Histology

Routine tissue examination consists of two parts. First, the pathologist examines the tissue macroscopically performing a gross examination. After the tissue has been processed and prepared, a microscopic examination is then performed.

HANDLING AND FIXATION

Tissues submitted for routine surgical pathology should be placed in 10% buffered formalin as soon as possible after the surgical removal. 10% formalin is the most commonly used fixative. Pre-filled containers with formalin and/or containers of formalin are provided by WPM at no extra charge.

Fixation of tissue is the foundation for subsequent stages in the preparation of tissues for microscopic examination. Fixation alters tissues by stabilizing the protein so that it is resistant to further changes. Fixation also kills the tissue so that bacterial attack and autolysis (enzyme action) are prevented. The following items are essential to good quality tissue fixation:

1. Volume ratio: The formalin volume should be sufficient to adequately and entirely cover the tissue specimen.
2. Time: Ideally, tissue should be placed in formalin immediately after surgical removal. Small tissues, such as those from bladder biopsies and endometrium, can be ruined in a short time by drying out.

A MSDS for formalin will be provided or made available upon request.

STORAGE

Room temperature storage of fixed tissues is suitable. Refrigeration is not necessary. Do NOT freeze. Tissue fixed in formalin is stable indefinitely. Tissue held over the weekends or holidays do not need further handling or storage requirements if fixed in formalin.

LABELING

All tissue specimens received must be properly identified with the following information:

1. Patient’s first and last name
2. Source of specimen
3. Physician
4. Date of specimen collection

This information should be written on the specimen label, not on the lid.

In addition, there are adhesive labels on each requisition that can be placed on the tissue specimen container for added identification. These are especially helpful for specimens with multiple parts. There is a space on each label to indicate the specimen source or site. The number on the label (# 1, # 2, etc.) should match the corresponding specimen number on the requisition.

SPECIMEN COLLECTION, HANDLING AND TRANSPORTATION

All specimens should be placed into a proper, leak-proof primary container with a secure leak-proof lid that is tightly screwed on or fastened. Select the appropriate size of specimen container for the tissue size to be submitted. WPM Pathology Laboratory provides a variety of containers for specimen collection. Care should be taken by the person collecting the specimen not to contaminate the outside of the primary container.

Properly identify the tissue specimen using the instructions listed above.

When multiple specimens are to be examined and diagnosed individually, each specimen must be submitted in a separate, appropriately labeled container.

Before being transported to WPM, the primary container is to be placed into a secondary container (specimen transport bag), which will contain the specimen if the primary container breaks or leaks in transit to the laboratory, unless the specimen is too large. WPM provides specimen transport bags (biohazard) for use as a secondary container.

Transport to WPM, along with a properly filled out WPM SURGICAL PATHOLOGY & NON-CYTOLOGY request form.

Universal health precautions should always be followed when handling specimens and fixatives.

TURN-AROUND-TIME

Specimens are processed upon receipt, and test results are reported to clients after they are released. Most routine surgical tissue specimens will have a report issued within one to two working days after tissue receipt. Specimens requiring special stains, immunohistochemistry, or outside pathology consultation will require more time.

Selected test results are telephoned as soon as they are available. Reports may be faxed as soon as they are available, and followed up with a hard copy via courier or mail. Reports also are available by secured Internet site for those wishing to use this option. Clinicians also may call for results.

SPECIMEN ACCEPTABILITY

In order to maintain specimen integrity and accuracy of identification, it is imperative that specimens submitted to WPM be properly and completely labeled. Specimens received mislabeled or unlabeled, may be cause for rejection. Since most tissue specimens cannot be collected again, WPM personnel will notify the sender to try and amend the labeling problem. However, in some instances it may be impossible to correctly identify the specimen with the correct patient and the specimen will be rejected. Care must be taken to insure that specimen containers are properly labeled, and that the test request forms match the specimen.

Skin or mucosal tissue are examined for presence of bound IgG, IgM, or IgA, third component of complement (C3), and fibrinogen. This is useful for confirming a diagnosis of pemphigus, bullous pemphigoid, dermatitis herpetiformis, or lupus erythematosus.

