Immunohistochemistry Examples of Use

Immunohistochemistry- examples of usage and explanation

• S-100 stains many things including cells of melanocytic origin and certain types of histiocytes. Example: Suspected choroidal melanoma without pigment. In Rosai-Dorfman disease emperipolesis will be easy to identify.


• HMB-45 stains melanocytes. Example as above or PAM with atypia to identify transepithelial migration.


• BCL-1 This is a cyclin D derived protein, one of the regulators of the G1 restriction point, which act in phosphorylation and inactivation of the Rb protein gene. BCL-1 is expressed in proliferating cells. BCL-1 may be over-expressed in mantle zone derived lymphomas but usually not in other low grade lymphomas.


• BCL-2 This is an anti-apoptotic protein that is integral to cell death pathways. It was first noted to be expressed in lymphomas, but is expressed by many cells.


• CD-1 stains certain types of histiocytes, activated T cells. Example used in combination with S-100 in Langerhans histiocytosis.


• CD-1a fairly specific for Langerhans cell histocytosis (LCH), dendritic cells in T cell lymphomas. Useful in Rosai Dorfman disease in which histiocytes are negative. Also negative in Erdheim-Chester Disease (ECD) , and juvenile xanthogranuloma (JXG).


• CD45 identifies cells hematopoietic origin


• CD68 identifies cells of histiocytic origin. Reactive in JXG, ECD.


• CD-5 e.g. B cell associated antigen, negative in MALT tumor cells and follicular lymphomas, positive in B cell CLL, mantle cell lymphoma


• CD-10 e.g. negative in MALT tumor cells, positive follicular lymphomas and a host of other things.


• CD-34 e.g. stains strongly the tumor cells of solitary fibrous tumor and in fact is part of the definition of that tumor.


• CD-43 e.g. Sialophorin is the antigen. B-cell antigens in chronic lymphocytic leukemia, stains normal T cells and activated B cells, granulocytes, monocytes. Co-expressed with CD 20 in SLL.


• CD-3 e.g. T cell marker, useful in most lymphoma evaluations


• CD-20 e.g. B cell marker useful in most lymphoma evaluations


• Kappa e.g. light chain restriction in B cell lymphoma evaluations


• Lamda e.g. light chain restriction in B cell lymphoma evaluations


• SMA (smooth muscle antigen) e.g. smooth muscle in leiomyomas of the ciliary body


• EMA (epithelial membrane antigen) e.g. stains sebaceous carcinoma but less often in basal cell carcinoma*; stains adenoid cystic carcinoma


• CKit (CD117) reacts with adenoid cystic carcinoma more strongly than basal cell carcinoma (but both express it) (see link).


• CAM 5.2- e.g. special keratin that is positive in sebaceous carcinoma (see link) but less often in squamous carcinoma*

Often panels of immunohistochemistry are most useful. For example:

JXG- positive for CD68, Fascin, XIIIa, alpha-1-antitrypsin but negative for CD1a.
ECD- positive for CD68, XIIIa, but negative for S100 and CD1a.
LCH- negative forCD68, positive for fascin, S100 and CD1a

Lymphangioma versus Hemangioma- podoplanin from lymphangioma. CD 31 stains both.

Conjunctival Nevus versus Melanoma- Ki-67 (proliferation marker), HMB-45 (perhaps more strongly positive in melanoma), MART-1 (defines all melanocytic cells), CD45- ( to distinguish Ki-67 positive inflammatory cells), pankeratin (to distinguish Ki-67 positive epithelial cells) . If possible do these on immediately adjacent sections. Fluorescence in situ hybridization (FISH) may be ordered for confirmation of certain tumors. For conjunctival melanoma, the cytogenetics lab offers 6p23(MYB), 11q13 (CCND1) and centromere 6 probes (CEP6) .

Conjunctiva and Orbital Lymphoma- CD3,CD5,CD10,CD20,CD43,kappa, lambda, BCl2, BCL6, BCl1

Squamous Carcinoma- positive for Ck5-6, P63, pancytokeratin; negative for BerEp4, EMA

*Sebaceous carcinoma is best documented by the presence of abundant lipid in tumor cells. However, the immunohistochemistry is helpful in cases that were not suspected and there is no wet tissue left of the specimen (all in the paraffin block). For the immunohistochemistry see: Sinard JH 1999 Archives of Ophthalmology

Examples of Embedding Instructions and Histochemical Stains

General comments: Difficult cases for embedding will be done by specific technicians and should be identified by name on the orders: e.g. Sandra to embed only!

Examples of embedding on cut edge: skin conjunctiva, cornea, iris, sometimes muscle.
Examples of embedding on flat face: muscle, soft tissue (fat, fibrous tissue, orbital.
Examples of embedding on cut end: temporal arteries, optic nerve.
Example embedding and sectioning instructions:

• eyelid for seborrheic keratosis: bisect, embed on cut edge 1 ribbon or section stained
• eye- Sandra to embed only! On cut face. Sections to include optic nerve. Stain with H&E and PAS.
• cornea for bullous keratopathy: embed on edge, 1 section H&E and 1 section PAS
• cornea for Salzmann's nodule: cornea, embed on cut edge 1 section H&E, 1 PAS, and one for oxidized aldehyde fuchsin stain
• conjunctiva for pterygium: Sandra to embed only flatten at embedding station, trisect parallel to the long axis, embed slices on edge, cut 1 section stained with H&E and 1 section stained with PAS
• temporal artery- initial sections: slide at 1 mm intervals and embed on cut end so that a cross section of the vessel is obtained, initial face and stain 1 section for H&E and one unstained.
• Temporal artery if initial section is negative: Deeper q 20th through block. (depends on thickness of the sections q 30th is adequate if sections are 4-5 microns).
  Typical use of commonly employed special stains:
• PAS- identify glycogen or basement membranes. Use for all corneas, conjunctiva, eyes.
• Verhoeff Van Gieson (EVG)- stains elastic tissue. Use for temporal arteries and vascular tumors.
• Infectious Package PAS, GMS, AFB and Gram stain. Use for suspected infections.

• Shown are the Gram stain in the figure to the left with black arrow one pointing to gram positive cocci. Notice the size of the organism compared to the adjacent red nuclei and the clustered arrangement of bacteria. This is a control slide and will often show both gram positive and gram negative organisms.

The Gomori methenamine silver stain (GMS) highlights fungal hyphae. Branching of the hyphal structures are helpful to narrow the diagnostic possibilities. Here 45 degree dichotomous branching is seen at red number 1. It is also important to look for septae which are visible as thin lines splitting the hyphal structures (red arrow 2).

The Ziehl- Neelsen stain is a carbol-fuchsin stain that is used for acid fast bacilli and show very small organisms that are red and elongated. Some are extracellular (lighted arrow 1) and some are intracellular (arrow 2). In this control slide the organisms are particularly abundant.



