Pathology Associates

I. TISSUE/SURGICAL PATHOLOGY SPECIMENS

In most cases, specimens submitted for routine histology should be placed in 10% formalin, cytopathology in a sterile container without fixative, and all accompanied by a filled out pathology requisition. For more detail and special cases, see below.

Correct identification of the tissue is essential. Proper identification should be securely fastened on the specimen container and should include: 1. Patient name. 2. Hospital number (if a hospital patient). 3. Date obtained. 4. Referring physician's name. 5. Presence of a biohazard (if known). 6. The label should indicate the source of the specimen or should be marked with an identifying letter (A, B, C, ...) or number (#1, #2, #3, ...). This identifying information should match the information on the specimen requisition form.

A specimen request form must accompany all specimens submitted. Patient demographic and billing information must be complete, accurate, and legible. A properly completed request form should include the following:

Patient Name

Patient Social Security Number

Date of Birth

Gender

Patient Phone Number

Medical Record Number (Note: If a hospital patient, patient type inpatient vs outpatient must be
clearly identified.)

Responsible Party name

Insured/Responsible Party Address

Mark Relationship Box

Medicare/Medicaid Number

Insurance Name and Address (other than Medicare/Medicaid)

Insurance Contract Number, Group Number

Secondary Insurance Name and Address

Secondary Contract Number, Group Number

Insured Social Security Number (if not the patient)

Employer Name/Phone Number

Record Collection Date/Time

Obtain Patient Release Authorization

Complete test request area:

Referring Physician

Source of Specimen

Patient Clinical History Clinical Diagnosis/ICD9 codes. (Frozen section diagnosis when applicable). Signs and Symptoms Patient History/Remarks

Attach a legible copy of insurance card if available. All identifying information on the specimen request form must match that on the specimen container.

All tissue specimens should be presumed to be infectious and universal safety precautions should be followed in handling all specimens.

All tissue specimens, except those submitted for frozen section, should be placed in fixative. The fixative used should be the one specified for the tissue in the following list. In most cases this will be 10% formalin. The amount of fixative should be adequate, and the specimen should be placed in a large enough container to completely cover the specimen with fixative.

If a frozen section is requested, the specimen must not be placed in fixative, but must be delivered promptly to the laboratory. Any biopsy submitted for frozen section should be placed on ice if any delay in reaching the laboratory is anticipated.

If a fine needle aspirate is requested, refer to Cytopathology Section.

The tissue specimens should be placed in the area designated for pick-up so that the courier can transport the specimen to the pathology laboratory in a timely fashion. Specimen pick-ups are normally made at regular intervals, but special pick-ups can be made.

If a frozen section is requested this should be indicated. Please contact Pathology Associates so that the specimen can be transported to the laboratory and a frozen section performed.

If a pathology report is requested RUSH, a report will be telephoned to the physician the next morning (except for Sundays and Holidays). Please mark the specimen RUSH. The specimen must arrive in the pathology laboratory the same day to allow for overnight processing. Please contact Pathology Associates for specimen transport if a RUSH is requested after the courier has made the last pick-up of the day.

Please contact Pathology Associates if special transportation to the laboratory is needed such as:

If special handling of a specimen is needed.

If an unscheduled specimen pick-up is needed.

If a specimen needs to be transported immediately following a scheduled surgical procedure.

Frozen section results are phoned to the requesting physician, by our pathologist, usually within 15 minutes of receiving the specimen. Written reports are delivered within 24-48 hours after receiving specimen, unless the completing of the case requires additional work-up. If you have a special need for faster service on a special case, indicate RUSH on the request. Our pathologists are always available to discuss cases with the referring physician.

Automatic faxing may also be a possibility at the request of the physician’s office or the hospital. If there is a problem with courier delivery of written reports, please contact us at 256-533-1480.

BIOCHEMICAL STUDIES:

Fixation: None.

Special instructions: If biochemical studies are requested, a sterile sample of the fluid or tissue should be put into a special transport tube. These transport tubes can be obtained from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the procedure to allow us time to deliver the transport tubes. If time does not allow for prior notification, place the biopsy or other tissue specimen, such as products of conception, into a sterile container with sterile saline or cell culture medium to keep it moist. The best results are obtained if the biochemical studies are started the same day as the tissue is obtained. If a delay is necessary, refrigerate the specimen until it can be transported.

BONE MARROW - BIOPSY OR PARTICLE PREPARATION

Fixation: Bouin's fixative and air dried smears.