NOTIFY WPM BEFORE SPECIMEN COLLECTION TO OBTAIN ZEUS TRANSPORT SOLUTION.

1. A punch biopsy (3-4mm) of involved or uninvolved skin is obtained.
2. Immediately place the specimen in a vial of Zeus transport solution. Seal tightly.
   NOTE: Assay CANNOT be performed on specimens fixed in formalin.
3. Label vial with patient’s name, biopsy site, and date.
4. Place specimen vial in a specimen transport bag.
5. Transport to WPM, along with a properly filled out WPM SURGICAL PATHOLOGY & NON-GYN CYTOLOGY request form. Under Special Requests: indicate [X] Other, and write in “cutaneous immunofluorescence”.

NOTE: Interpretation of results is facilitated by submitting the following clinical data on the patient: age, sex, clinical diagnosis, biopsy site (anatomic), exposure of site to sun (exposed, unexposed), and relationship to lesional skin (perilesional, involved, uninvolved).

FOR LYMPHOMA PHENOTYPE AND LYMPHOMA VS. REACTIVE HYPERPLASIA

FLOW CYTOMETRY

Flow cytometry studies are useful in distinguishing lymphoma from reactive lymphoid hyperplasia as well as immunophenotyping for classification within the new lymphoma classification system (revised Europe-American lymphoma classification).

1. A small slice of lymph node (minimum amount 0.5 X 0.5 X 0.2 cm) should be placed in a RPMI media tube for flow cytometry analysis. (RPMI 1640 culture media is available from WPM on request.)
   • The specimen should be stored refrigerated until submission to WPM.
   • It is best to schedule the biopsy so that we (WPM) receive it Monday thru Thursday to avoid deterioration of cells over a weekend.
   • Label specimen container for proper identification and place in a biohazard bag for transport to WPM, along with a properly filled out WPM SURGICAL PATHOLOGY and NON-GYN CYTOLOGY request form. Under Special Requests: mark [X] Flow Cytometry.
2. Place remainder of the specimen in formalin for routine pathology examination. However, if the lymph node specimen is small, submit the entire specimen.

TOUCH IMPRINTS

If adequate fresh tissue is available, touch imprints prepared from the fresh tissue is helpful.

1. Touch imprints or touch preps are prepared by placing the fresh (unfixed) tissue specimen on a sterile gauze to absorb excess blood and minimize damage to cells. A slide is then gently touched to the specimen, making several imprints of the tissue along the surface.
2. Four to six (4-6) slides may be made with two (2) of the slides fixed with cytologic spray fixative. The remainder of the slides may be air dried.
3. Label the slides as fixed or air dried.

NOTE: If the amount of tissue is limited, the priority is:

1. formalin-fixed tissue
2. tissue in RPMI media for flow cytometry

For further questions or information, call one of the pathologists at 785-823-7201.

Muscle biopsies are for the evaluation of muscle disease in terms of neurogenic atrophy, muscular dystrophies, myositis, myopathies, and enzyme deficiencies.

CONTACT WPM LABORATORIES BEFORE PERFORMING PROCEDURE

Due to transport of the specimen on dry ice, the biopsy must be performed on Monday, Tuesday, or Wednesday only.

1. Obtain the biopsy from a muscle that is definitely affected, but not so severely affected that much of it is replaced by fatty or fibrous connective tissue. This usually means a –1 to –2 rating on the Mayo Clinic manual muscle testing scale, or a +4 rating on the MRC scale. In addition, the involved muscle should not be one that has been traumatized by injection or by electromyographic (EMG) studies. Typically, the triceps, biceps, or vastus lateralis are chosen.
2. Excise a specimen approximately 1.0 cm x 0.5 cm with minimal trauma, dissected along the long axis of the muscle. Due to the nature of testing that is done, do not use electrocautery or a muscle clamp in removing the specimen.
3. Place the specimen, after removal, on saline dampened (not soaked) 4 x 4 gauze. Place in a plastic container on wet ice for immediate transport to the Histology Dept. at Salina Regional Health Center; Santa Fe Campus; Salina, Kansas. There the specimen will be flash frozen in 2-methyl butane. The specimen will then be sent on dry ice to Mayo Medical Laboratory.
4. Submit specimen with a properly filled out WPM SURGICAL PATHOLOGY & NON-GYN CYTOLOGY request form. Under Special Requests: indicate X Other, and write in “Muscle Biopsy”.