• Dystrophy Package: Alcian Blue pH 0.4, Congo Red, Trichrome. Use for suspected BIGH3 dystrophies and macular dystrophy of the cornea.
• Prussian Blue- stains iron. Use to stain basal epithelium in keratoconus or hemosiderin.
• Giemsa- excellent for air dried cellular elements. Use for air dried cornea swabs for acanthameba, fine needle aspirations of suspected lymphomas.
• Von Kossa- stains calcium black. Use for band keratopathy.
• Masson’s trichrome stains muscle red, tendon blue. Use for rhabdomyosarcoma, slipped tendons, vascular tumors that have smooth muscle (e.g. cavernous hemangioma).
• Mucicarmine stains neutral mucins pink. Use for suspected adenocarcinoma.
• Oil red O- stains lipids red in frozen section. Not useful for fixed tissue. In our lab this stain is largely supplanted by osmication and thick sections for toluidine blue which give far better quality of staining and sectioning. (Rarely do the plastic surgeons send the tissue fresh anyway). Lipids stain bright green with this stain (see arrows below).
  
  
  





• For other special stains see one of the many books in the lab such as the Laboratory Methods in Histotechnology, AFIP Manual, by Prophet, Mills Arrington and Sobin.

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Reference Phone Numbers for UCLA Labs

FREQUENTLY NEEDED PHONE NUMBERS:
Autopsy PADS-56470
Autopsy Suite-56844
Cytology Lab -51076 or 47135
Flow cytometry-58806
Histology-69696; 56926
Immunohistochemistry -50643
Information -56301; 66766
JSEI OR-55031
Lymphoma evaluations (Dr. Said)-56877; 51149
Microbiology (Dr. Wagar)-42708
Molecular Pathology (Dr. Grody)- 55648
Resident/Fellow Eye Pathology-56780
Surgical Path Reporting Office-58947
Transcription-47155
Electron Microscopy A3-231M-55737--
Pam Murillo (Path Computer Specialist) pmurillo@mednet.ucla.edu-56092; 91661---
Courier Service (Apollo Corp)-1-800-873-3444---
Cornea Division-53090--
Retina Division-54749--

To search the UCLA Directory: Click here

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ERRORS TO AVOID IN EYE PATHOLOGY

HERE IS A LIST OF ERRORS IN EYE PATHOLOGY THAT ARE ALL TOO COMMON AND A BRIEF EXPLANATION:
1. Specimens suspected of cancer failed to be properly oriented and inked. This is most common in outside cases because the small size of the specimen does not seem to bear credible margins to a general pathologist. A resident on the service should consider it necessary to ink all cases suspected of cancer unless otherwise determined by the attending pathologist.
2. Inadequate cleaning of the forceps between cases. It is possible to carry over tumor cells from one case to the next. Although this is generally obvious when the case is reviewed, it is much safer to prevent this by washing the forceps between cases. This is generally necessary to also remove the marking inks. At the end of each day the forceps and bench are cleaned with bleach.
3. Unfixed specimens are ruined because of a delay in fixation. This occurs most frequently when a specimen arrives without fixation as is frequently the case with vitreous aspirations and ocutome washings. The resident should recognize that the specimen does not have a formalin label on it and proceed with processing or ask for assistance from the attending pathologist.
4. Errors in reporting are too common and should be avoided. Here are the most common:
a. The patient's name is spelled incorrectly and another patient is assigned to the case. This occurs because the computer gives you accession choices from thousands of cases. This can be avoided by checking the date of birth and other identifying information carefully.
b. Erroneous or inadequate history may lead to a misdiagnosis. The resident must carefully verify the historical information and make sure it is complete before attempting to make a diagnosis.
c. Failure to record for the permanent record the orientation, or marking inks used. Since the computer generated report is will be the final permanent record, it is critical for the resident to accurately describe the orientation, plane of section and marking inks so that the slides can be interpreted years later by another.
d. In cases with multiple specimens, failure to describe the anatomic location of the specimens and refer to the specimen in this way throughout the report. This makes the report difficult for the surgeon to follow. Using the cassette letter to refer to the case is not acceptable since these letters do not necessarily corresponding to the specimen number ( if it does it is simply coincidence).
e. Typographical errors both of omission and commission. Please check your work so this does not happen.
f. The wrong date is recorded for specimen receipt. This occurs because the computer is programmed to default to the day before your date of accession for all specimens you are accessioning and examining. Obviously this is erroneous since you are generally examining the day of surgery for Jules Stein cases and often several days later for outside cases. Please be vigilant about recording the correct date for the surgical procedure that generated the specimen (also called the date of service), or alternatively the day the specimen arrived in our laboratory as the receipt date.

5. Errors in Gross examination. These generally occur because the resident attempts to gross the specimen without seeking the advice of the attending pathologist. Please remember that there is no rush to gross in specimens as they are processed once per day. Adequate supervision is available to answer any questions, the resident simply needs to inform the attending pathologist as to the existence of the specimen. Under no circumstances should the resident attempt to gross in a specimen in which they have not been properly instructed and trained.



6. Leaving portions of the report blank because you do not know the answer. Every single case is examined and signed out by the attending pathologist. In our system it is important that you attempt to make an effort to come to a diagnosis. This is key to the educational experience provided. The attending pathologist will correct erroneous diagnoses and go over errors with you. Nothing ventured, nothing gained!

7. Outside slides are not labeled in pencil with the UCLA surgical number. Labeling the slide with our number is critical as the slides and paperwork may be separated and in order to return the slides they must be referenced. As a double check we put the outside surgical number in our log book and as further insurance each report contains the exact labeling of the slide (see outside slide reports).

8. Failure to record the final surgical diagnosis in the log book. The resident is responsible for keeping this record as it serves an educational purpose. In cases in which the attending does not choose to review with the resident, the act of recording the diagnosis will provide positive reinforcement for the correct diagnosis or call attention to questions on a case that the residents may wish to ask on the attending pathologist. It also allows the resident to see the final wording of the case. In addition it provides an instant record from which to refer to inform the clinicians regarding unsuspected diagnoses.

9. Failure to order special tests on computer from the histology lab. It is critical that the resident both record their order on a paper copy as well as order the test on the computer. The computer record is received by the histology lab and they will do the special stains or deeper sections in the wee hours of the morning. The computer provides a record if for some reason there is a problem with the paperwork...(e.g. lost in transition by couriers).

10. Failure to record a case in the log book. Every single case must be logged in by hand to the specimen accession log book. This is necessary in case of computer crashes, is an integral part of resident education and is critical for medicolegal considerations.

11. Failure to write on cassette in Pencil! Ink is soluble in alcohol and organic solvents and washes right away. All of the labels on the cassettes may vanish and a disaster may ensue. It is critical to label cassettes in PENCIL and write clearly. Put the name of the patient on the side of the cassette.

11. Failure to turn off the microscope at the end of the day. The pointer light and microscope light should be turned off at the end each day.

12. Failure to check packages from outside consultations for blocks, paper work or additional slides. Remember the packages are kept until the case has been completely signed out by the attending pathologist.

13. Failure to use biopsy bags for small specimens may result in lost specimens.

14. Failure to fold the edges of the biopsy bag may result in lost specimens.

15. Failure to wet the embedding bag to retain its fold may result in lost specimens. The combination of 14 and 15 increases the likelihood of losing the specimens.

