Handling Special Stain Slides and Requests – When the special stain slides and request forms are received in the Special Stains Laboratory, the slides are placed in a 60o-65o C. oven.  The required information on the request forms is then recorded in the surgical, autopsy, cytology, or research record books.

6. Procedure for Performing Special Stains – Special stains are performed on liver, kidney, bone marrow, and other biopsies the same day they are received.  All acid-fast bacilli, G.M.S., and bacterial requests are done the day they are received.  Most of the surgical special stain requests are done on sections which have been cut by 11:00 a.m.  Autopsy and research requests are done when corresponding surgical special stains are performed or when they can be worked into the daily work schedule.  Cytology requests are done the same day they are received.

7. Procedure for Handling Rush Special Stain Requests – Requests for rush special stains are done on surgical and cytology specimens.  Most of these rush requests are for microorganisms such as fungi, pneumocystis carinii and acid fast bacteria.  Whenever a rush case is pending the supervisor or a senior technologist should be notified as soon as possible.  When the slides on the rush case are received the special stains are done as soon as possible.

8.        Procedure for Signing Out Special Stain Slides – The stained slides are checked with a microscope in order to determine if the quality of the stains are acceptable.  Any stain which does not meet the standards of quality control will be repeated.  All control slides are checked to make sure that they are positive.  Any stain on which a control slide comes out negative will be repeated.  A record is kept on all the stains that are repeated on the Special Stain Quality Assurance Monitoring Form.  The slides that are signed out in the record book are delivered to the person who made the request.  Rush requests are delivered when they are completed.

9. Procedure for Changing Solutions – The solutions (xylene, alcohols and distilled water) in the hydration and dehydration set-up are changed once a week (usually on Monday).  The alcohols and xylene in coplin jars are changed as needed.  The staining solutions in the coplin jars are changed on a regular basis.  Those changed weekly are Weigert’s iron hematoxylin, 2% sodium thiosulfate, 0.2% gold chloride, and 0.3% sodium borate.  The others are changed every other week except for Masson trichrome, cardiac trichrome, amyloid, Zenker’s, Schiff’s, and Kreyberg's which are changed once a month.  The Strong-Fast solutions are changed every other month.  All the solutions in coplin jars and those in the solution cabinets and refrigerator are dated when changed or prepared, and the initials of the persons who changed or prepared them are indicated on the labels.  Outdated solutions are discarded and fresh ones prepared.  The shelf-life of dye and chemical solutions is indicated in the section of Special Staining solutions.

10. Procedure for Changing, Revising, Adding and Deleting Staining Methods –

1. New staining methods are added when the need or request for them arises.  These methods are thoroughly tested on appropriate material and evaluated by the Laboratory Director and senior pathologists before they are implemented.

2. Old staining methods are deleted when they are no longer requested or are replaced by new or modified techniques.  The deleted methods are filed with the reason, date of deletion and approval of the Laboratory Director.

3. Continuing research is conducted to improve staining methods and/or reduce the time required to perform the techniques.  The modified staining methods are thoroughly tested to make sure that they perform at least as

well as the original procedures.  Many of these modified methods have been published in various journals.

11.        Quality Assurance in the Special Stains Laboratory –

1. The solutions used in the various staining procedures are changed at regular intervals.

2. Only reagent grade or high quality chemicals are used in preparing solutions.  Dyes which are certified by the Biological Stain Commission are used to prepare the staining solutions.  Many chemicals and solutions are stored in the refrigerator at 3°-6°C. or in the freezer at -15° to -18° C.

3. When a new dye or chemical is used in a staining method, the staining results are compared with those obtained with the old dye or chemical.  If they are satisfactory, the bottle is labeled with an S, the date used and the initials of the person who used it.

4. The temperature of the ovens, water baths and refrigerator are checked daily and recorded with date and initials of the person who checked them.

5. The technologists and trainees on special stains have their own copies of the staining methods.  Some of these are revised and updated from time to time.

6. Only those staining methods which give consistently reliable results are used.

7. Whenever problems occur with any of the special stains, the cause of the problem is investigated and the necessary corrective measures taken.  This is recorded on the Quality Assurance Monitoring Form.

8. Independent research to improve existing staining methods and develop new ones is maintained on a continuing basis.

9. Meetings are held with the Laboratory Director and members of the Biological Stain Commission regarding dyes, staining procedures, and problems with special stains.

10. Positive controls are included with stains for acid fast bacteria, aluminum, amyloid, argentaffin, argyrophil, bacteria, bile, calcium, central nervous system, chromaffin, copper, DNA, RNA, enzymes, fibrin, fungi, glycogen, hemoglobin, hepatitis B, surface antigen, iron (ferric), keratin, mast cells, reticulum, melanin, mercury, mucopolysaccharides, nerve fibers, neutral fats and lipids, Nissl substance, pancreatic islet cells, pituitary cells, pneumocystis carinii, and spirochetes. See pages 191A and 191B for a list of those stains that require controls.