Label specimen for proper identification.

NOTE: To assist in interpretation of the specimen, please submit pertinent information, such as the patient’s name, age, name of muscle, date of biopsy, and a brief clinical history on the Mayo Muscle Histochemistry Patient Information Sheet. Pertinent studies such as serum creatine kinase (CK) and electromyographic studies, and the suspected clinical diagnosis should be included.

CONTACT WPM LABORATORIES BEFORE PERFORMING PROCEDURE

Due to transport of the specimen on dry ice, the biopsy must be performed on Monday, Tuesday, or Wednesday only.

For further questions or information, call a WPM Pathologist at 785-823-7201.

Outside consultations are initiated by pathologists to assist in a diagnosis or to confirm a diagnosis on difficult or unusual surgical pathology cases. WPM uses pathology experts specializing in a specific area, such as urology or dermatology.

It is not uncommon for a patient or physician to request a second opinion on the surgical pathology diagnosis or to confirm the diagnosis, especially cancers. To request a second opinion, call the Histology Dept. at WPM. They will need to know the patient, who and where to send the referral specimens, and what is needed for referral (blocks and/or slides).

It is the responsibility of the physician’s office or patient to forward medical records, test reports, and x-rays, if necessary, to the second opinion physician or group.

Immunohistochemistry (IHC) assays make it possible to more accurately identify tumors whose cytologic and architectural characteristics are not, in themselves, sufficient to render a reliable diagnosis.

Immunohistochemistry testing aids the pathologist in establishing cellular origins to poorly differentiated tumors, classify lymphomas and leukemias, detect micrometastases in tissue, and identify and qualitatively estimate hormone receptor and prognostic marker distribution for tumor evaluation. This in turn makes it possible to more effectively predict the likely response to tumors to various possible treatments, and to thus optimally treat an affected patient.

An example would be performing immunoperoxidase stains for Estrogen Receptor (ER), Progesterone Receptor (PR), Ki-67, and HER2-neu (c-erb B2) on a breast biopsy with carcinoma.

WPM Pathology Laboratory performs many immunohistochemistry (IHC) assays in-house, thereby reducing turn-around-time. WPM Pathologists typically order which IHC assays are necessary to make a diagnosis, so providers don’t need to worry about which IHCs to order. For more information on IHC assays, contact a WPM Pathologist.

The College of American Pathologists (CAP) publishes cancer protocols, or checklists that pathologists can use to convey necessary prognostic factor and staging information to clinicians. These protocols reflect changes to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, Sixth Edition, and the most recent World Health Organization (WHO) tumor classification. The American College of Surgeons Commission on Cancer (ASC CoC) has recognized the value of the CAP Cancer Protocols in caring for cancer patients. In January 1, 2004, it mandated that pathologists at Commision on Cancer approved cancer programs include only scientifically validated or regularly used data elements of the checklists in their surgical pathology reports on cancer specimens.

WPM implements the CAP protocols in our surgical pathology reports for many cancer specimens.

For proper management of patients with recurrent stone formation, it is essential to have an accurate analysis of all crystal material present to guide therapy.

1. Submit entire specimen which has been filtered from urine. Place specimen in a clean, dry container. DO NOT tape the specimen to anything. Tape adhesive interferes with the analytical procedure.
2. Place specimen container in biohazard bag and transport to WPM Pathology Laboratory, along with a properly filled out WPM SURGICAL PATHOLOGY AND NON-GYN CYTOLOGY request form requesting: “Stone Analysis”. Label specimen for proper identification.

Helicobacter pylori has been shown to cause active chronic gastritis and has been implicated as a primary etiologic factor in duodenal ulcer disease, gastric ulcer and non-ulcer dyspepsia. By causing chronic inflammation, H. pylori may weaken the mucosal defenses and allow acid and pepsin to disrupt the epithelium.

H. pylori produces large amounts of urease enzymes. The CLOtest (Ballard Medical Products) detects the urease enzyme of H. pylori in gastric mucosal biopsies.

