Mid-Florida Pathology Specimen Collection Guide

 

GENERAL INFORMATION

Purpose

 

The purpose of this procedure is to establish proper protocol and provide guidelines for the collection and transportation of patient specimens for routine histological/cytological examination submitted to Mid-Florida Pathology Laboratory.

 

Hours of Operation

 

The laboratory is open to receive specimens from 8:00 a.m. to 8:00 p.m., Monday through Friday.  For technical assistance, please call or fax –

PHONE: 352-460-0292      FAX: 352-460-0785

 

Cytology Department: (Gyn and Non-Gyn Samples)

 

Submitting Specimens

 

1. All specimens must be accompanied by a completed Non-Gynecologic or Gynecologic requisition appropriate to the specimen. Failure to complete the form will delay processing of the specimen and possibly render the specimen inadequate for evaluation.  The Cytology requisition forms may be obtained from the Cytology Preparation area.
2. In addition, all cytologic specimens must be submitted with an appropriate ICD-10 code and a copy of the patient’s insurance information.  Failure to provide this information will delay processing of the specimen.
3. The specimen container and requisition slip must be clearly and correctly labeled:
4. The label on the specimen container must indicate:

• Patient's name
• Specimen type, including specific anatomic sample site
• Physician's name
• Date of specimen collection
• The requisition must indicate:
• Patient's name
• Patient's date of birth
• Patient's medical record number
• Patient's address or hospital location
• Physician's name
• Specimen type, including specific anatomic site sampled
• Date of specimen collection
• ICD-10 code appropriate to the procedure

 

NOTE:  Specimens lacking adequate identification, or which do not match in every respect the requisition accompanying them will not be accepted.

 

Specimens submitted by 6:00 p.m. will be reported on the next working day.

 

Criteria for specimen rejection

 

• Specimen submitted without appropriate requisition form
• Unlabeled specimen container
• Mislabeled specimen
• Improper specimen fixation
• Slide broken beyond repair
• Frozen specimen

 

Unlabeled specimens must be recollected, if recollection is possible. Otherwise, every attempt will be made by laboratory personnel to resolve the above problems before rejecting the specimen.

 

In addition to all the criteria above, all HPV, Gonorrhea, Chlamydia and Trichomonas testing, specimens will be rejected if there is an inadequate amount of material for testing. All tests require 1ml of fluid except for an endocervical/urethral swab and urine. Testing of all urine specimens requires 2ml of fresh urine to be received by the lab within 24 hours of collection. 

 

The Clinician or Clinician's office will be notified of the rejection either by phone or fax. Please refer to the "Specimen error or Rejection" documentation form All rejection forms and specimen requisition forms will be kept and filed according to the patient's last name.

 

There are two types of cytology specimens:

 

Gynecologic (Gyn) – Conventional pap smears or ThinPrep specimens: Place the specimen and requisition in separate compartments in a biohazard bag.  Place the bag in the basket on the door outside the Cytology preparation area.

 

Non-Gynecologic specimens (Non-Gyn): Secure the requisition around the specimen with a rubber band or place the specimen and requisition in separate compartments in a biohazard bag.  

 

Specimen transport:

 

• ThinPrep NonGyn Pap vial (green label)
• Sage metal screw top vial (Unfixed fluid specimens such as urine, sputum, bronchial washings, pleural, peritoneal, pericardial and similar fluids)
• Spill proof slide jars (Slides fixed in 95% ethanol)
• 15 or 20 ml evacuated stoppered tubes (Such as are used for blood collection: Pleural, peritoneal, pericardial and similar fluids)
• Plastic slide folders (Unfixed or spray fixed slides)
• FNA Collection Kit – Kit contains 5 slides, slide holder with alcohol, and 15ml tube with CytoLyt.