Special instructions: Air dried smears (no fixative) should be made from the bone marrow aspirate. Air dried touch preparation smears of the bone marrow biopsy should be made prior to placing the biopsy into Bouin's fixative. The marrow particles should be collected in a heparinized syringe to prevent clotting, and the blood should be filtered from the marrow particles prior to placing the particles into Bouin's fixative.

If bone marrow cultures are requested, a sterile sample of the marrow aspirate (approximately 2-3 ml) should be collected for culture on the appropriate media.

If cell marker (immunophenotype) studies are requested, a sterile sample of the marrow aspirate should be put into 1 ACD solution A anticoagulated tube (yellow stopper). Store and transport at room temperature.

If chromosome analysis is requested, a sterile sample of the marrow aspirate (approximately 3-4 ml) should be put into a special transport tube. These transport tubes can be obtained from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the procedure to allow us time to deliver the transport tubes.

CELL MARKER STUDIES (IMMUNOPHENOTYPE):

Fixation: None.

Special instructions:

A) For effusions, immediately following collection, add 10 units of injectable sodium heparin for each milliliter of fluid collected. Store and transport at room temperature.

B) For peripheral blood, submit 2 ACD solution A anticoagulated tubes (yellow stopper), and 1 EDTA anticoagulated tube (lavender stopper). Store and transport at room temperature.

C) For bone marrow, submit 1 ACD solution A anticoagulated tube (yellow stopper). Store and transport at room temperature.

D) For tissue, place approximately 0.5 grams of sterile tissue into RPMI transport media. Store and transport at 4 degrees C. Do not freeze. RPMI transport media can be obtained from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the biopsy to allow us time to delivery the RPMI transport media.

CHROMOSOME ANALYSIS:

Fixation: None.

Special instructions: If chromosome analysis is requested, a sterile sample of the fluid or tissue should be put into a special transport tube. These transport tubes can be obtained from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the procedure to allow us time to deliver the transport tubes. If time does not allow for prior notification, place the biopsy or other tissue specimen, such as products of conception, into a sterile container with sterile saline or cell culture medium to keep it moist. The best results are obtained if the analysis is started the same day as the tissue is obtained. If a delay is necessary, refrigerate the specimen until it can be transported.

CULTURES - BACTERIAL, FUNGAL, VIRAL AND CHLAMYDIAL:

Fixation: None.

Special instructions: Collect a pea sized (3-5 mm) portion of tissue. In general, for bacterial and fungal cultures, the tissue is ground in small fragments and inoculated into the appropriate culture media. For viral and chlamydial cultures, the tissue is placed into VCTM (viral-chlamydial transport medium) and refrigerated for storage and transportation.

DNA ANALYSIS:

Fixation: None.
Special instructions: Submit the tissue fresh on ice to the pathology laboratory as soon as possible. If a delay is unavoidable, the following can be done:

A) For breast tumor tissue, submit 1.0 gram of frozen tissue (trimmed free of fat, necrotic, and normal tissue). Freeze immediately. Store at -20 degrees C and transport on dry ice.

B) For other tissue, submit 0.5 gram in sterile RPMI transport medium. Store and transport at 4 degrees C. Do not freeze. RPMI transport media can be obtained from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the biopsy to allow us time to deliver the RPMI transport media.

C) For bladder washings, submit 150-200 mL of wash fluid in a sterile container. Store and transport at 4 degrees C. Do not freeze.

D) For effusions, immediately following collection, add 10 units of injectable sodium heparin for each milliliter of fluid collected. Store and transport at room temperature.

E) For peripheral blood, submit 2 ACD solution A anticoagulated tubes (yellow stopper), and 1 EDTA anticoagulated tube (lavender stopper). Store and transport at room temperature.

F) For bone marrow, submit 1 ACD solution A anticoagulated tube (yellow stopper). Store and transport at room temperature.

ELECTRON MICROSCOPY (EM):

Fixation: 2% glutaraldehyde.
Special instructions: Take a sliver of tissue (approximately 10x2x2 mm), mince into 1mm sized pieces, and put into 2% glutaraldehyde fixative.

FROZEN SECTION:

Fixation: None.

Special instructions: Send fresh specimen to Pathology Associates immediately. The specimen should be sent in an airtight container to prevent drying. Any biopsy specimens that may be delayed should be sent on ice.

IMMUNOFLUORESCENCE (IF):

Fixation: Immunofluorescence (IF) fixative.

Special instructions: If immunofluorescence studies are needed on a biopsy, a portion of the biopsy tissue should be put into IF fixative (Michel’s Solution). IF fixative can be obtained from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the biopsy to allow us time to delivery the IF fixative.

KIDNEY (RENAL) BIOPSY:

Fixation: 2% glutaraldehyde, Michel's fixative and 10% formalin.

