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1.0 TITLE: Sample Staining 2.0 POLICY:All gynecologic slides for cytologic examination should be stained by Papanicolaou.The Papanicolaou stain or onother appropriate permanent stain is used for non-gynecologic specimens. 3.0 PROCEDURE PAPANICOLAOU STAINING 3.1 MaterialsHematoxlin- Commercially prepared0.5 % Hcl (0.5 ml. Hcl and 95.5 ml. Distilled water.Absolute and 80% isopropanol (see preparation of graded alcohol)OG-6- Commercially prepared EA – 50- Commercially preparedXylene

3.2 Step by step Staining procedure: (Automated multistainer)1. 80% isopropanol 10 minutes (fixation)2. Rinse in tap water3. Harris or Gill Hematoxylin in 2-3 minutes (time vary with selection of hematoxylin solution)4. Rinse in tap water or Scott’s tap water5. 80% isopropanol 10 dips6. OG-6 stain for 1.5 minutes7. 80% isopropanol 10 dips8. EA-50, or modified EA-50 or EA-65 stain for 2.5 minutes9. 80% isopropanol 10 dips, 2 changes10. 95% isopropanol 1 minute11. Clear in 2 changes of xylene, 2 minutes each12. Mount with permanent mounting medium.Automated Multistainer Downtime: the same staining procedure is done manually.

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4.0 QUALITY CONTROL OF STAININGTo maintain the optimal quality of the cells staining for diagnostic interpretation.4.1 The stains are filtered each morning.4.2 Weekly stain changing and cleaning of set up is documented.4.3 One empty slide for each batch of stain is evaluated microscopically.4.4 Daily QC sheet is maintained for quality of staining on all specimens.4.5 All reagents are used within their indicated expiration date.4.6 Assigning the Expiry Date to any reagents that do not have a manufacturer-provided expiration date. The assigned expirationdate is 5 years from the opening date.4.7 Use of expired ReagentsExpired reagents are used only under the following circumstances:1. The reagents are unique, rare or difficult to obtain; or2. Delivery of new shipments of reagents is delayed through causes not under control of the laboratory.3. The laboratory has a strict quality control and documents validation of the performance of all reagents on Daily QC sheet.4. Fill the form (Gen. 023) for each expired reagents used. 5.0 ASSESSMENT OF STAINSCytology stains undergoing a daily technical quality review are exempt from annual assessment. Where applicable, expiration dateassigned by a manufacturer must be observed. However, most stains used in cytology laboratory are not subject to outdating, so thatassignment of expiration date may have no meaning. Procedure of stain assessment:1. Cytology stains are assessed regularly for proper storage and acceptable quality.The acceptable performance of such stains is confirmed each time a new batch of stain is started. (Technical assessment is done onactual case material and as part of the evaluation of cytopathology cases).A well stained Pap smear should demonstrate crisp blue/purple nuclei. Cytoplasmic staining should show a broad spectrum of colorranging from orange in highly keratinized cells through ranges of orange/pink in superficial cells and green/blue in intermediate andparabasal cells.2. Whenever a new batch of stain is started a test slide is stained and presented to senior Cytotech to assess stain quality.3. A record of daily QC sheet is maintained for regular check on quality of staining.

