Tuesday, December 27, 2005


Ocular Cytology Specimens

Vitreous / Lensectomy Washings --- use “Vitreous Washing Gross and Microscopic Description Forms”
The tissue removed from the eye vitrectomy or lensectomy surgical operations comes as tiny fragments suspended in the physiological fluid used to wash material out of the eye during the operation. This fluid readily supports microbiological growth and in a very short period will be contaminated (usually by bacteria). Therefore, it must be refrigerated and taken to cytology as soon as possible. Please follow the following steps:

• Record the amount and characteristics of the washing. If there is perfluorocarbon or silicone oil present, record as "dense bubbly material that sinks (floats) to the bottom (top) of the cassette.".
• Complete the Cytology Request Form with patient's information, S number, and indicate whether you want PAP or cell block, and the quantity (usually 1). In general, order cell block whenever there are numerous tissue fragments. If infection is suspected, special stains will be needed. Please discuss with the pathology faculty prior to ordering special stains. ALWAYS READ THE HISTORY BEFORE PROCEEDING. If Hx of endophthalmitis, a “cell block” must be ordered.
• Place the Cytology Request Form and the specimen in the refrigerator for courier pick up. The last routine pick up is at 3 pm. If you receive specimens after this time, deliver it to cytology lab (see map at the end of this manual) or call the courier for pick up immediately.
• Place the Intraocular Washing Form and billing sheet in the slot for "Awaiting Slides".
• The courier will deliver the slides to the pathology lab when they are ready. The slides for cytospin/PAP typically take one day; those for cell blocks take two days.
• Retrieve the paperwork from the slot for "Awaiting Slides". Record microscopic findings and diagnosis on the form. Cross out "sections from cell block" on the form if only cytospin/PAP was ordered, or vice versa.
• Submit completed report and slides to the attending pathologist inbox.
• Proof read the typed report and record diagnosis in the log book as for other surgical cases.

This is how the cytospin/PAP and the cell blocks are prepared. The fluid is treated by the technologist as a cytospin preparation and by staining slides as "Pap smears," or by putting it through a millipore or nucleopore filter, or if there are many tissue fragments, by centrifugation and embedment in paraffin for sectioning as with a solid piece of tissue. The procedure for processing is as follows:
1. Divide the specimen equally between 2 large centrifuge tubes and spin AT 1000 - 1500 RPM for 15 minutes
2. From one tube decant the supernatant and overly the sediment with fixative (ether-alcohol). Seal the top with a stopper.
3. From the other tube, decant the supernatant and prepare thin films of sediment on glass slides previously labeled. Fix in a Coplin jar immediately with 95% ethanol. Prepare 2 additional films and air dry for Wright's stain.

AIDS and CMV Vitreous / Lensectomy Washings:
If a vitreous washing is from a patient with AIDS and/or with CMV infection, please notify the pathology faculty. Request 2 unstained ethanol-fixed cytospin slides from the cytology lab. See below as well.

Vitreous Washing for suspected Acute Retinal Necrosis or Viral Infection: Several options are available for careful consideration. These include morphologic evaluation by preparing smears, cell block, cytospin preparation or fixation for immunohistochemistry and electron microscopy. In addition culture and PCR tests should be considered by the surgeon in the OR so the specimen does not get contaminated in transport or processing. These consideration depend on the amount of material provided. If the specimen no discernible cell fragments, then it may be appropriate to centrifuge the specimen in 50 ml tubes, combine the pellets and then make smears both alcohol fixed and air dried and/or send for cell block. Some alcohol fixed smears can be retained for immunohistochemistry. In this way a small portion, a few microliters may be saved for PCR analysis in a microfuge tube which can be frozen or to which acidified ethanol added. If there is a lot of material the specimen may simply be divided for each of the tests indicated. A complete clinical history will help guide the evaluation so obtain it before you begin.

Vitreous Washing for suspected malignant lymphoma: Again several options are available. The specimen and therefore all specimens may be sent to hematopathology for their evaluation. The risk is that the evaluation will not be as complete as below. If it is decided that eye pathology will evaluate the specimen, then similar considerations are in play as mentioned above. First obtain a complete history with clinical examination. Intraocular lymphomas have a characteristic clinical appearance that justifies the expensive and time consuming work-up. In general undiluted vitreous and vitreous from a cassette will be delivered from the OR. Depending on the cellular content these can be handled separately or combined. If a lot of material is evident (cloudiness) in the undiluted vitreous then air dried smears (see below ) are made as well. Ideally the following preparation should be made: 1. flow cytometry on fluid from the cassette (be warned that these specimens are often deemed insufficient by the flow lab). 2. ethanol fixed and air dried smears (6 and 2 respectively) from either the undiluted vitreous if cloudy or ul drops of the combined centrifuged pellet. Ethanol fixed slides are retained in 95% ethanol for immunohistochemistry. 3. cell block fixed in formalin and embedded in paraffin for morphology and immunohistochemistry 4. drops from the pellet of combined centrifuged material, fixed in acidified ethanol or frozen for PCR gene rearrangments. 5. material form the pellet fixed for electron microscopy in 4% paraformaldehyde.