11. Controls are not included for the following special stains: Gomori’s trichrome, Jones’ basement membrane, Masson’s trichrome, Periodic Acid-Schiff (PAS), and Verhoeff’s elastic.  The reason for not using controls for these stains is that they all contain the substance for which they are being stained which perform as internal controls.  Therefore, when these stains are viewed with a microscope it is easy to determine if they are properly stained.

12. When the special stain slides are completed they are viewed by the technologist or trainee and the supervisor or a  senior staff member with a dual-viewing microscope.  The following purposes are achieved by this joint viewing:

1. The slides are critically evaluated as to the quality of the special stains to determine if they meet the standards of acceptability.  If they are unacceptable the stains are repeated.

2. Control slides are determined to be either positive or negative.  After the stains and controls have been evaluated a record of the results is recorded in the Special Stains Record Book as follows:  S = satisfactory for those cases where the control is internal, * = control positive and delivered with the case, and 3= the control was positive but was delivered with another case stained at the same time.

3. Some basic histology is taught.

4. Some histopathology is taught.

12.        Laboratory Safety Procedures in the Special Stains Laboratory –

1. All workers are instructed in proper laboratory safety practices as required by the Occupational Safety and Health Administration (OSHA), New York State, and other agencies.

2. All workers are instructed in the procedure for handling biohazardous material as described in a memo dated December 1996.

3. Medical Emergencies

1. Acid or chemicals splashed into eyes.  Flush with water using the eye wash located next to the refrigerator.  Get medical treatment in the Emergency Department or University Health.

2. Glass in the eye.  Do not rub the eye.  Go immediately to the Emergency Department.

3. For all other medical emergency situations call Security at X13.

4. Chemicals, Dyes, and Solutions

1. Bottles containing chemicals, dyes, and solutions are clearly labeled.

2. Material Safety Data Sheets on chemicals and dyes are kept in the laboratory.

3. Reagents and solutions must contain a label clearly staining the contents with the date and initials of the person who prepared them and the expiration date.

4. Solutions must be pipetted by automatic pipetting devices.  Mouth pipetting is prohibited.

5. Acids are stored on the lower shelf in the Acid Storage Cabinet.

6. Acids, bases, and chemicals that will react with one another are stored separately.

7. Flammable liquids are stored in the Flammable Storage Cabinet located in room G-5405A.

8. Flammable liquids or materials are never stored in the refrigerator.

9. Disposal of chemical, flammable liquids, and waste solutions is accomplished through the Hazardous Waste Management Unit.

10. Gloves and lab coats are required when weighing chemicals and dyes, in the use of acids, and when preparing solutions.
11. The fume hood is required when working with acids and volatile solutions.

12. Trace amounts of acid, chemical, and dye solutions may be flushed down the drain using large amounts of water.

5. Acid or Chemical Spills

1. For large spills of acid or chemicals, call Health and Safety at X53241 and evacuate the area.

2. For small spills of acid treat with sodium bicarbonate (located under the test tube rack by the window) and then mop up with paper towels.  Call the Hazardous Waste Management Unit at X52056 for pickup and disposal.

6. Smoking and Open Flames

1. Smoking is not allowed in the laboratory and a No Smoking sign is posted.

2. Open flames are never used or allowed.

7. Food and Beverages

1. Food or beverages are never stored in the refrigerator.

2. The use or storage of food or beverages in the laboratory is prohibited.

8. Fume Hoods

1. Two fume hoods are available and are used daily.  Coverglassing of slides is done under one and the other houses the solutions (xylene and alcohol) used in the hydration and dehydration of the slides.

               b)        The fume hood housing the solutions is also used when working with acids and volatile liquids.

9. Broken Glassware

1. Broken glassware must never be placed in the regular trash cans.

2. Place broken glassware in the Glass Disposal Can.

10. Biohazardous Material

1. Gloves are required when handling biohazardous or unfixed slides.

2. Gloves and containers used in the transport and handling of biohazardous slides may only be disposed of in the Biohazardous Waste Can in room G-5405A.

3. For additional information see memo dated January 1992 – Procedure for Handling Biohazardous Material in the Special Stains Laboratory.

11. Maintenance of Equipment

1. All electrical equipment is checked annually by Engineering.

2. Fume hoods are tested annually for adequate face velocity by Medical Engineering.

3. Microwave ovens are checked annually for microwave leakage.

4. Balances are cleaned and adjusted annually.

5. Microscopes are cleaned and adjusted annually.

6. The temperatures of the refrigerator, waterbath, and ovens are checked and recorded daily with the date and initials of the person who checked them.