Eye Pathology Procedure Manual for Specimen Processing- A Guide for Residents

Teaching Eye Anatomy to Medical Students

noreply@blogger.com (Mission for Vision) — Sat, 03 Mar 2007 18:08:00 +0000

The resident on the eye pathology service teaches the medical students each Monday morning anatomy of the eye and pathology to start their required rotation in Ophthalmology (part of surgery). We employ a systematic method for teaching the students and the ophthalmology residents are to follow this system. Do not deviate from this method!!! It has been developed by the attending over the past 21 years and is highly effective. Historically the anatomy teaching session has been one of the most popular sessions of the week for the medical students (by their evaluations). Just as important it is a learning session for the resident in teaching anatomy and pathology from a human eye rather than some picture. This is not an opportunity for the resident to develop their own style of teaching. We want you to learn the power of the Socratic method. No notes, pictures or props are permitted. You are to teach from your knowledge and the human eye as your only resource. Likewise the students will not be permitted notes, books, or writing implements. They have been instructed to study the material on line prior to this session and now have the opportunity to demonstrate their knowledge. Here are the steps you will follow.
Set up the lab for teaching:
1. Put the 4-5 chairs, preferably the ones without wheels and others that are not particularly useful to use at the gross table on the side opposite the double headed microscope. You will be seated at the microscope. Clean off the table.
2. Pick out a gross specimen from the cabinet on the left. These will usually be marked "for teaching." The specimen will be 2 calottes of a horizontally sectioned human autopsy eye.
3. Clean a class petri dish carefully with soap and water. Then fill it to the top (at least at the top of the label on the dish) with 50% alcohol (in the brown container). There is a small metal mesh disc that works nicely for holding the eye. Put it in the Petri dish.
4. Clean off your forceps so they are free of any green ink.
5. Place the eye in the dish and put it in the center of the table where it will be visible to all students.
6. Also find your teaching glass slides in a wooden box in the cabinet above your desk. Put them next to the multiheaded microscope for now.
7. Obtain the examinations from the attending pathologist.
For the session:
1. Begin by administering the examination on anatomy. The attending will provide you with the test and answer sheets. The students have been told to study this material in advance and expect to be tested.
2. At the end of the test period (when the student are done ~ 20 minutes, you will grade the exams with the attending pathologist.
3. Those students who pass will now go on to view a dissected human eye and attend your session on pathology.
4. Those students whose knowledge base is unacceptable will be relegated to the library for further study of the basic material and will take another examination later in the week.

5. For those student who have passed and remain in the room with you will now sit at the chairs around the lab table.
6. You will follow the teaching of Socrates. You will not lecture to students. You will simply ask them questions and let them find the answers. The session begins by you learning each of the student's first names and ask what specialty they are interested in.
7. You will use your forceps as a pointer (do not touch the internal ocular structures or you will ruin the teaching specimen). You will point to an anatomic structure that you want them to recognize and ask them its name. If they don't know the name ask them to describe the location in detail and to create a name. This will be a test of your understanding of how anatomic structures are named (for example the derivation of conjunctiva from conjoining the eye and eyelids). If one student does not know the name move to the next student and so on until you reach a student that knows the name. Once the name of the structure has been identified ask all the students who were clueless to recite and spell the name. This will help to reinforce their knowledge. In fact this will go very quickly because they have already demonstrated their knowledge on the exam but will give them confidence.
8 . Move to the compound microscope multiheader. Using the glass slide of a normal eye point with a pencil successively to following structures and ask the students to name them. Here is convenient to just go around in a circle. If you have done your job properly the students will race through this section and you will be amazed at what you have taught them. Now place the slide under the the multiheaded compound microscope. Turn the pointer light on medium, not high or you will burn the bulb out and then have to teach without a bulb. This is an experience you would like to avoid. Ask the student to each take a seat at one of the head of the microscope and use the pointer to ask them to name the various ocular structures. Cover all the same structures that you did in the gross spending more time on the layers of the retina, ciliary body, iris, choroid, sclera, optic nerve. In each case the students are naming the structures. You simply point to them.
Ocular Pathology: Now move to the pathology slides. Generally this is best done by letting the each students look at one slide grossly. Ask them to take a moment to study it without a micrscope and try to make a diagnosis. The following diseases should be covered: melanoma, retinoblastoma, glaucoma, diabetic retinopathy. In most cases they will be able to do this by studying the size of the eye (retinoblastoma, glaucoma), identify the blue tumor (retinoblastoma, melanoma) or see a cupped optic nerve head (glaucoma) or tractional retinal detachment (diabetic retinopathy). Then you can show them the finer points under the microscope; all the time asking them to describe what feature you are pointing to and how it came to be. We are expanding the teaching collection to give the residents a broader depth of teaching.
9. Good luck and have fun.

Native Lenses

noreply@blogger.com (Mission for Vision) — Sun, 27 Aug 2006 17:32:00 +0000

A human lens submitted to the Ophthalmic Pathology Laboratory is generally the result of either a planned intracapsular cataract, planned extracapsular cataract without phacoemulsification, displacement of the lens posteriorly through a ruptured capsule, or complete zonular dehiscence posterior displacement.