Special instructions: The kidney biopsy should be delivered fresh to Pathology Associates immediately after being obtained for processing by a pathologist. It is important that a summary of the patient's clinical findings accompany the biopsy as this is needed for meaningful interpretation.

If the biopsy must be processed on-site, the following procedure should be followed:

The goal is to remove a portion of cortex (glomeruli) for Electron Microscopy (EM) and Immunofluorescence (IF), with the remainder of the tissue fixed for Light Microscopy (LM). There is no surefire way to accomplish this 100% of the time, but the following procedure nearly always works if there is a reasonable sample:

a. Cut a 1-2 mm piece of tissue from each end of the biopsy and put into 2% glutaraldehyde for EM.
b. Cut a further 2 mm piece of tissue from each end and put in Michel's fixative for IF.
c. Put the central piece into 10% formalin for LM.

LIVER TISSUE FOR QUANTITATIVE IRON OR COPPER:

Fixation: None.

Special Instructions: Specimen required 0.5 mm x 5.0 mm from a needle biopsy of the liver. A larger section is needed from a wedge biopsy. Send in special metal free tissue vials available upon request from Pathology Associates. Please call Pathology Associates at least 48 hours in advance of the biopsy to allow time for delivery of the vials.

LYMPH NODE:

Fixation: 2% glutaraldehyde, B-5 fixative, and 10% formalin.

Special instructions: The lymph node should be delivered fresh to Pathology Associates immediately after being obtained for processing by a pathologist. Keep it moist with a small amount of sterile saline.

If the node must be processed on-site, the following procedure should be followed:

The goal is to put adequately sized pieces of the lymph node into several different fixatives to allow for a complete study of the node. This is easy for a large node, but may be somewhat difficult for a small node. Touch preparations are made and then pieces of the node are put into 2% glutaraldehyde for Electron Microscopy (EM), B-5 fixative for Light Microscopy (LM), and 10% formalin, also for LM.

a. Slice open the node along its short axis to expose the center, and make several touch preparations of the node. This is done by gently pressing the cut surface of the node once with the flat surface of a glass slide. Make 4-6 touch preparation slides and let the slides air dry (no fixative).
b. From the center portion of the node, take a sliver of tissue (approximately 10x2x2 mm), mince into 1 mm sized pieces and put into 2% glutaraldehyde fixative for EM.
c. Take a slice of the node (at least 15x15x5 mm or larger depending on the size of the node) and put into B-5 fixative for LM.
d. Put the remainder of the node into 10% formalin for LM. Place approximately 0.5 gms. of tissue in RPM1 transport media (obtain from Pathology Associates). Please allow 48 hours for delivery of media.

e. Sentinel lymph node(s) is located by the surgeon using technesium labeled sulfur colloid and vital blue dye. The node(s) should be labeled as “sentinel node”, placed in formalin (or fresh if frozen section is requested), and then sent to P.A.

NERVE BIOPSY:

Fixation: None, submit fresh wrapped in saline moistened gauze.

Special instructions: Please call Pathology Associates at least 48 hours in advance to advise us of an impending nerve biopsy. The minimum nerve biopsy size should be about 3 cm in length. The specimen should be wrapped in saline moistened gauze and put into an airtight container to keep the specimen moist (do not immerse in saline). The specimen should be submitted fresh to Pathology Associates as soon as possible. A clinical diagnosis and brief history, including EMG findings, CPK levels etc. must accompany the specimen.

STONE (CALCULUS) - BLADDER, KIDNEY, URETER, GALLSTONE, ETC.:

Fixation: None.

Special instructions: Fixation is not necessary if no tissue is attached to or submitted with the stone.

II. NON-GYNECOLOGICAL CYTOLOGY

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A. General Information and Precautions

Proper collection techniques and immediate specimen processing are essential for optimal results. Since laboratory personnel must be able to process specimens immediately, it is best not to collect specimens in the very late afternoon, evenings, weekends, or holidays. If you must collect specimens during these times, notify the lab so that proper handling can be assured.

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B. Fixation

1. All fluid and urine specimens should be submitted in a clean container without fixation. If a delay in specimen transport is anticipated, the specimen should be refrigerated. This will maintain cell viability for up to 48-72 hours.

2. All non-gyn smeared slides should be submitted as follows: Half sprayed with cytology fixative and half air dried. Caution: Cytology fixative may be fatal or cause blindness if taken internally. Wash hands thoroughly after handling. Do not get into the eyes, on skin, or on clothing. If contact should occur, immediately flush with copious amounts of water. If swallowed, obtain medical attention immediately.

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C. List of Specimens

BODY CAVITY FLUIDS ( PERITONEAL,PLEURAL,ETC):

Fixation: None.
Special instructions: None.

