Wednesday, January 18, 2006


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?


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.


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

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 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. Simple examination reveals a white reflex if the native lens is in place; fixation results in precipitation of lens crystallins. A pseudophakic eye (plastic or silicone lens) and an aphakic eye (no lens) will appear clear the pupil will be dark. This is even more evident with proper transillumination. 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 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.