In the later instance, one must also consider the reason for the surgery, obtain clinical information to consider the diagnoses with weak, ruptured, or diseased zonular fibers. A history of trauma, metabolic abnormality such as Marfan's syndrome, or pseudoexfoliation syndrome should be actively sought. A careful gross examination should be performed to find correlative abnormalities. Microscopic examination can be performed to attempt a more definite diagnosis.

Don't assume that native lenses are "gross only" specimens.

What is an express shunt?

noreply@blogger.com (Mission for Vision) — Tue, 08 Aug 2006 19:53:00 +0000

An express shunt used in glaucoma filtration procedures is illustrated in 2 views. Click to enlarge to see the internal conduits (numbers 1 and 2) and the phalange (arrow 3) to hold the metal device in place.

Synthetic Intraocular Lenses

noreply@blogger.com (Mission for Vision) — Sat, 22 Jul 2006 18:01:00 +0000

Intraocular lenses have a variety of shapes and sizes. The type of lens should be identified whether it is an anterior chamber or posterior chamber lens and if it is intact. Sometimes the haptics are amputated or the optic is transected. If there is attached tissue and the specimen was fixed in formalin, consideration should be given to sectioning the tissue. There may be infections in the residual lens capsule that are subclinical such as P. acnes or there may be evidence of pseudoexfoliation syndrome which may lead to displacement because of weak zonular support. The lens should be described carefully. In the images there is a 6 mm optic (number 1), two positioning holes (number 2) located in the haptics and there is attached tissue (number 3). The first image is a plate style and the one below has clear C shaped haptics. Both are 1 piece lens. The texture of the lens should be described, as silicone lenses are soft and flexible but polymethymethacrylate lens are hard and rigid. Careful observation will reveal that the lens above has fibrous tissue remaining from the residual lens and the other has iris tissue from an anterior tuck. These findings should be correlated with the clinical history to understand the reason for removal of the lens. The color of the haptics should be described for the posterior chamber style PCIOL below. This particular lens is flexible and made of silicone, which must be proven by attempting to fold the optic..

One important lens is the RESTOR lens which is recognized by its apodized diffraction rings. This is best seen with reflected back illumination. It is easily missed without careful examination. The lens should be returned to the patient for a rebate if appropriate.

Close inspection of intraocular lenses under the dissecting microscope sometimes reveals some very interesting findings. For example here is a lens that was removed after cataract surgery for displacement into the vitreous. Note the pits in the IOL follow a somewhat circular distribution (arrows 1). However, one disruption of the plastic appears to have occurred in the substance of the lens (arrow 2).

Turning the lens at a slight angle reveals that the defect is fan shaped similar to the energy burst from an exit wound. The patient has had Yag capsulotomy and frequently there are a few shots that are slightly off focus. These findings should be recorded, particularly the location of the defects to the visual axis, in this case off axis.

The anterior chamber style intraocular lens is easily recognized by the distinctive shape of the haptics. Each haptic joins the optic to form an "S" (white arrows). The anterior chamber lens is placed in the eye when a posterior chamber lens is contraindicated. The most common reason is posterior capsule rupture during cataract surgery. The remaining lens capsule and zonule are not sufficient to support the intra capsular sulcus placement of the lens. Anterior chamber intraocular lenses are generally removed because an inappropriate size was placed in the anterior chamber. The direct trauma to the posterior cornea or the iris may produce endothelial cell loss or iridocyclitis. It is important to look for any vitreous clinging to the wound.

Phakic intraocular lenses, like the Artisan lens, have become somewhat popular and their insertion is considered a cosmetic procedure. The lenses may have a modification so that the lens can rest against the iris. They are partially flexible. However, endothelial cell loss has been well documented at about 1.8%/year with these lenses. The lens may be removed after the onset of corneal edema and bullous keratopathy as in this case. The phakic intraocular lens may be recognized by their distinctive shape.

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Glaucoma valvular devices

noreply@blogger.com (Mission for Vision) — Sat, 22 Jul 2006 17:37:00 +0000

The filtration devices are equipped with a tube (at the top of the photo) that protrudes from a base plate (white in the photo) with holes.
The device in the image is a Baerveldt implant. The description should be accurate and any obstruction of the tube should be noted. If there is unusual or exuberant soft tissue on part of the valve it should be submitted for sectioning.

Ahmed valves are clear plastic devices that contain a membrane type valve composed of 2 flaps that seal an opening. In the figure below an Ahmed valve is photographed face on or from above. In the overview, five holes lie at the periphery of the valve for positioning with suture and the trapezoidal valvular mechanism. The tube (missing in this picture would be at the base of the arrow and the direction of flow would follow the arrow. The inner workings of the valve are seen after longitudinally making a cross section of the trapezoidal chamber (in the plane of the red arrow). The aqueous flows from right to left in the blue colored channel (arrow 1). The leaves of the valve are relatively long and indicated by number 2. When the pressure in the anterior chamber is high, the valve leaves separate creating an open valve as depicted. When the pressure is low the leaves of the membrane have natural elasticity that keeps them together seen in the photograph below (valve closed arrow 3). It is important to examine the region of the valve carefully in case of Ahmed valve failure for evidence of fibrous tissue or obstruction of the valve.

An express shunt is a considerably more simple glaucoma filtration device. Click here.