 

Specimen Collection and Preservation

 

There are methods requested by the laboratory for proper collection and preservation of all specimens submitted for cytologic study.  Valid interpretation of cellular features depends on strict adherence to these techniques.  The method may vary depending upon the type or source of specimen to be examined.  In general:

 

1. Any specimen collected by a smear technique (i.e. initially smeared on a glass slide by the physician or other individual at the patient's side) must be immediately fixed by immersing in 95% ethyl alcohol or by spraying with the fixative provided. An exception to this general rule is the collection of fine needle aspirates (see Assisted Fine Needle Aspiration Procedure, Appendices A and B).
2. Any fluid specimen or specimen collected in non-preservative fluid medium must be submitted to the laboratory as soon as possible (preferably within one to two hours) after collection so that it may be processed before significant cellular degeneration occurs. It is most helpful that specimens of this type reach the laboratory prior to 2:00 p.m.  Monday through Friday.  If a specimen is unavoidably collected after laboratory hours, it should be refrigerated (not frozen) and delivered as soon as the laboratory opens.
3. All supplies for the collection of cytologic specimens are available from the Cytology Department, Sales Rep or Courier.
4. Review of collection and preservation procedures for the specific anatomic site and/or method of collection is recommended.

GUIDELINES FOR COLLECTION OF SPECIMENS FROM THE FEMALE GENITAL TRACT

 

Specimens from:

 

Cervix, endocervix, ectocervix, lateral vaginal wall, vaginal pool, vagina, vulva and/or endometrium

 

Patient Preparation: 

 

1. At the time of scheduling the patient for gynecologic examination, please inform her that the quality of her pap smear may be affected by factors under her control:
2. Request that the patient avoid sexual intercourse, douching and the use of vaginal contraceptives or suppositories at least 24 hours prior to examination.
3. Request that the patient schedule her appointment from day 4 to 15 of her menstrual cycle, the optimal time for sampling. Avoid collecting the smear while she is menstruating.
4. The smear should be taken prior to bimanual pelvic examination.

 

Pertinent Clinical Information

 

Items marked in bold are minimum acceptable clinical information (See Gynecologic Cytology Requisition)

 

Date of specimen collection

Source of specimen

Patient's age and date of her last menstrual period

Hormonal status (cyclic, pregnant, post-partum, post-menopausal, abnormal bleeding)

Hormone replacement

Antibiotic cream

Presence of IUD

DES exposure

History of cervical intraepithelial lesion, cervical malignancy or any extragenital malignancy

History of systemic chemotherapy

History of radiotherapy

History of gynecologic surgery: conization, hysterectomy (specify complete or partial), D&C, LEEP, cryosurgery, electrocautery, laser vaporization

Previous abnormal cytology - Please include date, diagnosis and AMH accession number if available.

Current abnormal findings or patient complaints, especially presence of a lesion or history of abnormal bleeding.

Last pap smear: Date and Results

 

Handling Conditions:

 

The preservative solution (PreservCyt) in the ThinPrep vial is antimicrobial and anti-viral and has been shown to cause greater than 99.999% inactivation within 15 minutes for the following microbes and viruses: Candida albicans, Eschericia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium tuberculosis, Rabbitpox, and Human Immunodeficiency virus (HIV).  Nevertheless:

 

All cytological specimens should be considered infectious until fixed with a germicidal fixative.  OBSERVE UNIVERSAL PRECAUTIONS WHEN HANDLING SPECIMENS FROM ALL PATIENTS.

 

Conventional specimens: Specimens must be immediately (within 5 seconds) preserved with the provided fixative. Once fixed, the specimen may be kept at room temperature indefinitely.  Avoid exposure to dust and surface abrasion

 

Thin Prep Specimens: The storage limit for cells in PreservCyt is 90 days at 4 to 37 degrees Celsius.

 

Absolute certainty of specimen identification must be maintained throughout acquisition and processing.  All slides must be labeled in pencil with the name of the patient from whom the specimen was obtained. 

 

 PROCEDURE:

 

Label the frosted end of the slide or ThinPrep vial, in pencil, with the patient's last name and first name or first initial. Indicate the anatomic source of the specimen: "C" - ectocervix, "E" - endocervix, "V" - vagina, "LVW" - lateral vaginal wall, "B" - brush, "VCE" - combination vaginal/cervical/endocervical.

 

Have fixative or vial open and readily available for use.

 

Remove talc from gloves before touching patient, slides or instruments.

 

Introduce the speculum without lubricant.  Warm saline may be used to facilitate introduction - do not use lubricant jelly.  Position the speculum so that the entire face of the cervix can be visualized.

 

Conventional Method:

 

It is important to obtain a smear that is not obscured by blood or inflammatory exudate.  Following positioning of the speculum, discharge may be gently removed with a ring forceps holding a folded gauze pad or with a dry proctoswab or scopette.  Inflammatory exudate may be removed by placing a dry 2" x 2" gauze pad or scopette over the cervix and peeling it away after it absorbs the exudate.  However, the cervix should not be cleansed by washing with saline as this may result in a relatively acellular smear.