PRODUCT DESCRIPTION AND STORAGE

CLOtest is a sealed plastic slide holding an agar gel, which contains urea and other components. If the urease enzyme of H. pylori is present in an inserted tissue sample, the resulting degradation of urea cause the pH to rise causing a reaction of the gel, which is interpreted by the Pathologist.

CLOtest should be stored in the refrigerator at 2 – 8ºC / 36 – 48ºF. CLOtest has a shelf-life of 18 months when properly refrigerated.

PREPARATION OF THE PATIENT

Patients should not have taken antibiotics or bismuth salts for at least 3 weeks prior to endoscopy. Suppression of H. pylori by these agents makes the organism difficult to detect by any means, and re-growth of H. pylori patchy, leading to false negative results in the first few weeks after treatment.

PREPARATION OF THE CLOtest

Before use, the CLOtest should be examined to make sure that the well is full and is a yellow color. If a CLOtest is any color but yellow, it should not be used. If this should happen, proceed with another CLOtest slide or call WPM Pathology Laboratory.

Before the endoscopy, warm the slide to room temperature (20 – 30ºC). Warming will help the test work faster.

TAKING AND INSERTING THE BIOPSY

1. A biopsy sample for CLOtest may be taken as soon as the physician has examined the stomach. The usual area to biopsy is the antrum, along the greater curve.
2. Biopsy area of normal looking tissue rather than an area affected by erosions or ulceration. This is because H. pylori may be present in smaller numbers if the epithelium is eroded or the mucus layer is denuded. The standard biopsy forceps will provide a specimen of sufficient size (1 – 3mm diameter).
3. If the biopsy specimen appears to be very small, it may be worthwhile taking a second biopsy and inserting both specimens into the CLOtest. Be careful not to contaminate the second specimen with blood from the first biopsy site.

CLOtest PROCEDURE

1. Turn the CLOtest slide so that the label is facing up and you do not see the well with the yellow gel.
2. Peel the label away from the plastic slide so that you can see the yellow gel. Do not remove the label.
3. With a sterile needle, take away the biopsy sample from the biopsy forceps and push it into the yellow CLOtest gel. Make certain that the biopsy sample is buried in the gel so that it will have the most contact with the gel.
4. Re-seal the CLOtest label by pressing the label back on the plastic slide, so that the gel is covered.
5. Write the name of the patient, the date, and the time that the biopsy sample was placed in the CLOtest gel, on the label.

TRANSPORTATION

Submit the CLOtest slide, along with a properly filled out WPM SURGICAL PATHOLOGY AND NON-GYN CYTOLOGY request form. Under Special Requests: indicate [X] Other, and write in “CLOtest”. Place in specimen transport bag for transportation to WPM Pathology Laboratory.

RESULTS

A “SURGICAL PATHOLOGY REPORT” will be issued by a Pathologist with the results from the CLOtest. A positive report indicates, with a 97% specificity, that H. pylori was present in the biopsy specimen.

False negatives may occur when very low numbers of H. pylori are present, or the bacterium has a patchy distribution (not present in the antrum), or with widespread intestinal metaplasia.

PRODUCTS OF CONCEPTION:
1. Place fetal tissue in RPMI media or sterile saline. DO NOT PLACE IN FORMALIN.
Label the specimen.
In case of fetal demise, fetal tissue such as lung, kidney, liver, or periumbilical skin
are suitable for submission.
In cases of fetal demise, the placenta maintains cell, viability longer due to
maternal blood circulation; therefore, placental specimens are more likely to
yield cell growth and therefore a cytogenetic result.

2. Maintain at room temperature. If transit is to be delayed for more than one day,
refrigerate the sample. DO NOT FREEZE.
PLACENTAL SAMPLING:
1. Collect the placental tissue sample from near the umbilical cord insertion site
beneath the amnion. Remove 5 mm3 of placenta tissue with a sterile surgical knife
and dissecting forceps.
2. Place sample in RPMI media or sterile saline. DO NOT PLACE IN FORMALIN.
Label the specimen.
3. Maintain at room temperature. If transit is to be delayed for more than one day,
refrigerate the sample. DO NOT FREEZE.