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Extraocular Muscle

noreply@blogger.com (Mission for Vision) — Fri, 07 Jul 2006 03:07:00 +0000

Rectus Muscle for ID only
How do you examine rectus muscles?
Rectus muscles receive a gross description after viewing under the dissecting microscope. The high magnification under the dissecting microscope is of the same order of magnitude (usually greater) than the low magnification at the compound microscope. During the gross examination the resident should try to identify tendon which is white and usually quite abundant with striations that run parallel to the apparent short axis (yellow arrow 2 in the figure above ). Muscle appears tan and is usually most abundant at one edge (white arrows above and arrowhead 2 in the figures). The length of the muscle removed needs to be documented accurately. Since the extraocular muscle is quite wide in vivo (about 9 mm) but the length resected quite small (usually 2-4 mm), the width that the pathologist sees in the gross is really the length of the muscle. This is especially evident when one sees the striations running in the short axis of the specimen. In the figure striations are obvious both in muscle and tendon (arrows 1 and 2) and therefore the muscle length is actually the distance between the arrowheads marked 3. In the body the long axis of the muscle is oriented parallel to arrow 3. An accurate measurement has ramifications medico-legally if the patient is under or overcorrected. So be careful!

How do you write up the gross description of an extraocular muscle?
Gross description:
Specimen 1, in formalin, labeled "right lateral rectus muscle" consists of one rectangular red and white fragment measuring 9 x 2 x 2mm in greatest dimensions. Under the dissecting microscope the external surface has fibrous white strands which are parallel and longitudinally oriented, adipose tissue, and blood vessels.
FINAL DIAGNOSIS: Muscle and fibrous tissue, "right lateral rectus muscle" (resection) - consistent with muscle and tendon

Very often a short length of muscle tendon is removed at its scleral insertion. Knowledge of the lengths of the extraocular muscle tendons is essential to determining whether it is likely that there is any muscle in the specimen at all. The medial rectus tendon is only about 3.8 mm in length and the lateral rectus tendon can be about 8 mm in length (see link for others). Without any muscle tissue the diagnosis would read:
FINAL DIAGNOSIS: Fibrous tissue, "right lateral rectus muscle" (resection) - consistent with tendon

When should you send an extraocular muscle for microscopic sections? Extraocular muscle may be sent for sectioning whenever it is necessary for the diagnosis. For example if a "slipped" muscle is suspected the surgeon wants to muscle or tendon versus a fibrotic pseudotendon. In this case microscopic sections are mandatory and a trichrome stain will highlight muscle fibers in red and collagen fibers (fibrosis and tendon in blue). Tendon appears as a compact lamellar array of fibers oriented in parallel.

Another example of an extraocular muscle that should be sent for sectioning is in cases of suspected chronic progressive external ophthalmoplegia. Here frozen sections for muscle enzymes, immunohistochemistry stains done in panels for specific proteins, and electron microscopy are necessary for the diagnosis. The surgeon needs to be wary of the needs of the pathologist to make the diagnosis, lest information will be lost without the proper fixation. The infamous ragged red fiber of CPEO is often not diagnosable in a specimen largely composed of tendon. A notable lawsuit was awarded against the surgeon that dumped a muscle biopsy into formalin in a patient with a suspected muscle dystrophy.

There are many other example when microscopy may be needed. Graves ophthalmopathy is associated with muscle enlargement, chronic inflammation, and Alcian blue mucopolysaccharide deposition.

SELF TEST FOR EYE SPECIMEN GROSS EXAMINATION

noreply@blogger.com (Mission for Vision) — Thu, 19 Jan 2006 02:47:00 +0000

Self Test
The purpose of this examination is to confront you with common problems that occur in the activities of processing specimens. Please use the procedure manual to find the answers to the questions.

Part I: Tissue Processing:

1. What are the purposes of inking a specimen in the laboratory?

2. An eyelid specimen was received without history. It was grossed in by the Ophthalmology resident who accessioned it, measured it and described it as an elliptical fragment with a nodule in the center. Without further ado he sent it for processing in a green cassette. The case settled out of court for an undisclosed sum. What was the nature of the lawsuit? If you were a lawyer for the plaintiff, what mistakes would you expose in this case?

3. Describe the steps involved in processing tissue for routine sectioning. Assume tissue is in a cassette in formalin. How does it get to the slide ? Describe all the steps including a brief description of all the solutions it must traverse.

4. Describe the difference between processing techniques for a cytospin and a cell block for cytology specimens.

5. On the cytospin requisition there is a box for unstained fixed slides. Give 3 possible uses for this unstained material other than histochemical stains.

6. Which of the following is the best preservative to prepare material when you are in doubt of the diagnosis and uncertain what tests you should get on a fragment of tissue?
a. 10% formalin
b .4% paraformaldehyde
c. 2% paraformaldehyde; 2% glutaraldehyde
d. rapid freezing in liquid nitrogen

7. Name the fixative that you would use for:
(a). a specimen for electron microscopy(b). routine paraffin embedding
(c). gene rearrangement work up(d). a conjunctiva specimen for bullous pemphigoid
(e). immunohistochemistry
8. Imagine you are a technician receiving a specimen at 5:30 AM from the technicon in paraffin for embedding. Each edge of the specimen is marked with a different color: yellow, green, orange, black, and blue. By convention, which edge will you put down in the very bottom of the metal mold unless otherwise instructed? Which dye was used to mark that edge?