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6.0 PROCEDURE: IMPORTANT FACTORS INFLUENCING STAINING RESULTS OF PAPANICOLAOU STAIN6.1 Maintenance of Solutions and StainsSolutions may be used over a longer period of time if the slide carrier is rested on several thickness of paper toweling for a fewseconds after removing it from the solutions. The life expectancy of stains may be increased by storing them in dark bottles when notin use and in keeping staining dishes covered.The frequency of replacement of solutions required to ensure crisp, well-stained slides, is dependent on the volume of slidesprocessed daily. Daily microscopic checks are recommended. The following and nature of material processed.6.2 HematoxylinRemains relatively constant in staining characteristics and seldom requires discarding if small amounts of fresh stain are added daily toreplace stain loss due to evaporation. However, the use of coating or Carbowax fixatives may result in contamination, makingfrequent changes necessary.6.3 OG-EALose strength more rapidly than Hematoxylin and should be replaced each week or as soon as the cells appear gray, dull or withoutcrisp contrasting colors.6.4 Water Rinses: Should be changed after each use. Alcohols used during the rehydrating and dehydrating process prior to thecytoplasmic stains should be replaced weekly or may be discarded each day to avoid the necessity of filtering these solutions. Thealcohol rinses following the cytoplasmic stains are usually changed on a rotating basis after each use.6.5 Xylene Should be changed as soon as it appears tinted with any of the cytoplasmic stains. Water in the xylene will make the solution appearslightly milky. The clearing process may be disturbed, and tiny drops of water can be seen microscopically on a plane above the cellon a slide.6.6 Dipping Slides:Agitation of the slides by dipping is necessary to remove excess dye. If slides are not rinsed properly, a dull rather than sharp, crisppicture results. Slides should be dipped gently to avoid cell loss, and the slide carrier should not hit the bottom of the staining dish. Each dip should last approximately one second. Dipping too slowly will result in too much decolorization. However, one or twodips more or less will not affect results.6.7 Intensity of Staining Reaction:The desired intensity of nuclear and cytoplasmic stains is one of personal preference and varies with different cell samples. Individualexperimentation is necessary. The quality of the stained slides is also dependent on the solubility, percentage of dye concentration,

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experimentation is necessary. The quality of the stained slides is also dependent on the solubility, percentage of dye concentration,etc., of the dyes used in making EA, OG, and Hematoxylin, as discussed in the section of this chapter devoted to stain preparation. Factors other than timing, however, may influence the nuclear and cytoplasmic staining intensity, quality of stain, aging, storagecondition, temperature, etc…….6.8 Nuclear Stain Too Pale:Under staining of the nucleus may occur for one or more of the following reasons:

1. Contamination of Hematoxylin with Carbowax or coating fixatives.2. Time in Hematoxylin is not increased for Carbowax-fixed specimens wherein.3. Smears may have been permitted to air-dry before fixation.4. The pH of the tap water not sufficiently alkaline to blue properly.5. Single cells may appear understained if thick areas of the smears are correctly stained.6. Stain may become diluted if water is not drained from racks prior to immersion in Hematoxylin.7. If the timing of staining in Hematoxylin is based on material collected in intensity of staining.8. Expiration date of commercially prepared stain may have been overlooked.9. Stain may be too old and should be replaced.

10. Inadequate mixing of the contents of aerosol and spray fixatives can result in poorly distributed fixation. Staining may beuneven and spray can result in poorly distributed fixation. Staining may be uneven and muddy in appearance. Shake allfixatives well prior to use.

11. Slides sprayed with aerosol fixatives at too close or too far a range result in pale, poorly stained slides. 12. Waxes and oils from hairspray fixatives alter staining reactions if not adequately removed. Some brands may require the

soaking of slides overnight in 80% isopropanol alcohol, rather than merely rinsing them in alcohol prior to staining.6.9 Nuclear Stain Too DarkOver staining of the nucleus may occur for one or more of the following reasons:1. Cells fixed for a few minutes in modified Carnoy’s shrink, causing some chromatin condensation. Therefore, staining time inHematoxylin must be decreased.2. If time is based on staining slides prepared from fresh material, less time must be used for prefixed material.3. If single cells are well stained, thick areas of the slide may appear overstained.4. Nuclepore filters that are dissolved prior to staining sometimes require less staining time in Hematoxylin.5. The smears may have been prepared directly from very bloody or high-protein fluids. The sediments from these fluids should bewashed with a balanced salt solution prior to slide preparation.