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OUTSIDE SLIDE CONSULTATIONS

Outside Slide Review
In general, outside slides are sent from other institutions for review or a second opinion. They require a precise description of the number of slides received and the label on each slide. DESCRIBE EVERTYHING ON THE LABELS OF THE SLIDES, INCLUDING HOSPITAL NAMES, ADDRESS, PHONE NUMBER, SPECIAL STAINS, ETC.. Everything! This is because only a portion of the total slides may have been sent. Mention whether there is or is not an accompanying report and whether it corresponds to the slides. For example:

GROSS DESCRIPTION: Received for review are 4 slides and the corresponding pathology report. All slides are labeled " Department of Pathology, Named Hospital, City, California, 90002 L01-341". Individual slides are labeled "A", "B", "C", and "D”, respectively.

MICROSCOPIC: Slides from the specimen designated “periocular tissue” in the accompanying report show dense connective tissue with moderate chronic inflammatory infiltrate consisting of lymphocytes, plasma cells, and occasional eosinophils with no involvement of vessels.

Slides from the specimen designated “lacrimal gland" in the accompanying report show dense connective tissue and lacrimal gland with moderate chronic inflammatory infiltrate consisting of lymphocytes, plasma cells, and occasional eosinophils without involvement of vessels.

FINAL DIAGNOSIS: Soft tissue, "periocular tissue" (OSR-biopsy)- chronic inflammation
FINAL DIAGNOSIS: Lacrimal gland, "lacrimal gland " (OSR-biopsy)- chronic inflammation

In some cases the slide labeling may be complicated by the large number of slides and the type of biopsy and special stains. In this case it is possible to consolidate the slides as shown:

GROSS DESCRIPTION: Received for review are 24 slides and the corresponding pathology report. All 24 slides are labeled "Jane Doe, Department of Pathology, Named Hospital, City, Arizona, S11111". 14 of the 24 slides are additionally labeled "H&E" and individually labeled "A", "B", "C", and "D”, respectively. 10 of the 24 slides are additionally labeled "X-lab" and are individually labeled "MART-1", "S-100", "HMB-45", "Ki67", "panker”, "PC "MART-1", "PC S-100", "PC HMB-45", "Contro Ki67", and "Control AE-1-3”, respectively.

MICROSCOPIC DESCRIPTION: The slides show a tumor composed of nests and sheets of epithelioid cells that infiltrate into fat, muscle and fibrous tissue. There is extensive necrosis and individual cells show marked pleomorphism, a plasmacytoid shape, intranuclear cytoplasmic invaginations, nuclear atypia, prominent nucleoli and numerous abnormal mitotic figures. The slides that are indicated as taken from the margins of resection in the accompanying report are negative for tumor. The immunohistochemical stains show the tumor cells are reactive for anti-MART-1 and -S100. In the MART-1 reactive cells 32% react to anti- HMB-45, and 8% react to anti-Ki67. However the tumor cells are non-reactive to pan-keratin.


R and S cases are similar but EY cases (rarely used) have a letter format. But the general principle to indicate everything on the submitted slides holds true on these cases as well.

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MULTIPLE SPECIMENS-SAME CASE

Multiple specimens for one patient
Frequently residents are confused how to handle multiple specimens from the same patient. They all have the same accession number because they are from the same case. However, the cassettes are labeled in succession as needed but with the suffix of letters, which is strictly an in-lab system. Each specimen must be designated the way the surgeon intended so that the surgeon can determine the diagnosis associated with each specimen from its anatomic designated location.
Example:
GROSS DESCRIPTION: Specimen #1, in formalin, labeled "right cheek" consists of one elliptical fragment measuring 9.5 mm x 5 mm x 3 mm in greatest dimensions. The external surface appears tan and irregular. There is a 3 mm pigmented area at one edge with numerous cilia. A slight depression is seen on the surface. The specimen was inked and bisected. Cut sections show diffuse pigment below the surface. The specimen was submitted in cassette A.

Specimen #2, in formalin, labeled "left cheek", consists of one fragment measuring 7 mm x 4 mm x 2 mm in greatest dimensions. The external surface appears irregular with a 3.5 mm papillomatous elevation. There are numerous cilia. The specimen was inked and bisected. The cut surface shows a pigmented line below the surface with numerous white nodules. The specimen was submitted in cassette B.

MICROSCOPIC: Sections of the specimen labeled "right cheek" show skin and dermal appendages with scattered pigmented melanocytes, melanophages, and extracellular dark brown to black pigment. There are numerous collections of histiocytes and lymphocytes in a perivascular distribution.

Sections of the specimen labeled "left cheek" show skin with nests of nevus cells in the dermis. The individual cells have round to oval shaped nuclei of uniform size and shape and are widely spaces. Rare cells contain fine granular pigment. Cells in the superficial dermis are larger than those seen in the deeper aspect of the dermis.

FINAL DIAGNOSIS: Skin, "right cheek" (excision) -extravasated foreign pigment consistent with tattoo
-chronic inflammation

FINAL DIAGNOSIS: Skin, "left cheek" (excision) -intradermal nevus

If the microscopic examination of two specimens are identical they can be combined but gross descriptions and diagnoses are always listed separately.

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OCULAR CYTOLOGY SPECIMENS

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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EMBEDDING INSTRUCTIONS

EMBEDDING INSTRUCTIONS

The pathology attending usually gives instructions to the technician regarding embedding. We do that with written instructions and on the laboratory computer. .

As you know, all specimens must be accompanied by a routing slip which indicates where the specimen should be sent (cytology, immunohistochemistry, etc.). Instructions for embedment must be written on the routing slip or if no directions for embedment are given to the technologist, the tissue will be embedded on the flat surface rather than any edge. Thus, if any other method of embedment for sectioning is desired (on edge, on end, etc.), the edge or end must be marked with an orange dye (mercurochrome works well) which will remain after dehydration and paraffin infiltration to guide the technologist during embedment (see next page). An example of this would be the cornea, which must be sectioned perpendicular to the surface in order to study all layers at a given level and in a given section. Complicated specimens require diagrams and verbal communication with the technologist. Very complicated specimens should be embedded by with the aid of the technologist. Do not underestimate the importance of proper orientation of the specimen.

KEY ELEMENTS OF PROPER EMBEDDING (click on Figure 1 to ENLARGE in a new window!)

1. As stated above, the surface to be cut can be marked with mercurochrome.

2. Skin or mucosa also may be oriented by putting India ink on the epithelial surface and let the technologist know (by written instructions or tag) that the specimen is "skin". The specimen will then be cut perpendicular to the mark.

3. A flat piece of tissue may also be cut on the edge by instructing "cut on edge" (either orally or by instructions on the routing slip).

4. Surgical margins of all specimens with, or suspected of having neoplasms, must be marked with ink (India ink, Mrs. Stewart's Bluing, green, and yellow). Both will go through processing, be visible in stained sections, and be helpful in the assessment of surgical margins for tumor. Methods of blocking tissue for assessment of tumor in the surgical margins are shown in the following Figure. Please call the attending pathologist for questions.

5. Extremely small specimens (e.g. trabeculectomies and epiretinal membranes) should be marked with tissue dye (to permit visualization by technologist) and then placed carefully in an embedding bag.