Part II Specific Tissues
9. Conjunctiva Imagine yourself in your third year of residency preparing to remove a 4 mm pigmented lesion from the conjunctiva. Describe how you will prepare the tissue to send to pathology. How wide should your surgical margins be?

10. Conjunctiva The specimen is received as described above. How will you gross it in? Describe the way you will cut it, embed it, and what type of sectioning you will order from the technologist? Draw a diagram and show where you would make your cut. Show with arrows which edge or face it would be embedded on?

11. PKP Describe the steps for processing a penetrating keratoplasty specimen removed for aphakic bullous keratopathy (gross considerations and special fluids in processing).

12. Unknown corneal disease. Describe for a new disease, a suspected stromal dystrophy. It has never been described. What is the best way to maximize the information for processing? What is the most versatile choice for fixative?

13. A small corneal biopsy Dr. Cornea wants to do a corneal biopsy on an AIDS patient with a corneal ulcer. The clinical laboratory has reported S. epidermis on a scraping, but this ulcer has been progressively worsening for 2 months despite therapy. Dr. Cornea wants an answer in the shortest time because the patient is losing vision.
(a) What is the shortest turnaround time offered for a biopsy of this kind?
(b) What special stains are appropriate and when should you order them (at the time of sectioning or after seeing the H&E).
(c) The specimen is only .3mm in diameter, What can you do to make sure that the biopsy specimen will not be lost in processing.
(d) What type of sectioning will you order? (i.e deep cuts, step sections, etc....???)

14. Temporal Artery You received a 40mm segment of temporal artery. Describe how you will gross this in.
(a) What thickness will your slices of artery will you plan for the technician?
(b) Will you instruct the technician to slice along the long axis of the vessel or in cross section?
(c) How will you instruct the histology technologist to embed the specimen?(d) How many sections will you order initially? What will your instructions be to the histotechnologist?
(e) In the classic paper(s) on temporal arteritis, how big is a skip lesion? Give your answer in microns.

15. Eyelid A pentagonal resection of the eyelid is removed for a basal cell carcinoma at the medial canthus.
(a) Draw a theoretical diagram of what this might look like. On the diagram indicate how you will mark and cut the specimen.
(b) Indicate the plane of section of the tissue to obtain adequate margins.

16. Retina Dr. Retina is doing a retinal biopsy for a suspected acute retinal necrosis syndrome.
(a) What are the possible viruses ?
(b) List the ways commercially available to detect these viruses.
(c) What will you recommend to Dr. Retina for fixation and Laboratory tests?

17. Vitreous Washing The retina service sent you a cytospin specimen from a vitrectomy on a patient with AIDS. You examine the bottle record the volume, and order 1 pap stain cytospin slide and take it to the pathology lab.
(a) What diagnoses will you miss?
(b) What errors in judgment were made?
18. Foreign bodies You collect a specimen from the operating room that is labeled foreign body. It was removed during vitrectomy surgery. What is the appropriate procedure for handling, processing and storing this specimen.

19. Retinoblastoma The oncology service is going to send you an eye with a mass suspected of being a retinoblastoma. There is a family history of retinoblastoma. They want to know how to fix the eye before they send it to you. By the way they will want "genetic studies". The attending pathologist is ill. What will you recommend?
20. Lymphoma work up As a private practitioner you have a patient with suspected orbital lymphoma. You have decided to do an orbital biopsy in the operating room. When the specimen is removed, the nurse asks you what tests do you want? Your reply is "send it to pathology for a lymphoma workup". What specific things will be done in pathology as part of that work up? What is the best way to send the specimen (i.e. fixative, preservative etc.)
21. Blepharoplasty Describe the usual method in which we process blepharoblasty specimens? What are the exceptions to this method?

22. Autopsy eyes Describe how you would cut the autopsy eyes with the following features:
(a) a nevus in the periphery a 5’oclock.
(b) a surgically aphakic eye.
(c) a pseudophakic eye.
(d) an eye of a 9 month old baby.

23. The most common mistake.... You are cutting autopsy eyes in the standard fashion and the razor blade easily passes through the sclera above the optic nerve all the way to the lens. As you turn the knife to cut anteriorly, you hear a resounding “crunch”. Describe the gross features of the eye that if recognized would have allowed you to avoid this mistake?

24. What is the length of optic nerve that is to be left after preparing an autopsy eye?

25. When are residual optic nerve sections discarded?

26. How do you process epiretinal specimens?

27. What are the types of embedding bags? Which one do you use for very small specimens such as Descemet's membrane removal?

28. A Descemetectomy is performed and the specimen placed in an embedding bag. The histology lab staff report the bag is empty. The resident and the attending pathologist go over and examine the bag. There is indeed no specimen in the bag. From a quick observation of the bag the attending immediately and correctly deduces that the resident is the culprit. What mistake did the attending correctly predict was made in processing by the resident?

29. In the case above, where would you now look for the specimen? What special procedures are required on a daily basis by the residents to avoid lost specimens that slip out of cassettes?