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6. Slides were fixed in higher concentration of alcohol than is normally used.7. Waxes and oils from hairspray fixatives alter staining reactions if not adequately removed. Some brands may require the soakingof slides overnight in 95% alcohol rather than merely rinsing them in alcohol poured to staining. 6.10Contamination ControlHematoxylin, EA, and OG-6 should be filtered at least once daily and after staining any slides containing known cancer cells. Thealcohols used for rehydration and dehydration, the absolute alcohols and xylenes must also be filtered or replaced daily. To avoidcross-contamination from one slide to another in the same staining rack, it is recommended that those specimens notorious forshedding cells be stained in different staining dishes or at different times. It is generally recommended that gynecologic and non-gynecologic material be stained separately. Other fluids, particularly those, wet fixed in alcohol, must be stained in separate racks and the stains filtered after each run. A goodquality, medium-speed filter paper, such as Whattman #1, removes most cells. Effusions containing numerous cancer cells mostfrequently shed tumor cells that may attach to other slides. For this reason, quick preparation using a drop of sediment mixed withToluidine blue and examined immediately can be used to screen out these fluids, which should be stained at the end of the day in aseparate rack.Regardless of the care used in staining, cross contamination of slides may occur, and it is particularly disturbing if “malignant floaters”occur. If this happens, all solutions and stains should be immediately filtered or discarded. It is also wise to make a microscopiccheck of the mounting medium at this time to eliminate the possibility of its being a source of contamination.Following tables 2 to 6 summarizes the Papaninolaou staining problems and their remedies:

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7.0 PROCEDURE DIFF-QUICK STAINING:"Diff-Quick" is a proprietory brand of a Romanowski stain. The Romanowski group of stains are defined as being the black

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precipitate formed from the addition of aqueous solutions of methylene blue and eosin, dissolved in methanol. The variants of theRomanowski group differ in the degree of oxidation (polychroming) of the methylene blue stain prior to the precipitation.The stain class was originally designed to incorporate cytoplasmic (pink) staining with nuclear (blue) staining and fixation as a singlestep for smears and thin films of cells. Minor modifications of working stain concentration and staining time have been made over theyears for fixed tissue sections and Cytology smears. Reagent Formulae: “Rapid-Chrome Kwik-Diff” solution I/II/ 111 or kits (commercially prepared)Step by Step1. Prepare a slide(s) and allow drying.2. Prepare three containers (eq. coplin jars, or staining dishes). Fill one container with Kwik-Diff Reagent #1(Fixative), the secondwith Kwik-Diff Reagent #2, and a third container with Kwik-Diff Reagent #3. Pour adequate amounts of each solution into thestaining dishes.3. Dip slide or rack of slides five times (1 second per dip) into Reagent #1. Allow excess to drain into jar or dish and blot edge onabsorbent paper.4. Dip slide or rack of slides five times (1 second per dip) into Reagent #2. Allow excess to drain into jar or dish and blot edge onabsorbent paper.5. Dip slide or rack of slides five times (1 second per dip) into Reagent #3. Allow excess to drain into jar or dish and blot edge onabsorbent paper.6. Rinse slide or rack of slides by dipping or swishing in distilled water or deionized water.7. Air dry slide(s) or use warm air blower before mounting with oil and reading.Technical Points (Step 6) - Exposure to water should be as brief as possible to prevent excessive decolourisation.

ResultsBackground

light blue

Platelets violet to purpleNeutrophils nucleus cytoplasm

dark blue pale pink

Eosinophils nucleus cytoplasm granules

bluebluered to red/orange

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Basophils nucleus granules

purple or dark bluedark purple/black

Monocytes nucleus cytoplasm

Violet sky blue

Epithelial cells Nucleus Cytoplasm

Dark Bluelight Blue

8.0 PROCEDURES PREVENTION OF CROSS-CONTAMINATION DURING STAINING:8.1 Papanicolaou Staining:1. Discard/ Filter hematoxylin, cytostain, each day before beginning the staining procedure.2. Waters are replaced after each staining procedure.3. All solutions/ stains are replaced weekly and more frequently according to the following situations.4. Include an empty slide in each batch of slides for Pap stain and check after each batch for any floaters. If there is a suspicionshow it to Senior Cytotechnologist to check under the microscope for any floaters as source of cross contamination.8.2 Gynecologic cytology: Gynecologic cytology slides are stained in SurePath.8.3 Non- Gynecology:

Non- Gynecology slides are stained separately according to the following criteria

1. Cytospin and less cellular thin smears are stained together in a separate batch.2. Thick smears made from the Sediment or highly cellular FNA smears are stained separately after the thin smears mentionedabove.3. Examine the empty slide (already included in the batch under the microscope, for any floaters, and in case of any suspicion ofcross contamination show it to the senior Cytotechnologist for confirmation.4. Filter the staining solutions if needed. 9.0 PROCEDURE FOR REHYDRATION OF SLIDES9.1 Materials Needed0.9% sodium Chloride (Normal Saline)80 % isopropanol9.2 Step by step procedure:

1. Dip air dried smears in 0.9% sodium chloride (normal saline) for 30 seconds(up to 5 minutes maximum).

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(up to 5 minutes maximum).2. Fix 80% isopropanol alcohol for 5 minutes.3. Stain the slides with Pap stain in the usual manner.