 

The specimen may be obtained using an Ayre spatula to sample the ectocervix in combination with a Cytobrush to sample the endocervix OR a Papette (broom) may be used to sample both areas simultaneously.  (The use of the Cytobrush is controversial in pregnant patients.  A papette device (broom) may be substituted if clinically indicated.)

 

Each method requires attention to unique aspects of technique to optimize the resulting sample.

Ayre Spatula/Endocervical Brush Technique:

 

Insert the spatula in the cervical os with the small protrusion at the 9 o'clock position.  Rotate 360 degrees in a clockwise direction so that all material is collected on one side of the spatula.   Smear the collected material on the slide from the frosted end toward the bottom.  Spray fix thoroughly from a distance of 12 inches within 3-5 seconds.  (If using a one-slide preparation method, hold this material on the spatula while rapidly obtaining the endocervical sample.)

 

Insert the Cytobrush in the os leaving the bristles closest to the handle exposed (no more than one cm). Limiting the depth of insertion reduces inadvertent sampling of the endometrium. Rotate the brush gently 90 to 180 degrees.  Reduced rotation of the brush minimizes bleeding which may dilute or obscure the collected sample.  Roll and twist the brush across the length of the glass slide, bending bristles slightly.  (Admix the material held on the Ayre spatula if using one slide method.)  Spray fix thoroughly from 12 inches within 3-5 seconds.

 

Papette (Broom) Technique:

 

Gently insert the central long bristles of the Papette device into the cervical os until the lateral bristles bend fully against the ectocervix.  Rotate 1800 degrees (five full rotations) in only one direction by rolling the handle between the thumb and forefinger.

 

NOTE:  Do not insert the shoulders of the Papette device into the cervical canal.  Insertion of the shoulders of the device into the cervix may cause trauma to the cervix and/or may cause the head of the device to release in the endocervical canal when extraction is attempted.

 

Transfer the sample with a single paint stroke action from the frosted end of the slide toward the bottom.  Turn the Papette device over and repeat the paint stroke action over exactly the same area.  Spray fix thoroughly from a distance of 12 inches within 3-5 seconds.

 

Allow the fixed specimen(s) to dry before placing in a plastic folder or slide box for transport to the laboratory.

 

Complete a Gynecologic Cytology Requisition form or enter a Cytology order, as indicated below, in the SCM system.  Complete the patient's name, identification number, date of birth, date of specimen collection and all pertinent clinical history including the date of last menstrual period.

 

Thin Prep method:

 

Obtain samples as described for the conventional method.

 

Rinse the Papette device in the PreservCyt vial by pushing the broom into the bottom of the vial 10 times, forcing the bristles apart.  Swirl the broom vigorously to further release material, Discard the collection device

 

OR

 

Rinse the spatula into the PreservCyt solution vial by swirling the spatula vigorously in the vial ten times.  Discard the spatula. Rinse the brush in the PreservCyt solution by rotating the device in the solution ten times while pushing against the PreservCyt vial wall.  Swirl the brush vigorously to further release material.  Discard the brush.

 

Tighten the cap so that the torque line on the cap passes the torque line on the vial.

 

Place the labeled specimen in its plastic slide mailer in a plastic biohazard specimen bag.  Place the transmittal sheet in the pocket provided on the bag or attach the sheet securely to the specimen if no bag is available.  Specimens received without a transmittal sheet will be rejected for processing.

 

Educational Note:

 

The Pap smear (whether conventional or liquid-based) is a screening technique to aid in the detection of cervical cancer and its precursors. It is of proven value as part of a regularly scheduled screening program.  It is not a diagnostic procedure.  Both false-negative and false-positive results may occur.  Accordingly, positive results should be confirmed with additional studies, and any lesion should be biopsied as clinically indicated this is a decision that you should discuss with your patient.