INTERNAL (GROSS) EXAMINATION OF THE EYE

noreply@blogger.com (Mission for Vision) — Wed, 18 Jan 2006 21:50:00 +0000

Eyes may be incised for internal examination in other ways (see figure to the left) Usually this involves a posteroanterior incision in different meridians. For example, eyes that have had cataract extractions may be incised in a vertical plane nasal to the optic nerve in order to study the corneal wound and its complications. Surgical eyes with intraocular tumors must be incised in a plane to slightly intersect the tumor to facilitate study. The mechanics of the operation remain the same, the only difference being the degree of rotation of the incision from the horizontal meridian. All surgically aphakic and pseudophakic eyes will be opened with an incision at the equator in a coronal plane.

The "grossing in" or internal examination is carried out with a stereomicroscope and a deep Petri dish containing 50% ethanol; submersion of calottes in ethanol eliminates most reflections from the vitreous body and permits detailed examination of the inner surfaces of the eye. However, the eye must have been equilibrated in ethanol in the steps prior to the examination. The concentration of the alcohol in which the eye is examined must be precisely that in which the eye has been stored, otherwise the vitreous becomes "refractive" and examination of deeper structures is virtually impossible. Alternatively, the calottes may be stored overnight in the new alcohol (to achieve equilibration). Some important observations concerning the vitreous body (posterior vitreous detachment and the degree of synchysis), however, is best studied while suspending the calottes in air.

When manipulating the calottes in the Petri dish, care must be taken to grasp only episcleral tissue, extraocular muscles, or the like, since artifactual changes in the retina and uvea will occur readily with direct application of forceps. The inner aspects of the calottes are now studied systematically using low power (7x) and, if indicated high magnification. A variety of lighting techniques must be used, including: diffuse vs. focused, direct vs. oblique, direct vs. transillumination, etc...In effect the light is always moving, since many lesions can only be characterized completely with combinations of lighting and magnification.

Transillumination of the calottes can also be accomplished readily with the table light, during the internal examination. Subtle conditions of the vitreous can often be seen best with oblique Illumination that casts a shadow under the lesion. Subtle epiretinal or preretinal conditions deep in the calotte may only be seen by focusing the light on the adjacent retina as a secondary (indirect) light source. With proper examination techniques, gross examination of the optic nerve will yield almost as much information as time-consuming (and delayed) examination of microscopic tissue sections, even when special stains are employed. This is especially true of lesions causing demyelinization axon bundles. The light must be focused and oblique while examining the nerve on the submerged large calotte.

Findings from stereomicroscopic examination of the opened globe are drawn to scale on the gross sheet and labeled outside the drawings of the eye. (The semi-diagrammatic drawings of the opened globe on the sheet show the small (superior) calotte on the right and the large (inferior) calotte on the left. These drawings are three (3) times the normal size and all findings should be drawn to scale in their relative topographical location. The large calotte has been drawn as if it is slightly tipped upward in the front so that one can see the optic disc and the macula. One should record in the lower one-third of sheet only the important gross findings: if some categories are negative, indicate so. The bottom also constitutes a check-list for the examiner.

TECHNIQUE FOR EYE BISECTION

noreply@blogger.com (Mission for Vision) — Wed, 18 Jan 2006 21:45:00 +0000

Opening of the eye for internal examination is performed with a broad razor blade beginning posteriorly slightly above the optic nerve and cutting anteriorly in the horizontal (transverse) plane. The fixed adult lens is too hard to cut through without dislocating it and injuring adjacent structures in the anterior segment. Accordingly, the incision anteriorly must continue in the same plane but extend around the lens, leaving it in situ in the inferior calotte when the superior calotte (cap) is removed. For the right-handed person, this best accomplished as follows:

(1) The eye is held with the cornea down (against the table surface) and with the 12 o'clock position to the right; beginning slightly to the right of (i.e. above) the optic nerve, the incision is made with broad heel-to-toe strokes of the razor blade parallel to the 3 and 6 o'clock meridians (using the long posterior ciliary artery and nerves as a guide) to the level of the lens.
(2) Leaving the blade in position, the eye is repositioned with the incisional plane now parallel to the table surface and with the cornea facing to your right; the blade is then regrasped with the thumb and index finger at either end, and is slightly withdrawn to continue the incision around the lens.

(3) The incision is continued in the same plane by rotating the eye around a vertical axis in a counter-clockwise direction, while making oscillating strokes with the blade against the lens surface and maintaining the plane of the incision to pass precisely through the upper margin of the pupil.

(4) When the encircling incision is complete, the eye is repositioned with the 12 o'clock meridian facing up and the cornea facing away from the operator; with thumb forceps in the left hand, the episcleral tissue of the small calotte is grasped near the edge of the posterior incision and the calotte is slowly elevated.

(5) While raising the posterior aspect of the small calotte, the lens is held in place with the razor blade in the right hand by making a shallow bite into its posterior capsule; if the incision has been properly made, only the zonular attachments to the superior aspect of the lens with remain. These zonular attachments will rupture without incident as the small calotte is separated and at this time any uncut vitreous (especially within vitreous base) can be severed with a sweeping stroke of the blade.

The positions of the blade are shown as it cuts arounds the eyes using the step described above.