10.0PROCEDURE FOR DE-STAINING SLIDES:10.1Material: Xylene, 100% Isopropanol, HCL, 80% Isopropanol.10.2Reagent Preparation: 0.5% HCL (1ml concentrated HCL + 199ml distilled water).10.3Step by step procedure

1. Saturate slide in xylene until the cover slip comes off. (Frequent shaking will expedite the removal of cover slip.Note: Forcing cover slip may remove the cells–Do not force (Wait until cover slip comes off).2. Saturate the slide in xylene until the mounting medium becomes soft, and cover slip comes off (about 20 minutes or untilclear)3. Dip slide 5min – 1hours times in the 0.5 HCL to remove hematoxylin.4. Check slides microscopically for de-coloration.5. Rinse the slide in gently running tap water for 10-15mins to remove acids.6. Place the slide in Scott’s tap water.7. Rinse the slide again in gently running tap water8. Slide is now ready to be stained in the usual routine manner.

11.0PROCEDURE MAINTENANCE OF CYTOLOGY STAINS AND SOLUTIONS: The general guidelines to consider when staining slides are:

1. Staining times are set, and should be followed, using the provided staining schedule.2. The use of graded concentrations of alcohol in the staining process will minimize cell distortion and possible cell shedding.This protocol incorporates the general staining guidelines stated above, and the following specific recommendations:1. If slides have been spray-fixed, then remove the spray fixative by soaking slide in a standard 80% isopropanol fixative forat least ten minutes.2. Stain the slides with a standard modified Papanicolaou stain, by using the staining schedule provided by the cytologist.3. The hematoxylin, OG-6, EA-50 are filtered daily or as needed, covered when not in use and replaced weekly, or asneeded. 4. A daily record of these events is logged on the Staining Maintenance documentation chart.5. All cytology stains are ordered, prepared from the vendor.

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5. All cytology stains are ordered, prepared from the vendor.6. When it is time to order stains, the cytopreparatory technician tells his/her supervisor that the cytology stains, alcohols,and mounting medium need to be ordered.7. Non-gynecologic slides are stained in separate racks, and in a separate batch thus preventing cross-contamination.

12.0PROCEDURE MOUNTING THE CELL SAMPLE

1. Turn on fume guard / safety cabinet.2. Place slides to be coverslipped and all necessary materials near fume guard.3. Drain excess xylene form the slide with cotton gauze.

Spread horizontal drops of mounting media directly onto the coverslip or slide. Use the smallest amount of mounting media necessaryto completely cover the area without evaporating after air drying.

4. The coverslip is then placed carefully on the slide or vise versa over the cells so as to prevent air bubbles. If there is abubble, gently teased out using applicator stick or forcep.5. After coverslipping, wipe the excess mounting media from the slide with absorbent paper or gauze.

PrecautionsCare must be taken when handling the glass slide and coverslip to prevent minor cuts. Since the mounting media is placed on thecoverslip, the dropper should never come in contact with the surface of the slide (specimen) which could contaminate the dropper. 13.0Responsibility:

Applies to clinicians or laboratory staff responsible for handling the specimens.

14.0Attachment: Pap Staining MonitoringForm01

15.0Distributions:- LMD Administration - Cytology Laboratory - All Clinical Departments

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16.0References:

- Koss, Leopold G. – Koss’s diagnostic Cytology and its histopathological bases, 1992.- Comprehensive Cytopathology, edited by Marluce Bibbo, 1991. - Manual of Cytotechnology edited by Catherine M. Keebler, American Society of Clinical Pathologist, 1983.- Cytology- Diagnostic Principles and Clinical Correlates Third Edition. By Edmund S. Cibas and Barbara S. Ducatman- Manual for Cytology, National Cancer Control Programme, Directorate General of Health Services. Ministry of Health and Family Welfare. Government of India 2005