6. Finally, one can instruct the technologist to "call me when embedding" and thereby control the embedment and sectioning.

NOTE: ALWAYS KEEP THE PAPER WORK, (routing form) WITH THE SPECIMEN. IF THE PAPER WORK IS SUBMITTED FOR TYPING, MAKE SURE A COPY STAYS WITH THE SPECIMEN. Your sections will return with the routing form. Keep slides and paperwork together until after sign out.

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SUSPECTED LYMPHOMA- PROCESSING

Lymphoproliferative Lesions:
Lymphoproliferative lesions as a class constitute a significant diagnostic challenge to the hematopathologist, and routine preparations are often difficult to adequately characterize. Accordingly, any lesion suspected on clinical grounds to be a lymphoma, "pseudolymphoma", or lymphoproliferative lesion must be processed under supervision of the eye pathology attending. In general, the specimens will be sent to the hematopathology service but check.
Prioritize tissue collection for lymphomas as follows:
1. In the OR, wrap the tissue in gauze soaked in phosphate buffered saline (PBS) over wet ice. Do not immerse in buffer!
2. Tissue for routine paraffin embedment - In the lab, fix a thin slice in neutral buffered formalin, B-5 or paraformaldahyde.
3. Touch preps - Prepare 2 air-dried, unstained glass slides for later fixation and staining with May/Grunwald/Geimsa (MGG) by gently touching the freshly cut surface of the tumor on 3-4 spots on the slide. 2 more slides, prepared the same way, should be rapidly fixed in 95% ethanol for staining with H & E or PAP.
4. Immunohistochemistry / Gene rearrangement studies - Select a large piece of fresh tissue. Partially fill the well of a plastic blocking mold with O.C.T. compound (third drawer under the autopsy eyes). Place the specimen in the well and then fill the well with O.C.T. compound. Immediately place it in 95% ethanol (previously cooled to near freezing by dissolving dry ice in it) and place over a block of dry ice. Fill out the immunohistochemistry request form/These are then sent to the Immunochemistry lab in main pathology, CHS 13-240, x50643. Gene rearrangement studies are sent to the molecular biology lab 332B CHS, x55648 or x65294.
5. Electron microscopy --- Dice a small bit of tissue and place it in Formaldehyde-Glutaraldehyde fixative (2% each). Hold tissue in fixative until H & E sections from #1 above are done.
6. Flow Cytometry --- Fresh tissue is submitted for flow cytometry in a special culture media prepared by Flow Cytometry in Room AL 154, x55806 (x56877). Call them to obtain appropriate media and requistion forms. The specimen must be delivered promptly.
Orders for Immunohistochemistry: Our routine for MALT and orbital lymphoma includes antibodies to: CD3,CD5,CD10,CD20, CD43, BCl2, BCL1, kappa, lambda, BCL6, and IgM. Additional orders per attending.

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EVISCERATION AND EXENTERATIONS

Eviscerations and Exenterations:

Evisceration operations are used virtually exclusively for blind eyes that are painful. The operation removes the cornea and the ocular contents, leaving behind the scleral shell to retain a plastic implant. Evisceration specimens are usually processed like a complex surgical biopsy, blocking separately the cornea and as many blocks as required to study the ocular contents.

Example: EVISCERATION WITH PHTHISIS BULBI
Gross: The specimen, received in formalin and labeled, "intraocular contents OD," consists of an evisceration specimen which measures 15 x 15 x 14 mm in greatest dimensions. The external surface appears opaque and vascularized. The cornea is calcified. The rest of the ocular contents appear reddish-brown with dense calcification. After 1 week of decalcification the specimen was bisected. Cut section reveals corneal stromal scarring, peripheral anterior synechiae, a scarred and atrophic iris, a cyclitic membrane, a disorganized and collapsed uvea, total retinal detachment, fat droplets, and white tissue posteriorly (probably bone). The section was submitted in cassette A.

Micro: Sections of "intraocular contents OD" show cornea with hydropic epithelial changes, marked destruction and fragmentation of a calcified Bowman's layer with overlying extensive pannus formation, stromal edema, deep vascularization, scarring, and bone formation paracentral calcification below the epithelium. The Descemet's membrane is partially stripped away from the stroma. The endothelium is attenuated. The anterior chamber shows hemorrhage. The lens is calcified and shows marked cataractous changes. The iris is calcified atrophic and shows numerous clump cells. There is marked anterior synechiae that cover the anterior chamber angle. The ciliary body is atrophic. There is bone complete with marrow fat posteriorly and the retina is gliotic detached and focally calcified.

FINAL DIAGNOSIS: Intraocular contents, "OD" (evisceration)-- changes consistent with phthisis bulbi
-extensive calcification of cornea, iris, lens, retina and choroid
-retinal detachment and gliosis
-anterior synechiae with angle closure

Exenteration operations have been used primarily in ophthalmology to treat the following conditions: neoplasms of the eyelids that have either invaded the orbit or are too extensive to salvage (or replace) the eyelids: primary tumors of the orbit, secondary tumors of the orbit (e.g. extension of intraocular tumors), and severe inflammatory of infectious lesions (e.g. aspergillosis or mucormycosis). The eye cannot be salvaged in these cases because too much orbital tissue has been sacrificed in the operation to permit survival of the eye, or the primary process (whether neoplasm or infection) significantly involves the eye. As we have learned more concerning the survival of patients who have undergone exenterations of the orbit, the operation in ophthalmology for many conditions has lost favor. Such specimens currently are more likely to originate in the head and neck region, where the exenteration is combined with more extensive operations for neoplasms. Exenteration specimens are of major importance and each one needs to be discussed with the attending pathologist prior to operation, dissection and processing. The general procedure for these cases is to:

1. Obtain adequate history one day in advance

2. Inform the attend pathologist of the case one day in advance so that special solutions and arrangements can be made to collect the specimen.

3. Check to see if margins have been taken at frozen section or what margins in the exenteration specimen are pertinent. If margins exist then the specimen must be inked and sections taken for margins, (e.g. posterior, medial, lateral, superior and inferior margins as well as skin margins if necessary).

4. Provide adequate fixation if the attending pathologist does not want any tissue fresh for analysis (e.g. sebaceous carcinoma, research, melanoma, etc). This may involve removing the eye from the specimen.

5. Obtain adequate sections of the exenteration specimen to look for the pertinent pathologic findings (history and diagnosis dependent).

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ENUCLEATED EYES-Processing

ENUCLEATED SURGICAL EYES

NOTE: For all surgically enucleated eyes, call the attending pathologist to determine the best way to work up the case. Never proceed on your own without consultation.
If a normal eye, or eyes with suspected melanoma or retinoblastoma, are being removed, it is imperative to have tissue frozen, so alert the attending pathologist when you anticipate a specimen being removed. It is very poor form to call the pathologist after the eye has been received or fixed. In certain cases, patients may have legal grounds for a law suit if the appropriate studies are not performed because of mishandling.
If eyes arrive in formalin and have been fixed for 2 days, wash them in water to remove the formalin ( 2 changes about 5 minutes each) and place them in enough 50% ethanol to cover the eye. Let the eye equilibrate overnight. Change the alcohol the next day and equilibrate for a second day. The eye should return to a normal volume and should not be indented or shrunken. For sectioning the eye it is best to wait 2 days with the eye in 50% ethanol .
Surgically-enucleated eyes (as well as eviscerations and exenterations) must be accompanied by a detailed written clinical history before the work-up is undertaken. Eyes that are enucleated in the JSEI O.R. will be accompanied by a complete history. Be sure that you have sufficient clinical history prior to processing the eye.