DETAILED TECHNIQUES FOR EVALUATION OF SURGICAL AND AUTOPSY EYES

noreply@blogger.com (Mission for Vision) — Wed, 18 Jan 2006 20:34:00 +0000

Detailed Techniques for Evaluation of Surgical and Autopsy Eyes

General
Autopsy eyes provide a unique experience for residents. Here you will be able to correlate clinical findings with direct visualization of lesions, and enhance understanding of dimensions, anatomical relationships and structural alterations. The techniques demonstrated here were developed by my predescessors Drs. Ray Allen and Robert Foos. Some procedures have been modified slightly.

The following eyes / bottles should never be removed from the autopsy area:
-unlabeled bottles. Notify autopsy technical staff
-bottles containing more than one pair of eyes . Morgue staff should be notified
-bottles or eyes with a definitive or questionable diagnosis of unknown dementia
e.g. CJD
After at least 48 hours in fixative (10% formalin buffered with marble chips), the eyes are washed for 10 minutes twice in tap water and then placed in 50% ethanol. They will be stored in 50% ethanol thereafter. In a detailed study by a former JSEI resident Michele Lim, two days of equilibration was found to be necessary to re-expand the eyes; the eyes shrink in formalin.

Gross Work-up:

Preparation. Findings are recorded on a Gross Description Form, graphically and descriptively. Note: select the proper type of gross description sheet that reflects and the proper cut for the eye (standard with native lenses vs. coronal with pseudo- or aphakia). Always double check the pupil exam to confirm the proper form and cut for each eye. The eye is cleaned with forceps and blunt scissors. Beginning anteriorly, cut off the bulbar conjunctiva, leaving at least 3 mm in width on the globe if possible. Then, the rectus muscles are removed at their insertions. Episcleral tissue is stripped from anterior to posterior with thumb forceps and then severed. The superior and inferior oblique muscles are identified and removed leaving approximately 3 mm of tendon attached to the eye. The optic nerve is incised transversely with the razor blade in a coronal plane, leaving 2-3 mm attached to the eye. When removing the optic nerve, be sure to use a single continuous stroke in order that the transversely-cut surface will be smooth and interpretable. . The cut surface of the optic nerve will be examined later with oblique illumination. You should leave some episcleral tissue around the optic nerve at its entrance since the short posterior ciliary arteries enter here and may be important in the diagnosis of vascular disease.

External examination is done initially with the unaided eye and with a stereomicroscope when suspicious lesions are encountered. The eye should be reexamined to confirm the natural lens is present. Examination should proceed anatomically and include the conjunctiva, cornea, sclera, vortex veins, optic nerve, etc. Findings should be recorded on the gross sheet.

First you will need to determine which eye is the right and which is the left. This can be done by examining the muscular insertions. The globe is measured using the vernier calipers to the nearest one-tenth of a millimeter. Using the inferior oblique muscle as a handle, one can measure all three ocular diameters without changing grasp. Similarly, using the pointed ends of calipers, one can measure the optic nerve and the diameters of the cornea and pupil without re-grasping the handle. These measurements are recorded in the appropriate place on the gross sheet.

Transillumination
Transillumination is accomplished in our laboratory using the examination light fitted with an aluminum box to hold the eye. The box contains two round holes: one 34 mm in diameter to fit over the end of the Illuminator, and the other 20mm diameter (adult size) to hold the eye while transilluminating. Visualization is facilitated by extinguishing the room lights. Transillumination opacities can be marked with a tissue marking pen, a small amount of well placed ink etc.

Immunohistochemistry Examples of Use

noreply@blogger.com (Mission for Vision) — Wed, 28 Dec 2005 15:48:00 +0000

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).
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

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.

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

noreply@blogger.com (Mission for Vision) — Wed, 28 Dec 2005 15:18:00 +0000

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

noreply@blogger.com (Mission for Vision) — Wed, 28 Dec 2005 13:27:00 +0000

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

noreply@blogger.com (Mission for Vision) — Wed, 28 Dec 2005 00:57:00 +0000

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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:35:00 +0000

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

R and S cases are similar but EY cases 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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:33:00 +0000

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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:32:00 +0000

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.

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)
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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:21:00 +0000

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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:20:00 +0000

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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:17:00 +0000

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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 14:16:00 +0000

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

noreply@blogger.com (Mission for Vision) — Tue, 27 Dec 2005 13:56:00 +0000

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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GROSS DESCRIPTIONS

noreply@blogger.com (Mission for Vision) — Sat, 22 Oct 2005 22:49:00 +0000

Templates for cases are complete with history, gross, microscopic and diagnosis. The following are examples of some gross only cases with an explanation of the goals in examination:

Rectus Muscle for ID only
Rectus muscles receive a gross description after viewing under the dissecting microscope. The high magnification under the dissecting microscope is of the same order of magnitude (usually greater) than the low magnification at the compound microscope. During the gross examination the resident should try to identify tendon which is white and usually quite abundant with striations that run parallel to the apparent short axis (arrow 1 in the figure). Muscle appears tan and is usually most abundant at one edge (arrowhead 2 in the figure. The length of the muscle removed needs to be documented accurately. Since the extraocular muscle is quite wide in vivo (about 9 mm) but the length resected quite small (usually 2-4 mm), the axis of the apparent width of the gross specimen corresponds to that of the length in the orbit. This is especially evident when one sees the striations running in the short axis of the specimen. In the figure striations are obvious both in muscle and tendon (arrows 1 and 2) and therefore the muscle length is actually the distance between the arrowheads marked 3. In the body the long axis of the muscle is oriented parallel to arrow 3.