 

Additional ‘Out of the Vial’ Testing:

 

The current testing panel available at Mid-Florida Pathology is:

 

1. HPV
2. HPV 16/18/45 reflex
3. CT/NG (Chlamydia Trachomatis/Neisseria Gonorrhea
4. Trichomonas

SITE SPECIFIC INSTRUCTIONS FOR NON-GYNECOLOGIC

SPECIMENS

 

• BILE DUCT/AMPULLARY BRUSHING

 

Proceed as for routine endoscopy.  Identify the lesion and brush firmly and briskly about five to ten times to penetrate the lamina propria.  Withdraw the brush.  Unsheath the brush and clip it into the vial of CytoLyt.  Label the vial with the patient's name and specimen type.

 

    BODY CAVITY FLUID

 

The preferred specimen is at least 50ml of fluid, however less sample volume can be processed and up to 1000 cc of fluid may be submitted. 

 

*Heparin maybe added to bloody specimens to reduce clotting.  Add 3 units of heparin/ml of specimen per capacity of collection container.  If green top tubes are available, the fluid can be collected in them.  Gravity agitate to thoroughly mix.

 

Collect the specimen in 50ml tube or vacutainer bottle.

 

If delivery of specimen to laboratory is delayed, greater than 2 hours or overnight, please refrigerate (DO NOT FREEZE).

 

-       Pertinent Clinical Information

 

Note the following clinical findings if present:

 

Documented malignancy

Radiotherapy to an adjacent site

Chemotherapy

Chronic disease (e.g. cirrhosis, tuberculosis)

Note if the effusion is spontaneous, post surgery or longstanding

 

• BREAST

 

Nipple secretion

 

Have patient hold open bottle of fixative below breast.  Gently strip the sub areolar area and nipple, using thumb and forefinger.  If secretion occurs, allow a drop the size of a pea to accumulate on the apex of the nipple.  Support the areola and nipple with one hand.  With the other hand, place the slide upon the nipple, touching the drop. Allow the drop to spread laterally, then draw slide quickly across the nipple and immediately drop into bottle of fixative.  Repeat complete procedure until all secretion obtainable from the nipple is utilized.

 

When no nipple secretion can be obtained but there is nipple erosion or ulceration, the specimen consists of fluid oozing from the lesion.  Saline or albumin can be added to the erosion fluid, gently mixed with the lesion to exfoliate cells and smears made as above.  A cotton swab soaked in albumin may be used to abrade the lesion and transfer the material to a slide.

 

Cyst aspirate

 

Aspirate all cyst fluid.  Expel the contents into a 50ml conical centrifuge tube containing 15ml of CytoLyt.

 

Fine needle aspirate (solid mass)

 

Locate lesion by palpation.  Wipe site over the lesion with antiseptic.  Immobilize the mass in one hand in a favorable position.  Insert 21 or 22-gauge needle in the mass.  Retract the plunger as far as possible, creating a vacuum, while the needle proceeds in a straight line through the lesion.  Move the needle back and forth in the mass four or five times in various directions, maintaining negative pressure.

 

When the aspiration is complete, release the plunger and allow equalization of pressure before withdrawing the needle.  Withdraw the needle.  Disconnect the barrel of the syringe from the needle.  Express the contents of the needle onto slides as necessary.  Align the edge of a second slide perpendicular to the long axis of the first slide at the edge of the drop of fluid.  Allow the drop to spread a bit laterally, then gently draw the top slide toward the bottom of the first slide, creating a thin smear.  Immediately spray fix the prepared slide.  If additional slides are prepared, alternately spray-fix and air-dry slides, as they are prepared.  Rinse the needle into a 50ml conical centrifuge tube containing 15ml of CytoLyt.

 

Pertinent Clinical Information

 

Note the presence of a palpable mass (size, consistency) or abnormal findings on mammography or ultrasound

Note familial history of breast carcinoma

Note if patient is pregnant, lactating, receiving hormonal therapy, radiation or chemotherapy

 

    BRONCHIAL BRUSHING

 

Proceed as for routine bronchoscopy.  Identify the lesion and brush firmly and briskly.   Withdraw the brush.  The specimen may be submitted on glass slides or in a tube of CytoLyt solution as follows:

 

Smear the material obtained on the brush on the labeled slide.  Rotate the brush from side to side in an area the size of a nickel.  An attempt to spread the material over too large an area may result in increased artifactual distortion of the cellular material due to air-drying.  Place slide in 95% ethyl alcohol in a Coplin jar or leak proof slide jar.

 

AND/OR

 

Unsheath the brush and clip it into a vial of CytoLyt.  Label the vial with the patient's name and specimen type.