Gross The gross description should include the following:
a. complete a diagram by filling out a Gross Description Form for the appropriate eye. This will be invaluable for your description. Put appropriate measurements of the eye, tumor or pathologic processes on this form. There is a different form for coronal cuts.
b. completion of a standard Enucleation Form. (Note that there are separate forms for melanoma and retinoblastoma)
c. photography will often be required

Micro
The standard format for microscopic description are included in the Enucleation Form. Examples of completed work-ups are provided. The work-up of a surgically enucleated eye is essentially a mini-autopsy and the following few tips may be helpful. Study the sections with a clear understanding of the clinical history (you will virtually always need more information than provided on the Pathology Consultation Request form), the gross features, and how the eye was microsectioned (step levels, etc., see technologist). Your written narrative description should follow this general plan:

(1) Begin with a comment of the condition of the eye as viewed without optical aids (e.g. normal size, shape, large tumor, retinal detachment, etc.).
(2) Next, describe the most important lesion in the eye (e.g. tumor, retinal detachment, wound, etc.) beginning with low power observations, and adding additional details as needed with higher magnification.
(3) Add descriptions of secondary lesions.
(4) Briefly mention other important ocular structures or areas, even if negative.
(5) If the eye has some general condition which is diffusely involved (for example, in phthisis bulbi) one may elect to sequentially discuss ocular structures or areas (e.g. cornea, anterior chamber, chamber angle, etc.).

Diagnosis
This should be comprehensive and should begin with a succinct clinical summary. Remember that in old cases only the diagnosis is stored in the computer: Therefore, one must repeat in the diagnosis all the important aspects already discussed in the narrative description. For example:

FINAL DIAGNOSIS: Eye, "OS" (enucleation)
- Atrophy with shrinkage and disorganization (Phthisis bulbi)
- Corneoscleral scar, superotemporal, healed
- Peripheral anterior synechiae, 360, old
- Rubeosis iridis and ectropion uvea, moderately severe
- Retinal detachment, total, with both epiretinal and retroretinal membranes.


Surgically-enucleated eyes suspected of harboring neoplasms are treated in the following manner:

Melanomas --- use “Melanoma Form”.

A careful external examination must be done before any extraocular tissue is disturbed or removed. Remember if eyes come to you in fixative, you must first wash them in water to remove the formalin and place them in 50% ethanol for at least 30 minutes before external examination; 30 minutes is not ideal but can be used to expedite processing if absolutely necessary. However, the eye should have equilibrated to a normal volume and should not be indented or shrunken. For sectioning the eye it is best to wait 2 days with the eye in 50% ethanol . Alcohol will restore natural color and will facilitate distinguishing pigmented tumor tissue from blood clot. You may decide to do a careful dissection of the episcleral tissue outlying the known location of the tumor, but all tissue should be saved. In general, melanomas reach the episclera and the orbit through scleral canals that have a fairly predictable location (anterior ciliary vessels, vortex veins, short and long posterior ciliary arteries and nerves). These regions should receive especially close examination with the stereomicroscope. See below for processing vortex veins.

Transillumination should be done next. This is accomplished in our laboratory using the examination light fitted with a cardboard box to hold the eye. Examination is facilitated by turning off the room lights. When the intraocular tumor has been located, its margins may be marked with a ball-point pen, with delicate dots of India ink, or with dots of marking ink. These marks will assist you in planning an incision to transect the tumor when the room lights are on. In most cases, the incision to open the eye will be made postero-anteriorly in a plane that will pass through the edge of the tumor.

In general when the eye is to be processed rapidly in order to obtain a preliminary diagnosis, the vortex veins are removed from the episclera and the optic is transected 2 mm from the sclera and these pieces are sent separately. Sometimes, a small piece of tumor in the small calotte can be submitted immediately in order that tissue diagnosis may be rendered the following day. The large calotte can be held for photography, teaching, etc. The brief preliminary report from the microscopic examination of the piece of tumor in the small calotte will amount to little more than a tissue diagnosis. The report is written with a very short gross description, a brief microscopic description of the tumor, and a diagnosis, e.g.;

FINAL DIAGNOSIS: Eye, "OS" (enucleation)- choroidal malignant melanoma (spindle-cell type)

Comment- Complete report of the gross and microscopic findings in the large calotte will follow.

Once the preliminary report of the tumor in the small calotte is completed, the work-up of the remainder of the eye may be conducted at a more leisurely pace, and the specimen may be used for individual instruction and the daily gross conference. When the work-up of the large calotte has been completed and reviewed (signed) by the senior pathologist, the eye is submitted for step-level sectioning completely through the tumor.

Microscopic study should include an analysis of all step-levels (which the technologist will number for reference), but the narrative will be written as a synthesis of the information gained by step-level study. During the gross and microscopic study, the pathologist must anticipate the important questions asked by the surgeon and answer these questions with positive or negative statements in the narrative work-up.

The diagnosis for whole surgical eyes is necessarily more comprehensive than for simpler specimens. It is initiated with a succinct clinical history containing relevant information. In the case of eyes with melanomas, one should then elaborate on the preliminary diagnosis to include the predominant cell type, spindle or epithelioid cells in the tumor (if any), relative size of the tumor, presence or absence of extraocular extension, the vascular pattern (or pattern of vasculogenic mimicry), retinal invasion, emissarial invasion, vitreous involvement etc. Incidental diagnoses (retinal detachment, etc.) should be succinctly characterized to complete the diagnosis.


Example of a melanoma work-up:
Gross Description: Specimen, in formalin, labeled "left eye" consists of an intact left eye measuring 24.0 mm (AP) x 23.5 mm (H) x 25 mm (V) with 1.5 mm of attached optic nerve. The cornea measures 13.0 (H) x 12.0 (V), and the pupil is round and measures 7 mm in diameter. No melanin pigmentation is seen on the episcleral surface. Vortex veins are slightly dilated directly over the tumor, but no hyperpigmentation lesions are seen over these vessels. Transillumination reveals a tumor measuring 19 mm in greatest diameter and extending 7 mm from the optic nerve anteriorly to the pars plana. Horizontal section above the optic nerve reveals a pigmented tumor, 19 mm (diameter) x 12 mm (elevation) in the ciliary body and choroid temporal to the optic nerve. A large serous retinal detachment obscures the posterior boundaries of the tumor. The macula is markedly distorted and the optic nerve is obscured. The tumor lies adjacent to and touches the cataractous lens. The anterior chamber is shallow. There is pigment on the posterior lens surface and a Vossius ring on the anterior capsule. The retina adjacent to the tumor shows areas of punctate superficial pigmentation that follow the distribution of retinal vessels. There is a large coronal adenoma in the ciliary process at 6 o'clock. The peripheral retina shows typical cystoid degeneration.