Gross description:
Specimen 1, in formalin, labeled "right lateral rectus muscle" consists of one rectangular red and white fragment measuring 9 x 2 x 2mm in greatest dimensions. Under the dissecting microscope the external surface has fibrous white strands which are parallel and longitudinally oriented, adipose tissue, and blood vessels.
Dx: Muscle and fibrous tissue, "right lateral rectus muscle" (resection) - muscle and tendon

For more detail see this link.

Intraocular Lenses
An accurate diagram of the IOL should be drawn out. Note the type of lens if you know it; otherwise make your description accurate enough that one could draw a picture from your words. Note any tissue adherent to the lens and process tissue for appropriate histologic sections. Describe the shape, size, and color of optic and note any defects. The flexibility of the lens gives a clue whether it is made of polymethylmethacrylate (PMMA is hard) or silcone (soft). This should be indicated. Note the color, shape, size, and position of haptics (L-shaped, closed loop, etc. for anterior chamber lenses; C-shaped, J-shaped for posterior lenses). Note evidence of broken / amputated haptic or transected optic (often done to get the lens out of the eye). Note positioning holes, their size and position. Intraocular lenses are stored for 6-9 months! Restor Lenses should be saved for possible return to the patient for rebate.

Gross Description:
Specimen 1, in formalin, labeled "IOL, OS" consists of a clear synthetic hard device measuring 13 mm in overall length with a round clear optic that measures 6 x 5 x 1 mm. There is a positioning hole in the periphery. The lens is a 3 piece style; two blue J-shaped haptics extend from the periphery of the optic 180 degrees apart. The optic shows some pitting on the posterior surface.

Diagnosis: Synthetic device, "IOL, OS" (removal) - synthetic intraocular lens, posterior chamber style

Lenses (native)
Cataractous lenses are carefully examined with a stereomicroscope. It is important to note the color (yellow, yellow-white, or brunescent), the degree of opacification, and the diameter of the lens. You can calibrate the degree of opacification by trying to read newsprint through the lens. The lenses are then shelved for temporary storage (3 months), then discarded. No histologic sections need to be taken unless specifically requested or if there is an interesting finding such as a congenital anomaly, infection (like rubella, fungus, or bacteria) or tissue attached to the lens that would be important to document.

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SURGICAL IMPLANTS, METALLIC FOREIGN BODIES, HARDWARE

noreply@blogger.com (Mission for Vision) — Sat, 22 Oct 2005 22:48:00 +0000

Unless otherwise ordered, the tissue or material for gross examination (identification) will be stored temporarily (i.e. 3 months) and then discarded. Exercise caution in the following instances:

Despite the instructions "ID ONLY" by the surgeon the pathologist must use his own judgment. If a lesion of possible clinical significance is encountered, it must be submitted and processed for microscopic diagnosis.

If a piece of tissue, "hardware", or the like has potential medico-legal significance (e.g. intraocular foreign body), the specimen must be marked "store permanent". Hardware of this type are stored indefinitely in either fixative, alcohol, or dry in a plastic bag. ALL NON-METALLIC AND METALLIC FOREIGN BODIES SHOULD BE "STORED". Bullet fragments are routinely requested by the police months to years after their removal from the eye. All metal fragments must be tested with a magnet. The report must indicate whether the fragment is magnetic. While the storage is not “permanent” it should be in keeping with institutional standards compliant with state regulations.

A procedure sometimes used by the surgical pathologist is the "block only" category. Such specimens are embedded in paraffin, plastic, etc. and stored as such without microsectioning. This might be used for example when one had a large tumor of possible future teaching value or again, tissue of potential medico-legal significance. When blocked in paraffin or plastic the tissue can be stored virtually "forever".

If you choose a gross only work up for a piece of tissue, your description should include the phrase "under the dissecting microscope" and should contain enough information to indicate you accurately described the tissue. For example, "tan tissue with parallel longitudinally oriented fibers that merge with tendinous tissue" describes muscle. If you cannot describe the tissue in this way, it is probably not normal and microscopic sectioning is warranted. Cases with a history of both CPEO (chronic progressive external ophthalmoplegia) and ptosis repair should be brought to the attending pathologist's attention immediately, as the tissue will need to be processed for electron microscopy as well as routine light microscopy.

Gross descriptions should be accurate and give enough detail so that the reader could diagram the specimen with measurements from your report. Be sure to detail specific margins that were inked with different colors if appropriate. Document if the specimen was placed in an embedding bag. Clearly indicate when specimens from 2 cassettes, each inked a different color, are combined for embedding in one paraffin block (we do this occasionally in cases such as Mullerectomy specimens for efficiency).

SCLERAL BUCKLE: Scleral buckling devices are removed from cases of repaired retinal detachment when they extrude and/or become infected. A complete and accurate description of the size and shape of each piece as well as a description of any bands or grooves should be reported. A microscopic examination is not performed and the specimen is not processed but stored temporarily. See the example below.

