Normal Processing of Muscle and Nerve Specimens and After Hours Policy

***Remember to complete and enclose the CONSULTATION REQUEST FORM and the CLINICAL HISTORY FORM with all specimens.***

I. Muscle

A. Flow of normal processing 
Both in-house and outside specimens are received and processed by Histology (UPMC Clinical Lab Building, 2nd Floor Room 2031, 3477 Euler Way, Pittsburgh, PA 15213– call 647-6503 or 647-7660) during routine hours (6:00 AM – 2:30 PM). Specimens inadvertently sent to neuropathology or the Pathology PAs should be sent to Histology. 

B. Specimens from within UPMC Presbyterian and Local Specimens received within 2 hours
Ideally, the surgeon will obtain three 1 x 1 cm pieces of muscle obtained along the longitudinal orientation of the muscle fibers away from tendon insertions with at least one piece being placed on a muscle clamp. The tissue should be submitted immediately in the fresh state on a saline-moistened (not soaked) Telfa pad in a closed container. 

If the muscle is coming from a local institution within 2 hours, the specimen container with the muscle should be placed in a cooler containing cold (not frozen) ice water and delivered ASAP to the Histopathology Laboratory at the UPMC Clinical Lab Building.

C. Specimens Delivered from Outside the Local Area 
If the muscle specimen will not be delivered within 2 hours, part of the specimen (approximately 1 cm x 1 cm x 1.5 cm) should be snap frozen on site in isopentane or methylbutane cooled by liquid nitrogen and shipped on dry ice in a Styrofoam container via overnight mail. It is crucial the specimen remains frozen. If the specimen is mounted in gum tragacanth on a chuck, the tissue should be frozen before it sinks down in the gum. In a separate container maintained at room temperature, send several 1 mm x 1 mm fragments in a small vial of EM fixative (glutaraldehyde 3% in phosphate buffer) and send the remaining tissue (approximately 1 cm x 1 cm x 1 cm) in formalin after stretching the tissue over a piece of tongue depressor using sutures to tie each end to assist in proper orientation upon arrival at the Medical Center. (If the surgeon provides you with "free" and clamped specimens, use the clamped specimens for EM and formalin-fixative and freeze the free ones).

Depending on the number of muscle pieces submitted, the following procedures are performed by the Histology technicians:

  1. If there are two clamped specimens and one “free” specimen, one clamped and one free specimen are frozen in methyl butane, cooled by liquid nitrogen after mounting on a chuck using gum tragacanth. The other clamped specimen is submitted in formalin for paraffin embedding (cross and longitudinal section), except for a small piece that is placed in Karnovsky’s fixative for electron microscopy.
  2. If there are two pieces, only one piece is frozen. If there is only one piece, the specimen is divided for the above procedures. Two sets of glass slides are made from each specimen. If more than one site is sampled, then only one set of slides per specimen is made. Routine stains performed on cryostat sections are the following:
  • H&E
  • Gomori trichrome
  • PAS
  • Oil-red-O
  • ATPase pHs at 9.4, 4.6, 4.3
  • Congo red
  • Esterase 
  • SDH
  • Cox
  • MHC-1 & Negative

     3. Two H&E-stained paraffin sections (both cross-section and longitudinal) are also made.

D. After hours processing of muscle biopsy specimens

  • If specimens are received after routine hours, a single piece of muscle tissue is frozen in the deep freeze.
  • If there is more than one piece of tissue, one of these pieces is placed in formalin and the additional piece(s) is frozen.
  • Histology must be notified (647-6503) the next morning so that the specimen can be thawed out and snap-frozen, and then processed as above in Section I A.

II. Nerve

A. Flow of normal processing -- During routine hours (6:00 AM – 2:30 PM), both in-house and outside cases are processed by Histology (647-6503 or 647-7660).  Click here to view instructions for surgeons.

  1. If a 2 cm. or greater length of nerve is received, the specimen will be split in the following manner:

- 1 cm. is pinned on dental wax and placed in formalin overnight and then split longitudinally in half the next day, and then processed for teased fibers.
- 1 cm. is kept in formalin for paraffin embedding.
- Approximately 0.3 cm. are placed in Karnovsky’s fixative for plastic sectioning.

                 2. If the specimen is less than 2 cm., teased fibers will not be assessed.
                 3. Routine staining performed on paraffin sections include the following:

- Two H&E
- Two elastic trichrome
- One Congo red
- One CD3 by immunohistochemistry

     4. Approximately two sections per slide should be cut and the specimens should be embedded in paraffin for both cross-section and longitudinal processing.

B. After-hours processing -- If the specimen is received after hours, it should be kept in formalin in its entirety. If possible, the nerve should be stretched over a tongue blade and then transported to the Histology Lab the next morning. (Call 647-6503 or 647-7660 for pick-up.) 

C. Instructions for shipping and receiving outside specimens
Nerve biopsies should be fixed in a 10% buffered formalin and the nerve should be left intact-- (at least 2 cm in length). DO NOT CUT to allow for tease fiber preparation and paraffin embedding. If ultrastructural analysis is required , 0.5 cm of nerve must be submitted in 3% glutaraldehyde in phosphate buffer. If the specimen arrives within one hour, it can be submitted fresh, on a saline-sealed Telfa pad in an airtight container.