Microscopic: Sections show a large choroidal tumor composed of predominantly spindled cells with small areas of epithelioid cells containing brown pigment. The spindle cells are arranged in fascicles and contain oval nuclei with small nucleoli. There are occasional areas that contain cells with more abundant cytoplasm and round nuclei with prominent nucleoli. There is broad front invasion of the sclera. The vortex vein centered over the tumor contains extensive melanoma attached to its wall and following both its intrascleral and episcleral course. The retina overlying the tumor shows a serous detachment and exhibits extensive degeneration of photoreceptors including inner and outer segments. There are numerous macrophages with foamy cytoplasm in this area. Within the tumor blood vessels form a pattern of back to back loops. Retinal pigment epithelial drusen are present. Incidental findings include corneal epithelial edema, extensive actinic degeneration of collagen at the limbus, a tumor composed of non pigmented epithelial cells and myxoid stroma confined to the ciliary processes, and typical cystoid degeneration.

FINAL DIAGNOSIS: Eye, "OS" (enucleation)- Choroidal malignant melanoma, mixed cell type (spindle predominant).
-Extensive tumor present in vortex vein
-Serous retinal detachment
-Coronal adenoma of pars plicata

Retinoblastomas -- use “Retinoblastoma Form”

The work-up for eyes harboring retinoblastomas is identical to that used for melanomas or any other intraocular tumor except:
1) If the history is suggestive and/or the patient's family and physician wishes, special genetic studies are done to determine the likelihood of a new or inherited defect. These tests are done on specially-prepared samples and they must be fresh. This is done in conjunction with the attending pathologist.
2) Since the retinoblastoma tumor is often friable, one should measure and remove the optic nerve before opening the eye to prevent floating tumor fragments from "contaminating" the nerve.

A piece of the tumor (usually from the small calotte) and the optic nerve should be sent through in paraffin on the day (or the day following) the enucleation. The entire optic nerve should be embedded, and sectioning should assess the nerve 1-2mm behind the eye and at the cranial (surgical) margin, if indicated. If the intraocular tumor involves the optic disc or choroid (extensively), the large calotte should be sent through soon after the gross work-up.

There is a special data sheet for eyes with retinoblastoma that will serve as a check list to be sure that all morphological features important for the prognosis are assessed.

Endophthalmitis
An example:
Gross: The specimen received in formalin, labeled left eye, consists of a left eye that measures 25mm (V) x 25 mm (H) x 25 mm (AP). The cornea measures 10 mm (V) x 9 mm (H). The pupil is not visible and 4 mm of optic nerve is attached. The eye shows poor transillumination. The anterior chamber is not visible externally and the cornea is opaque. The conjunctiva is markedly elevated superiorly with numerous dilated vessels. The cross section of the optic nerve is distorted by crush artifact. The eye is cut vertically temporal to the optic nerve. Descemet's membrane is wrinkled superiorly. The anterior chamber is filled with a fibrous and fibrinous exudate the obscures ocular structures. The iris and ciliary body are covered by a fibrous membrane that extends into the vitreous cavity. No intraocular lens is visible. The vitreous cavity is filled with pus. The retina is detached posteriorly and there is a thick white subretinal exudate. There is a focal area of pigmentation in this area. The retina is not discernible in many areas. The ciliary body is thickened in focal areas.
Micro: Microscopic sections show severe acute and chronic inflammation in the anterior chamber, cornea, conjunctiva and sclera. The cornea shows absence of the corneal epithelium, neutrophils present between stromal lamellae, and focal marked necrosis. Descemet's membrane is detached in areas and there is a large wound gape superiorly in which lies inflammatory tissue composed of neutrophils, lymphocytes, plasma cells, mast cells, histiocytes, and fibrovascular tissue. The conjunctiva covers the separation of the corneal wound edges. The cornea is focally necrotic at the edges of the wound gape. The anterior chamber is distorted and filled with acute and chronic inflammatory cells, and granulation tissue. The iris is disrupted and necrotic. The intraocular lens and haptics are located in the posterior chamber and surrounded by inflammatory tissue. The lens capsule surrounds the synthetic intraocular lens. There are multinucleated giant cells and focal granulomatous inflammation surrounding the capsule. Blue staining amorphous material is present at the cataract wound site and in the vitreous. The ciliary body is markedly inflamed with acute inflammatory cells. The vitreous cavity is filled with fibrinous exudate and inflammatory cells. There are numerous ghost cells and foamy macrophages in the vitreous cavity. The retina shows complete exudative detachment and marked necrosis with complete loss of photoreceptors in most areas. There is a extensive subretinal fibrous membrane. The pigment epithelium and Bruch's membrane are focally disrupted. The choroid shows marked infiltration by lymphocytes, plasma cells and histiocytes. Numerous lymphocytes are present along blood vessels (Virchow Robin spaces) in the optic nerve posterior to the lamina cribrosa. Gram stains show occasional gram positive clusters of coccoid forms and a few clusters of gram negative forms in the inflamed tissue of the anterior segment.
Diagnosis: Eye, "OS" (enucleation)- Clinical history of cataract surgery, one month prior to enucleation--Severe acute and chronic endophthalmitis
-Exudative retinal detachment
-Subretinal fibrous membrane
-Wound gape
-Pseudophakia, Intraocular posterior chamber lens

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Common Specimens in Eye Pathology-Gross

Examination of Common Specimens in Eye Pathology: Cornea, Conjunctiva, Epiretinal Membrane, Eyelids, Temporal arteries

Cornea --
Place the specimen in water and examine it under the dissecting microscope. Scoop the cornea out with blunt curved forceps other than pinching it between the teeth of the forceps. Note scars, vessels, or lesions on the surface. Note depth of lesions. Measure the diameter. Then place cornea on a flat surface with epithelium facing down (in order to protect the endothelium). Do not place it on paper towel as this will easily peel the surface layers off! Bisect the cornea with a blade (in drawer under the sharp box) and record the central and peripheral thickness. Submit half for processing and save the other half in formalin. Order PAS stain on all corneas.

The microscopic description should encompass the findings of the epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane and endothelium. Example:
Gross Description: Specimen, in formalin, labeled "OS cornea," consists of a translucent disc measuring 7 mm in diameter and 1.2 mm in central thickness and 1.4 mm in peripheral thickness. The epithelial surface is irregular and shows tiny blebs. Centrally the epithelium is missing. The specimen was bisected and cut sections reveal thickening centrally to the stroma. The specimen was submitted in cassette A.
Microscopic: Section shows epithelium with hydropic change, subepithelial bullae, and an irregular Bowman's layer. The epithelium is detached in many areas from Bowman's layer. There is stromal edema, mild inflammatory infiltrate consisting of lymphocytes and neutrophils, and stromal scarring. Descemet's membrane is artifactually absent in one large area due to tangential sectioning. The endothelium is absent.