 

Important Clinical Information

 

Note the following clinical findings if present:

 

Documented carcinoma (primary site, cell type)

Hemoptysis

Long-standing pulmonary disease (e.g. COPD, TB)

Results of radiologic or bronchoscopic examination

If biopsy or resection previously performed, histologic diagnosis

Radiotherapy or chemotherapy

If viral or fungal disease is suspected, note evidence of infectious disease, suspected pathogen, and if the patient is immunosuppressed

 

    BRONCHIAL WASHINGS OR BRONCHOALVEOLAR LAVAGE

 

Collect aspirates and washings obtained at the time of bronchoscopy in a Lukens trap.  The preferred washing solution is CytoLyt.  Do not add alcohol or other fixatives to the specimen.

 

Important Clinical Information

Note the following clinical findings if present:

Documented carcinoma (primary site, cell type)

Hemoptysis

Long-standing pulmonary disease (e.g. COPD, TB)

Results of radiologic or bronchoscopic examination

If biopsy or resection previously performed, histologic diagnosis

Radiotherapy or chemotherapy

If viral or fungal disease is suspected, note evidence of infectious disease, suspected pathogen, and if the patient is immunosuppressed

 

 

    CEREBROSPINAL FLUID (note: please give any history of CJD)

 

The preferred specimen is cerebrospinal fluid collected without anticoagulant or fixative. The preferred sample size is 5ml, however less sample volume is acceptable. 

 

The specimen should be brought to the Laboratory for processing as soon as possible since cells degenerate rapidly in cerebrospinal fluid. If immediate transport is not possible, refrigerate until delivery to the laboratory.

 

• COLON BRUSHING

 

Proceed as for routine colonoscopy.  Identify the lesion and brush firmly and briskly about five to ten times to penetrate the lamina propria.  Withdraw the brush.  Unsheath the brush and clip it into the tube of CytoLyt.  Label the vial with the patient's name and specimen type.

 

• ESOPHAGEAL BRUSHING

 

Proceed as for routine esophagoscopy.  Identify the lesion and brush firmly and briskly about five to ten times to penetrate the lamina propria.  Withdraw the brush.  Unsheath the brush and clip it into the vial of PreservCyt solution.

 

 

• FINE NEEDLE ASPIRATION

 

BREAST See Appendix A

THYROID See Appendix A

LYMPH NODE See Appendix A

NECK See Appendix A

PAROTID GLAND See Appendix A

SALIVARY GLAND See Appendix A

KIDNEY See Appendix B

LIVER See Appendix B

LUNG See Appendix B

PANCREAS See Appendix B

PELVIC OR PERITONEAL See Appendix B

RETROPERITONEAL See Appendix B

 

 

 

• GASTRIC BRUSHING

 

Proceed as for routine gastroscopy.  Identify the lesion and brush firmly and briskly about five to ten times to penetrate the lamina propria.  Withdraw the brush. Unsheath the brush and clip it into the tube of CytoLyt solution.

 

• PNEUMOCYSTIS CARINII

 

The preferred specimen for identification of Pneumocystis carinii is bronchoalveolar lavage.  In decreasing order of sensitivity in detection of P. carinii, bronchial washings, bronchial brushings or induced sputum may yield diagnostic organisms. Expectorated sputum is not acceptable for identification of P. carinii.

 

Fresh, unfixed specimen must be submitted.  No fixatives are to be added.  Refrigerate if delay in transport to laboratory is necessary.

 

Indicate ‘Silver stain’ on the requisition

 

• SPUTUM, EXPECTORATED

 

Patient preparation

 

Prior to collection of specimen, provide a container labeled with the patient's name, physician and specimen type.  Instruct the patient to collect the specimen the following morning as follows:

 

Sleep on your back, if possible, the night before collecting the specimen.

 

Collect specimen in the morning, before the morning meal.  Upon awakening, clean the mouth with water, gargle and expectorate the water.

 

Inhale air to the full capacity of the lungs, and then exhale air with an expulsive cough.  Expectorate all sputum raised from the chest into the container provided.  Avoid collecting clear, watery saliva or thick, ropy secretions from post-nasal drip.  Repeat and continue until an adequate specimen is collected (at least one cc, preferably 3 to 5 cc.)

 

Early morning specimens provide the greatest yield of diagnostic cells, however all pulmonary material should be collected.  To avoid food contamination, instruct the patient to gargle and rinse the mouth after meals.