BREAST SMEAR (BREAST OR NIPPLE SECRETIONS):

Gently massage subareolar area and nipple with thumb and forefinger. When secretion occurs, allow only a pea-sized (3-5 mm) drop to accumulate. Touch a clean slide to the nipple and withdraw slide quickly. Using a second glass slide, smear the fluid between the two slides. Immediately spray one slide with cytology fixative and allow the other to air dry. Repeat procedure until all secretions from nipple are collected on two or more slides.

BRUSHINGS(BRONCHIAL,BILE DUCT,PANCREATIC DUCT,ETC.):

Brushings are obtained by inserting a small brush through the cope and brushing the desired area. Slides are prepared by gently swirling brush onto two to four slides. Fix half of the slides with cytology fixative and allow the other half to air dry. Place the brush into a separate container with liquid cytology fixative.

BRONCHIAL WASHINGS:

Fixation: None.
Special Instructions: None.

CEREBROSPINAL FLUID (CSF):

Fixation: None.
Special Instructions: As large a volume as possible should be collected, at least 3 to 5ml.

CYST ASPIRATION:

Fixation: None.
Special Instructions:
Following aspiration express cyst contents into clean container.

FINE NEEDLE ASPIRATION:

A 25 or 22-gauge needle is attached to 20-ml syringe. The lesion is aspirated by means of inserting the needle into the mass, and the plunger of the syringe is retracted to create a negative pressure. The needle is moved back and forth in the lesion. Negative pressure is released in the syringe and the needle is withdrawn. During aspiration the majority of the specimen should remain in the needle with only a small "flash" observed in the hub. Specimen or blood (with the exception of cyst aspiration) withdrawn all the way into the syringe is not desirable. The specimen is then expressed from the needle in one of two ways:

1.Direct smears and cytology fixative.

The direct preparation of smears is the preferred method of slide preparation if one is familiar and comfortable with making smears. A small drop of fluid or aspirated material is placed at one end of the slide. Another slide is placed on top of the drop and is smeared as in the preparation of a peripheral blood smear. Fix half of the slides with spray cytology fixative (or immerse in 95% ethanol) and allow the other half to air dry. The needle is then irrigated and washed with cytology fixative solution with the rinsings being placed back into the cytology fixative container.

2. Cytology Fixative Only

The other approach is to simply irrigate the syringe and needle with cytology fixative following the needle aspirate. Slides are not directly prepared. The aspirated material is placed directly into cytology fixative by washing and irrigating the syringe with cytology fixative solution.

GASTRIC WASHING:

Fixation: None.

Special Instructions: Washings should be obtained from a clean (food and debris free) stomach. (Gastric aspirations usually produce unsatisfactory cytologic specimens.) The stomach should be washed with 200 ml to 300 ml cold saline. A French Levine tube is passed into the stomach. The saline is then aspirated and reinjected several times to produce a vigorous flushing action against the gastric mucosa. As much of the original volume of saline solution as possible should be aspirated, kept in an ice bath and submitted to the laboratory immediately.

SKIN LESIONS:

Scrape the lesion with the end of a glass slide and smear specimen onto two to four additional glass slides. Spray half of the slides with cytology fixative and allow the other half to air dry.

SPUTUM:

Fixation: None.

Special Instructions: Optimal specimens (those containing alveolar macrophages and bronchial epithelium) are best obtained from deep coughing that may be assisted by saline aerosol and early morning collection.

URINE/BLADDER WASHINGS:

Fixation: None.

Special Instructions: Designate whether specimen is a voided urine or bladder washing.

III. AUTOPSY PROCEDURES

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This includes patients who die outside of the hospital as well as those patients who die in or on route to the hospital's emergency room, but who were not admitted to the hospital.

1. Call Pathology Associates to see if PA will accept the case.

2. Obtain valid written legal consent from the next of kin. This is done by filling out the Pathology Associates autopsy consent form, which must be signed and witnessed.

3. Please note that the next of kin must accept a charge for the autopsy to be done. The current charge can be obtained by calling Pathology Associates.

4. If Pathology Associates agrees to do the autopsy, the body must be transported to the Huntsville Hospital morgue at the next of kin's expense. The patient must be properly identified with armband or toe tag. The autopsy permit, any available chart, and papers for disposition of the body must be received by Pathology Associates before the case will begin. These may accompany the body to the Huntsville Hospital Morgue.

5. The autopsy will be done the same day if the body and all appropriate paperwork and identification are delivered to the Huntsville Hospital morgue by 4 p.m. After 4 p.m. the autopsy will be done the next day.

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