FINAL DIAGNOSIS: cornea, "OS" (penetrating keratoplasty)
- bullous keratopathy with acute and chronic stromal keratitis
-severe endothelial attenuation


Descemet's membrane (DESEK specimens):
Samples that are surgically removed from stripping of Descemet's membrane appear as a translucent sheet that will probably be invisible in solution. The specimen remains invisible under a dissecting microscope, so prior to even examining the specimen simple steps can be taken to visualize the sample. The steps in processing (see reference) are to add an orange tissue binding dye (eosin or mecurochrome) to the solution and make it visible. DESEK may be received from the operating room in saline or formalin. If the specimen arrives in saline then formalin is simply added to the specimen container with a drop of mecurochrome. After 4-8 hours the orange membrane is removed carefully without tearing and placed in the middle of one side of a tea bag. Gently place the tea bag side with the membraneon water and the specimen will miraculously unfold. If not then submerge the tea bag and the membrane will float and unfold. The tea bag can be used to pick up the unfolded membrane for processing. Be sure to look carefully under the dissecting microscope for guttata (tangential lighting helpful).
Fold the edges of the tea bag; the edges away from the specimen. Place the folded tea bag in a nylon bag (just to be sure it is not lost in process). Instruct the technician to bisect the membrane after processing and to place on the embedded edge.
Conjunctiva:
Surgical preparation of conjunctival biopsies-- Biopsies of the mucous membranes tend to curl when placed unsupported in fixative which makes it difficult to embed the specimen. To prevent curling, it should be gently spread on a small piece of paper towel, moistened with saline, with the stromal side against the paper, before placing it in fixative. The specimen, now adherent to the paper, is then gently floated on the surface of the fixative, making certain that the epithelial side is down and submerged in fixative. In a few hours, the specimen will be fixed flat and can either be removed for processing or can be processed while still adherent to the paper (excess paper can be trimmed before further processing). This procedure has the further advantage that any orientation information for the pathologist can be written by the surgeon on the paper (using ball-point pen or pencil). A suture can be used to help orient the specimen.

Conjunctiva is grossed in with the attention to margins. If a neoplastic process is even remotely possible, then ink the margins. If the specimen is less than 1 mm in diameter, it can be submitted without bisection. An accurate diagram should be made of the lesion before showing it to the pathology faculty.

If the conjunctiva comes with an orienting suture and is greater than 2 mm in length, then the attending pathologist will instruct the resident in preservation of proper orientation for microscopy. The principles are exactly the same as for skin biopsies with provided orientation (see below).

For pterygia, the specimens can be flattened and sectioned in a plane to capture the cornea in section. Requesting the histotechnologist to slice the specimen every mm after paraffin infiltration but prior to embedding will reduce the need to take interval sections (deeper sections) with the microtome and will greatly reduce the workload of the technician. Slicing after paraffin infiltration also results in properly oriented sections if the conjunctiva has been unfolded on the hot plate (melting the paraffin) before embedding. The histotechnologist should be trained in unfolding the conjunctiva at the embedding station prior to making these 1 mm slides.

For a diagnosis of suspected ocular cicatricial pemphigoid the conjunctival biopsy should preserved in Zeus media, rather than fixed, and processed by rapid freezing, followed by frozen section, and direct immunofluorescence for C3, IgG, IgA, IgM and fibrinogen.

GROSS DESCRIPTION: The specimen on the patient above was received in formalin, labeled "L. caruncle lesion biopsy," measuring 4 mm x 3 mm x 2 mm in maximal dimensions. The external surface shows hair follicles, an irregular cystic and brown pigmented surface. The undersurface is irregular yellow and waxy white in appearance. On cross section pigmented tissue is present beneath the epithelium. The specimen is inked (green), bisected, and submitted in cassette A.

MICROSCOPIC: Sections show conjunctiva with nonkeratinized stratified squamous epithelium, with goblet cells, hair shafts and sebaceous glands. There are nests of pigmented cells confined to the substantia propria with oval to round nuclei. Cells deeper in the lesion are smaller than those in the superficial portion.

FINAL DIAGNOSIS: Conjunctiva, "left caruncle" (biopsy)- subepithelial nevus


Epiretinal Membrane
The specimen usually arrives in saline. At least four times the volume of formalin must be added to the bottle. If the specimen is free-floating in fixative and difficult to see, place one small drop of mecurochrome dye in the fixative and wait for 3-24 hours to color it. These steps will aid both you and the technicians processing the specimen. After overnight fixation, remove the specimen, place it in an embedding bag, and submit it for sectioning.

Orbital Biopsies
Contact the attending for all orbital tumors. If the surgeons have not discussed the case with the pathology staff, call and get information on the clinical diagnosis and operation planned. If a biopsy of the orbit is suspected to be a lymphoid lesion, a complete lymphoma work up is indicated. See "Procedures for Handling Lymphoproliferative Lesions". Other orbital biopsies should be measured and described in detail with attention given to the surface and encapsulated architecture. The specimen is inked (if the surgeon attempted a complete removal) and bisected. A portion of the bisected specimen is saved in case electron microscopy is desired. Another portion is submitted for paraffin embedding.

Eyelid Lesions (the wedge resection)
Measure specimen carefully and examine for any lesions. Full thickness specimens require precise orientation and marking inks to preserve orientation after processing. If tumor is clinically suspected, discuss with the pathology faculty before processing.
In general, pentagonal wedge excisions have 3 margins that need to be inked: lateral, medial and posterior, i.e. where the surgeon has made cuts in the eyelid. The tarsal conjunctival surface and eyelid skin surface are not margins and do not need to be inked. The most common mistake made by those unfamiliar with anatomy of the eyelid is to ink the conjunctival surface thinking this is a posterior margin. Covered by epithelium, obviously it is not a margin at all. The closest margins to the tumor should be determined grossly. Several options exist to proceed thereafter; here are 4 examples.
1. Each margin, medial, lateral and posterior can be inked, removed in strips and processed separately. If there is tumor in the strips then step sectioning may give an idea of how close the tumor is from the margin but it is not as accurate as method 3 below. This is technically difficult, requires excellent fixation and should be done only after consulting the attending pathologist. It is the method of choice for very large tumors where there is good clearance from all edges and the strips are likely to be negative.
2. Each margin can be inked a different color, the specimen bisected in an anterior to posterior direction and the bisected section edge inked with orange dye for the histotechnologist to embed down in the cassette. Step sections by the histotechnologist will go through the tumor to approach the margins. This preserves of anatomic features of the eyelid. To be sure that the ink holds fast, it is best to bisect the specimen after processing. If the ink does not hold the orientation can still be preserved but this will require the input of the experienced pathologist and histotechnician. This technique is most commonly used because it is relatively simple to remove a thin slide from the center for possible electron microscopy. It also has the disadvantage of giving only relative distances that the tumor resides from the margin. This is an excellent method for small tumors in a small specimen where the margins will be clear in just a few sections.
3. Each margin can be inked a different color and the specimen bisected medial to lateral and embedded on the bisected edge. This has the advantage of providing the distance of the tumor from the medial to lateral margins but has the disadvantage of receiving sections without standard anatomic features. In this method step sections are taken to examine these margins that are usually of the most interest. It is the method of choice for tumors that obviously approach the medial and lateral margins and one needs precise distances to report to the surgeon.
4. After inking each margin a different color the specimen is processed for paraffin and embedding and may be breadloafed in a plane parallel to the nearest margins and embedded on the cut edge. In this case it is critical to embed the specimen in the same orientation and to retain in the block the order that it has been cut at the gross table. For the best fidelity this is best cut and embedded after processing. A crack histotechnolgist is quite capable of handling this with a bit of training. This is the most cost effective way to examine the entire eyelid (all breadloafed sections) in one slide, provided the specimen has been carefully oriented and the tumor does not approach the margin. But if the specimen is embedded inaccurately, then all margin analysis may be lost. This is not a good method when the histology lab is off site or the technician du jour is doing the embedding rather than one dedicated to eye specimens.
5. Hybrid techniques. Although a bit advanced for this forum, there are very efficient ways to preserve the anatomy, give excellent distances measurements to the tumor with high fidelity, and allow central sections to be processed for ultrastructure. Involved in these methods are special gross sectioning in a number of planes, re-embedding the specimen, and a very knowledgeable histotechnologist and a lot of free time.