 

 

The diagnosis of primary bronchogenic carcinoma may be increased when multiple, rather than single, specimens are examined. Collect three separate specimens on three consecutive mornings. Do not pool specimens.  Each specimen is to be submitted in a separate, properly labeled container with a separate requisition.

 

Important Clinical Information

 

• Note the following clinical findings if present:

 

Documented carcinoma (primary site, cell type)

Hemoptysis

Long-standing pulmonary disease (e.g. COPD, tuberculosis)

Results of radiologic or bronchoscopic examination

If biopsy or resection previously performed, histologic diagnosis

Radiotherapy

Chemotherapy

In cases where viral or fungal disease is suspected, note evidence of infectious disease, suspected pathogen, and if the patient is immunosuppressed

 

 

• TZANCK SMEAR

 

Label the slides with the patient’s name in pencil.

Remove crust or dome of the lesion.

Scrape the margin of the ulceration with a wooden tongue depressor.

Smear material onto a clean glass slide and fix immediately with spray fixative OR swirl tongue depressor in a vial of PreservCyt solution

 

 

 

• URINE, URETERAL OR URETHRAL WASHING

 

Patient preparation

 

If urinary output is low, the patient should be hydrated by oral or parenteral administration of fluids, unless there are clinical contraindications.

 

Instruct the patient the night before in the method of collecting a clean-catch specimen.  Explain that he/she may void in the morning upon awakening.

 

The preferred specimen is a clean catch second voided or catheterized urine consisting of 30 cc of fluid.  Do not add fixative or preservative of any kind if the specimen cannot reach the laboratory in 1 to 2 hours.  Otherwise, add CytoLyt, 1ml to 4ml of specimen.  Refrigerate and transport ASAP.

 

Pertinent clinical information

 

The following clinical information should be noted if present:

 

Documented malignancy: primary site and type

Hematuria

Urinary tract calculi

 

Other known urologic pathology (stricture, non-functioning kidney, ileal conduit, etc.)

Note if specimen is obtained by voiding or catheterization

 

APPENDIX A

 

GUIDELINES FOR FINE NEEDLE ASPIRATION OF PALPABLE LESIONS

 

1. Palpate the target lesion and estimate its distance from the point of entry on the skin.

 

2. Sterilize the skin with an alcohol or Betadine swab.

 

3. Fix the target with one hand, between the index finger and thumb.

 

4. Introduce the needle gently but firmly into the subcutaneous tissue, then into the target. A distinct change in consistency of the tissue should be felt when the target is entered.

 

5. With the needle firmly within the lesion, loosen cells from the target tissue by repeated short movements of the needle tip. Aspirate by exercising a negative pressure on the plunger of the syringe.

 

6. Without releasing the negative pressure, redirect the needle within the target by withdrawing it 2 to 5 mm and reinserting at a slightly different angle. Do not withdraw the needle beyond the boundaries of the target lesion.

 

7. When the aspiration is completed, release the plunger and allow equalization of pressure prior to withdrawing the needle from the lesion.

 

8. Inject the aspirated material into 15ml of CytoLyt and/ or prepare smears as follows:

 

9. Label the frosted end of the glass slide with the patient’s name and date of birth.

 

10. Avoid drawing the aspirated material into the barrel of the syringe: remove the needle from the barrel of the syringe. Aspirate air into the barrel.  Reattach the needle and carefully express the contents of the needle onto the labeled glass slide.

 

11. Using a second slide held perpendicularly to the longitudinal axis of the slide containing specimen, gently but firmly spread the aspirate along the slide as for a blood smear.

 

12. Allow one slide to air dry and spray fix the more cellular slide immediately (within 5 seconds). Repeat as necessary. 

 

13. Rinse needle by aspirating a few ml’s of CytoLyt through the needle and expelling the fluid into the centrifuge tube containing 15ml of CytoLyt.

 

14. If flow cytometry is desired, obtain a separate pass and eject the material into RPMI. Keep the specimen at room temperature and transport immediately to the Laboratory. Submit a flow cytometry request for the desired immunophenotyping panel(s).