Anterior Chamber / Vitreous Taps
The pathology faculty should be notified when anticipating these specimens. They should be transported on ice to the pathology lab for processing as soon as they are obtained in the operating room. Both air-dried and ethanol-fixed slides are needed. Several fixed, unstained slides should be requested in addition to routine slides for possible future stains. In addition to the fixed unstained slides, the following should be requested:

Lymphoma: air-dried smear for May-Grunwald Giemsa (MGG) - see above for other considerations
Squamous dysplasia/carcinoma: Papanicolaou

Chlamydial conjunctivitis: air-dried Giemsa or ethanol-fixed material with direct fluorescent antibody

H&E, Gram's, GMS, and other special stains may be requested for the fixed slides either at the time of initial processing based on clinical problem or requested subsequently. Always request additional fixed unstained slides at initial processing of specimen.

Fine Needle Aspirations
Fine needle aspirations are always done with the attending pathologist. Residents are expected to participate in seeing the patient, and in some resident cases, will have an opportunity to perform the biopsy. In general smears are made and immediately fixed in 95% ethanol or in cases in which a lymphoma is suspected they may be air dried and mixed with a Giemsa preparation (MGG). Below is an example of a typical report for an orbital fine needle aspiration. It should include an accurate clinical summary and exactly what was done. For a fine needle aspiration performed in the operating room, the precise role the pathologist should be stated in the Gross (e.g the smears were prepared, stained, and interpreted intraoperatively by the attending pathologist).

Clinical History: John Doe who has a history of an expanding right orbital mass over the past several weeks. The ultrasonographic studies indicate that the lesion has low reflectivity and is 27 mm in AP length.

Gross: A single fine needle aspiration was performed with a 30 gauge needle. Slides were stained with MGG and H&E.

Microscopic: Smears stained with H&E show a small cell tumor composed of single cells not forming epithelial groups. The nuclei are round, densely hyperchromatic, and have scant cytoplasm. There are occasional macrophages present. Some cells have markedly enlarged nuclei with prominent nucleoli.

Diagnosis: Mass, right orbit, (fine needle aspiration biopsy)- Small cell tumor suggestive of malignant lymphoma (see note)

Note: The tumor cells are most consistent with the morphology of atypical lymphocytes. A very thorough clinical evaluation is warranted to search for other sites of disease. Inflammatory conditions and other small cell tumors should be considered in the evaluation.

Intraoperative Intraocular Fine Needle Aspirations
Currently the ocular tumor service at JSEI frequently obtains fine needle aspiration material from cases suspected of being uveal malignant melanoma. The resident responsibility for these cases will be to confirm the time the case is actually starting and to label the glass slides, take 95% ethanol for fixation to the operating room and obtain a complete history the day prior to the case. The resident will attend these aspirations and observe the procedure, accession the case, view the slides and construct a report. These aspirates are reported verbally intraoperatively and the resident will report this to the attending in writing in a timely fashion.

'Conjunctival Smears:
Conjunctival smears for diagnostic study must be accompanied by a requisition form. If they are mistakenly sent to General Cytology, they will be interpreted by the general cytologist.

Preparation of the smears in the clinic will depend upon the information needed. Three diagnostic questions are usually asked:

1. Chlamydia: Three smears should be prepared. Two are rapidly air dried (one for Giemsa, one for reaction with direct fluorescent antisera); a third must be placed immediately in 95% ethanol (less than 1 second after smear is made) and stained with Papanicolaou's technique. Material can also be cultured; swab is placed directly in transport media and accessioned in the Clinical Virology Laboratory (Rm AL-233A, CHS) (x56215). See them for swabs and media

2. Inflammatory cell type: One rapidly air dried smear.

3. Dysplasia/Carcinoma: Place smear immediately (less than one second after preparation) in 95% ethanol for Papanicolaou's stain (PAP).

In general remember that Papanicolaou and H&E are used with 95% ethanol fixed smears in cytology. MGG is used with air-dried smears (no fixative at the time of the aspirate). Ethanol fixed slides stained with H&E or PAP smears are ideal for melanoma because pigment and nucleoli are obvious. PAP stain is excellent for squamous lesions. Air dried smears are excellent for lymphoma.



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