Skin Biopsies with Provided Orientation

Occasionally skin ellipses or biopsies may come with orientation for margin analysis. These will generally be oriented with a suture at 12:00 o'clock and the requisition will ask for margins. In order to provide orientation, consult the attending pathologist. Our standard practice is to ink the entire undersurface green and the edge containing the suture (superior) with yellow ink. An adjacent side is inked black. The suture is left in place until after paraffin infiltration. At that stage the specimen is checked to see if the marking ink remains after processing. If the ink is present all of the margins can be identified by the absence or presence of yellow or black ink. The specimen is then cut along the dotted line or in multiple planes parallel to the dotted line and aligned in order in a cassette (see embedding instructions). Inking opposite sides different colors, the typical neophyte's approach, will not permit adequate orientation. In the example to the left the skin ellipse is marked with a suture at 12:00 o'clock. The lesion is denoted as tan. Green has been used to ink the entire undersurface but in this view is only visible on the left edge because black and yellow ink have been used to cover adjacent edges and are overlayed on the green. When the specimen is sectioned in the plane of the dotted line, the left edge will be always identified as having only the green ink. The right edge is always identified as having the black ink. The superior edge will contain the yellow ink. In this way the orientation is preserved through microscopy. However, if by mistake the left edge is inked yellow and right edge black, one will be unable to tell the superior margin from the inferior margin in microscopic sections. This is a concept that may be difficult for the beginning Ophthalmology resident and Eye Pathology fellow until they learn to think in three dimensions.

Temporal Arteries
Temporal arteries are submitted to confirm the clinical diagnosis of temporal arteritis. A negative biopsy does not exclude the disease. The benefit of confirming the diagnosis by biopsy may make the procedure very worthwhile in the long term if the clinical diagnosis should be questioned later. Because the pathologic lesions of temporal arteritis are often focal with skip areas it is important that the artery be completely sampled. Our current standard procedure at the gross bench is to measure the length and diameter of the artery and make one clean bisection. The cross section is examined under the dissecting microscope to identify a narrow lumen or nodular thickening (signs of granulomatous arterities). The artery, now bisected is then sent to the histology lab for processing. Instructions for the histology technician include: Artery, make cross sections every 1 mm (no greater than every 2 mm), and embed on the cut edges. Make 1 section and stain for hematoxylin and eosin. Once the artery has been processed and infiltrated with paraffin the histology lab technician will make thin cross sections, no greater than every 2 mm (and preferably 1 mm). All cross sections are placed on the cut edge in a metal embedding cassette and a single paraffin block is made. A single slide is cut from this block, which will sample the entire artery at 1-2 mm intervals. For expediency this section will be stained and examined. Most positive cases will be evident on this first slide and can be immediately reported. If no arteritis is seen, then more sections are taken according to the following instructions to the histology technician. Make step sections deeper every 50th section (five-micron thick section) through the block. This allows sampling every 250 microns (or .25 mm), which should pick up skip lesions without difficulty and leave a large margin of error. In our recent experience the number of sections received is exactly the number that would be calculated from the length of the vessel. (See the classic work of Campbell's group at the Mayo Clinic). We monitor this in every case as will be evident below.
Indications for RUSH processing for the biopsy. If necessary the histologic processing can be ordered RUSH!, which will result in the sections returning by the next day or even sooner. Several factors generally preclude any need to send the cases for rapid or "rush" processing. First, there is some controversy as to the value of temporal artery biopsies in affecting clinical treatment. Several studies have shown that clinical treatment is unaffected. The patients have usually been started on steroids by the time the biopsy is performed. Therapy the will not be withdrawn immediately because a clinical response must be measured. Additionally, the sensitivity of temporal artery biopsy in not 100%; sensitivity in this disorder is hard to estimate because there is no gold standard for the diagnosis. However, Bayesian analysis of bilateral temporal artery biopsies suggests sensitivity could approach about 87% at best , (IOVS 2007;48:675-80). Therefore a negative temporal artery biopsy does not exclude the disorder. Also, the step sections will take additional processing time, so rushed initial sections will not always expedite the case. There are instances when rush processing may be desired. For example, if for some reason an irregularity in judgement has lead the clinician to perform the biospy and at the same time withhold treatment until a result is available (generally not the standard of care), and the clinician informs you of this, then by all means proceed with rush processing. If a long holiday is coming up (the laboratory will be closed) or a patient is going on a trip and needs to have instruction for dosing immediately then rush processing may help. Each instance must be considered on individual basis. It is important to have clinical input to make this decision. The need for a rush case should be discussed with the attending pathologist and orchestrated by talking to the lead histology technician.
Sample report:
CLINICAL DESCRIPTION: 75 year old male with history of headache for 2 months. Exam shows tenderness of the temporal artery. ESR= 75 mm
GROSS DESCRIPTION: Specimen, in formalin, labeled "biopsy from left temporal artery" consists of a tubular structure, measuring 10 mm (length) x 2.5 mm (diameter) and is bisected. Under the dissecting microscope the cross section shows narrowing of the lumen and nodular thickening of the vessel wall. The specimen is submitted after processing in cassette A.
MICROSCOPIC EXAM: Step sections at 250 micron intervals stained with EVG and Trichrome show a moderate sized artery with a narrowed lumen and severe mural granulomatous inflammation. The inflammation involves the thickened intima, media and adventitia. The inflammatory infiltrate is composed of collections of histiocytes with accompanying lymphocytes, plasma cells and eosinophils. The EVG stain shows fragmentation of the internal elastic lamina, with obliteration of short segments. .
FINAL DIAGNOSIS: Artery, "left temporal" (biopsy)- granulomatous arteritis

How does one monitor intervals at which step sections are made by the laboratory? At the end of the step sectioning we simply add up the number of total sections that were made of the artery on all slides. We compare that number to the expected number, which we calculate. For example in a 10 mm artery that was cut at 250 micron intervals then 10,000 microns/250 microns/section= 40 sections are expected. The interval actually cut is given in our final report for cases in which step sections were performed.

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