CLINICAL INFORMATION: retinal detachment repair 2002, exposure
FINAL DIAGNOSIS: Synthetic device "right eye” (removal)- consistent with scleral buckling device
MICROSCOPIC EXAM: Not performed.
GROSS DESCRIPTION: Specimen #1, labeled “scleral buckle from right eye” consists of a translucent soft flexible synthetic bandshaped device measuring 16 mm in length x 2.5 mm in width and x .5 mm in thickness. There is a groove in the center of the band that measures 1 x .5 mm.

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BIOPSIES-GENERAL INSTRUCTIONS FOR PROCESSING

noreply@blogger.com (Mission for Vision) — Sat, 22 Oct 2005 22:47:00 +0000

ACCESSIONING CASES
Cases are accessioned in our system with a letter S if the biopsy originated from UCLA or JSEI and R if it is outside this facility. The resident must assign the R or S number according to the location. Some cases will be delivered from UCLA Pathology Outreach with R numbers already assigned. These cases have already been accessioned and will keep the R number assigned by outreach. The resident simply needs to log them in the book to complete the accessioning. The basic tenet is that every specimen that is received by the lab is entered in the computer and in the log book by the resident.

BIOPSIES

The work-up of surgical biopsies is only begun when adequate clinical information is available; the work-up contains 3 parts: a narrative gross description, a narrative microscopic description, and a diagnosis. Be sure to obtain adequate clinical information and type everything listed clinically on the requisition form. If the information on the requisition form is incomplete then request history by emailing the assigned physician using your UCLA email account. Alternatively, you can call them or review the patient's chart in the originating physician's suite with their permission. Abbreviations should be typed out as complete words. Mistyped or mis-spelled words can followed by [sic] to indicate the mistake. Some use [sic] to follow the corrected error. Either usage seems to acceptable although most grammar texts use to follow the error left in place.

Microscopic Examination.
Microscopic examination is performed on most surgical specimens (except most gross only cases) and some autopsy eyes. The residents will learn to evaluate microscopic sections of most common ocular disorders during this rotation.

Microscopic descriptions should be written in a concise and systematic manner to include, sequentially, the architecture of the lesion and the cytologic features. Do not begin your description until you have thoroughly read up on your case. 95% of the diagnoses can be found on line at MissionforVision.org or in the books referenced in this manual. Get these references from the Biomedical Library or the bookstore.

For a simple case with one block and one section, begin your description with "Sections show". In this case you do not need to indicate a location or specimen since there is only one. For cases involving more than one specimen or cases in which more elaborate sectioning was done, you may begin by "Sections of the specimen labeled ‘x’ show” or Step sections through the block of the specimen labeled ‘x’show or Step sections of the specimen labeled ‘x’ show. Tailor your report to the way the specimen was sectioned. The term "sections show" is applied to a simple case where a ribbon of more than one section at the same level was taken of the biopsy. "Step sections show" refer to a case where levels were taken at 250 micron intevals to more thoroughly sample the tissue, e.g. deeper every 50th section x 5). "Step sections through the block" is used when sections were taken at 250 micron intervals all the way through the paraffin block exhausting all remaining tissue. This is often used when a neoplasm was strongly suspected and needs to be excluded. Include a description of all special stains that were performed in your report. If a case consists of more than one specimen, each specimen may be described separately. While specimens with the same microscopic findings may be described collectively, diagnoses are listed separately under "FINAL DIAGNOSIS" on our reports for each specimen (see example). Usually the main feature is detailed first (such as focus of cancer, ulceration, or the like), and then the secondary features are described. Occasionally, one may finish the description with pertinent negative statements (e.g. "granulomatous inflammation is lacking") or special features (e.g. "tumor is present in the lateral surgical margin"). In general pertinent negatives are not needed. Each specimen requires its own line and separate heading for "FINAL DIAGNOSIS". There are no exceptions. If a case has 23 specimens that all show the same thing, it may require a single microscopic description but there must be 23 listed separately each beginning with the heading "FINAL DIAGNOSIS" to include the respective anatomic sites. Be sure to comment on margins of all tumors.

Do not expect to be able to write perfect microscopic descriptions in the beginning of your rotation. Please always check your work to eliminate errors of omission, grammar, and orthography. You may use the descriptions in the work file or glossary as a guide for your gross and microscopic descriptions. You should expect that most of your work-ups will be significantly modified. The microscopic work up will reveal areas of ignorance, and is an excellent way for the pathology faculty to plot your progress through the rotation. Expect that you will be asked to rewrite the descriptions if they contain errors.

Format for Final Diagnosis.
In general the procedure for final diagnosis is the same as that for the specimens requiring the gross. The tissue type is that which you see under the microscope. So if there is keratinized stratified squamous epithelium with hair shafts and adnexal structures the tissue is "skin".

Next in quotation one puts either exactly the wording on the specimen label or that which conveys accurately the type of specimen submitted and laterality from the requisition. Next the procedure rendering the speciem is put in parentheses. Finally the diagnoses are given.

FINAL DIAGNOSIS: Tissue type, "specimen label on the bottle" (surgical procedure)-1st diagnosis
-additional diagnoses,from this first specimen

FINAL DIAGNOSIS: Tissue type, "specimen label on the bottle" (surgical procedure)-1st diagnosis
-additional diagnoses,rom this second pecimen
or e.g.:

FINAL DIAGNOSIS: Skin, "left lower eyelid" (biopsy)- seborrheic keratosis

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