 

15. If a needle core is obtained, immediately eject the core into a labeled vial of zinc formalin.

 

APPENDIX B

 

GUIDELINES FOR FINE NEEDLE ASPIRATION WITH RADIOLOGIC GUIDANCE

 

Schedule the procedure by calling Mid-Florida Pathology at: 

 

PHONE: 352-460-0292 FAX: 352-460-0785

 

1. Localize the target lesion and determine its distance from the point of entry on the skin.

 

2. Sterilize the skin with an alcohol or Betadine swab.

 

3. Localize the needle in the target lesion: Introduce the needle gently but firmly into the subcutaneous tissue, then into the target. A distinct change in consistency of the tissue should be felt when the target is entered.

 

4. With the needle firmly within the lesion, loosen cells from the target tissue by repeated short movements of the needle tip. Aspirate by exercising a negative pressure on the plunger of the syringe.

 

5. Without releasing the negative pressure, redirect the needle within the target by withdrawing it 2 to 5 mm and reinserting at a slightly different angle. Do not withdraw the needle beyond the boundaries of the target lesion.

 

6. When the aspiration is completed, release the plunger and allow equalization of pressure prior to withdrawing the needle from the lesion.

 

7. Inject the aspirated material into 15 ml of CytoLyt and/ or prepare smears as follows:

 

8. Label the frosted end of the glass slide with the patient’s name.

 

9. Avoid drawing the aspirated material into the barrel of the syringe: remove the needle from the barrel of the syringe. Aspirate air into the barrel.  Reattach the needle and carefully express the contents of the needle onto the labeled glass slide.

 

10. Using a second slide held perpendicularly to the longitudinal axis of the slide containing specimen, gently but firmly spread the aspirate along the slide as for a blood smear.

 

11. Allow one slide to air dry and spray fix the more cellular slide immediately (within 5 seconds). Repeat as necessary. 

 

12. Rinse needle by aspirating a few ml's of CytoLyt through the needle and expelling the fluid into the centrifuge tube containing 15ml of CytoLyt.

 

13. If flow cytometry is desired, obtain a separate pass and eject the material into RPMI. (RPMI is obtained from Hematology or Pathology). Keep the specimen at room temperature and transport immediately to the Laboratory. Submit a flow cytometry request for the desired immunophenotyping panel(s).

 

14. If a needle core is obtained, immediately eject the core into a labeled vial of zinc formalin.

 

 

Histology Department:

 

SPECIMEN COLLECTION - FIXATION

 

Histology Fixatives:  Because many histological samples cannot be recovered or repeated, it is critical for everyone involved with the tissue specimen collection process to have a working understanding of fixation. Specimens will be accompanied by individual lab requisition and/or a shipment list.

 

Fixatives for Histology:

 

10% Zinc Formalin

10% Neutral Buffered Formalin

 

Function of Fixatives:

 

1. Prevent autolysis (cell self-destruction from enzymes)
2. Prevent putrification (activities of decay, bacterial attack)
3. To harden the tissue (make tissue easier to handle)
4. To kill infectious agents.

 

PATIENT IDENTIFICATION:  

 

***Two patient identifiers are required on the labeled specimen container. ***

 

1. Patient name, medical record number or account number, date of birth and sex.
2. Requesting physician and phone number.
3. Date and time of collection.
4. Preoperative and postoperative diagnoses, clinical history, including previous biopsies, surgeries, prior therapy.
5. Specimen type and/or site.

**Location is not an acceptable identifier**

 

POINT OF COLLECTION: 

 

In order to maximize accurate results for tissue examination, the person handling the original specimen should:

 

1. Pre-label the biopsy containers with Name, DOB, Collection Date, and Collection Site.
2. Put the specimen in fixative as soon as it is removed from the patient. This must not wait until the   procedure is over.  Small specimens are especially susceptible to autolysis and putrification.
3. Completely cover the specimen with formalin. A 1:20 ratio (size of specimen to amount of formalin) is recommended.
4. Seal the container and check for leaks. Formalin is a caustic chemical and should be handled with care. 
5. Double check biopsy containers to confirm they are all labeled with the Patient Name, DOB, Collection Date, and Collection Site.
6. Fill out the Pathology Requisition.
7. Fill out the shipping log.
8. Place the container with the requisition in a biopsy or specimen transportation box and place in the designated specimen pick-up area.

 

  Fixation Times – Time is important with fixation in two respects:

 

1. Interval between interruption of blood supply and placement into fixative – This time period should be as short as possible.
2. Duration of fixation – Adequate fixation time is needed to prevent distortion in subsequent processing steps.  Approximately 6 hours of fixation should be allowed for most tissue specimens, although smaller biopsies can be processed sooner without a difference in the final outcome.

 

NOTE:  Breast samples may require further testing (Her2, etc) in which fixation times are essential for proper evaluation.  These specimens will be allowed to fix no less than 6 hours and no more than 48 hours.

 

SPECIFIC SPECIMEN TYPE GUIDELINES

                                                                                                                     

Bone Marrow Aspirate

 

1. Please perform bone marrow Aspirate Smears (5-6 air dried smears)

 

2. Bone Marrow Aspirate in 3 GREEN TOP Tube (Sodium Heparin), at least 1-2 ml in each tube. Dispense the aspirate into the green top tube, mix gently several times to avoid clotting (For FLOW CYTOMETRY, CYTOGENETICS, and FISH).

 

3. Bone Marrow Aspirate in LAVENDER TOP Tube (EDTA), at least 2 ml. Dispense a part of the aspirate into the lavender top tube, mix gently several times to avoid clotting (For MOLECULAR STUDIES).

 

4. Keep remaining aspirate in the syringe to clot.

 

5. Put the clot in (10% neutral buffered FORMALIN)

 

Bone Marrow Core Biopsy

 

Bone Marrow Core Biopsy, at least 1.5 cm in length (placed in 10% neutral buffered formalin)

 

Please include:

 

1. CBC Results (the most recent one)
2. Peripheral blood smear (if available)

 

 In case there is DRY TAP, please do the following:

 

1. Take a Core Biopsy, 1-2 cm in length; make Touch Preps of the Core Biopsy. Touch the biopsy to the clean glass slide (submit around 5-10 slides).

2. Place the above Core Biopsy in 10% neutral buffered FORMALIN container and label the container.
3. Take another Core Biopsy, 1-2 cm in length, and place in RPMI, for FLOW CYTOMETRY.
4. Take another Core Biopsy, 1-2 cm in length, and place in RPMI, for CYTOGENETICS.

 

Please include:

 

1. CBC Results (the most recent one)
2. Peripheral blood smear (if available)
3. Bone Marrow Microbiology Cultures
4. Place at least 2ml of bone marrow aspirate in a sterile tube (EDTA or Sodium Heparin Non-gel tubes).

 

Note:  It is recommended that the aspiration site for culture should be different from the bone marrow site used for routine studies.

 

SPECIMENS FOR IMMUNOFLUORESCENT TESTING

 

Place specimen in Michel's fixative (green top container). 

 

Note: Michel’s Solution is a tissue fixative used for preservation of skin biopsy specimens to be examined by direct immunofluorescent testing for demonstration of tissue-fixed immunoglobulins.

 

 

UNFIXED SPECIMENS

 

 Note: These specimens should be submitted to the lab on the same day that they are taken. If that is not possible they should be stored at 4ºC and transported to the lab within 24 hours.

 

1. The specimen must be in a sealed, airtight container.
2. The patient specimen container must have a minimum of two identifiers along with the date received and type of specimen.
3. The specimen cannot be stored in the refrigerator longer than 48 hours without a fixative.
4. Criteria for Fresh Specimens Submitted to Anatomic Pathology

 

• Sputum, bronchial aspirate, or mucosal fluid: Specimens with mucous content may be preserved for 12-24 hours, if refrigerated.
• Pleural, peritoneal, or pericardial fluids: Specimens with high protein content may be preserved for 24-48 hours without refrigeration.
• Urine or Cerebrospinal Fluid: Specimens with a low mucous or protein content will endure only a 1-2 hour delay before losing integrity, even if refrigerated.
• Gastric Materials: Specimens with a low pH must be collected on ice and prepared within minutes of collection to prevent cellular destruction and loss of integrity due to hydrochloric acids.

 

SLIDE TRANSPORTATION

 

1. Should be packed in appropriate containers for shipping, with complete requisition and labeled with at least two identifiers.
2. On arrival, the slides will be examined and their condition noted. If any slides are broken, it will be noted on the daily shipping log.  The client will be contacted and replacement slides will be ordered.

 

REFERENCE:  http://www.iuhealth.net/pdf/pathology-lab/compliance-regulations/compliance-bulletins/specimen_rejection.pdf