As Run By The UCSD Comparative Neuromuscular Laboratory:

 

What type of sample do we need to submit for a 2M antibody (masticatory muscle myositis) or acetylcholine receptor (AChR) antibody (myasthenia gravis) test ? How long does it take to get results back ?

Send 1-2 ml of serum (not whole blood) in a red top or serum separator tube by an overnight service. In the hot summer months it is good to include a cold pack, but in the cooler months the cold pack is not necessary. The turnaround time is 5-7 working days (does not include week-ends or holidays) from the day the sample arrives.

Can medications have an effect on the results of antibody testing for masticatory muscle myositis (2M antibody) or myasthenia gravis (AChR antibody) ?

Treatment with corticosteroids (prednisone, dexamethasone, and triamcinoline) at immunosuppressive doses (1mg/lb/day for longer than 7-10 days) can therapeutically lower antibody titers and result in a negative test for both myasthenia gravis and masticatory muscle myositis. It is ideal to collect the serum sample PRIOR to beginning corticosteroid therapy and store in the freezer for possible future testing.  Anticholinesterase drugs such as Mestinon will not have an effect on antibody testing.

What if the sample is lipemic or hemolyzed ?

For the antibody tests in general (2M and AChR antibody), lipemia and hemolysis do not affect the results of testing.  However, if a sample is severely lipemic (looks like cream) or hemolyzed (looks like port wine), please draw another sample.

Does the patient have to be fasted before drawing a blood sample ?

It is always best to have a non-lipemic blood sample, so an overnight fast is suggested.

If the serum sample is less than 1-2 ml can testing still be performed ?

The 2M and AChR antibody tests can be performed with as little as 500 µL of serum. However, package the sample well to prevent any leakage during shipping.

If it is necessary to draw a serum sample on Friday (laboratory is closed week-ends) or cannot be shipped the same or next day, how should the sample be stored ?

Serum samples can be stored for a week refrigerated. For longer storage, the sample should be frozen. When a previously frozen sample for 2M or AChR antibody testing is shipped, it does not have to remain frozen. In the summer months a cold pack should be included.

How long are serum samples stored in the laboratory ?

Serum samples are stored for 6 weeks then discarded.

How sensitive is the 2M antibody test ?

The 2M antibody test is an ELISA based test and is very sensitive and specific. A negative antibody titer may occur in end-stage masticatory muscle myositis (muscle biopsy needed), in polymyositis (muscle biopsy needed) or if the dog has been on immunosuppressive dosages of corticosteroids for longer than 7-10 days.

How should muscle samples be shipped ?

Two muscle biopsies should be collected from each site. Wrap the largest biopsy specimen (0.5 X 0.5 X 1.0 cm) in a saline dampened (NOT DRIPPING WET) gauze sponge and placed into a dry water-tight container (for example, a 10 ml red top tube) and keep cold until shipped. A second smaller biopsy specimen from next to the original biopsy should be placed directly into 10% buffered formalin. Place all muscle specimens into a Styrofoam box with cold packs and ship by an overnight night service so the samples arrive into the laboratory within 24-36 hours of collection. It is critical that the muscle biopsies remain cold so we receive the tissue in good condition.  A special collection technique is required for biopsy of a masticatory muscle.

Will corticosteroids or other medications affect the results of a muscle biopsy ?

Chronic corticosteroid therapy will result in type 2 muscle fiber atrophy, which can be determined in the biopsy sections by fiber typing. Other pathological changes should not be affected.

What collection methods are necessary for testing plasma lactate and pyruvate concentrations ? 

For plasma lactate analysis, blood samples should be collected pre-exercise, post 10 minutes of exercise, and after 30 minutes of rest into sodium fluoride/potassium oxalate (grey top) tubes. The plasma for lactate analysis should be separated, frozen and shipped on dry ice.

For plasma pyruvate analysis, an 8% solution of perchloric acid (PCA, Perchloric acid 60% Fisher Scientific A-228-6) or tricholoracetic acid (TCA) is needed for collection. An 8% solution is made by diluting 13.3 ml of 60% PCA or TCA to 100 ml in distilled water. This solution is stable if refrigerated for 6 months. A compounding pharmacist may be able to provide the 8% solution of either PCA or TCA. The solutions are stable if refrigerated for 6 months. Immediately after blood collection, add exactly 2 ml of blood to 2 ml of 8% PCA or TCA. Mix and place on ice for 10 minutes to insure complete precipitation of protein. Centrifuge, pull off the clear supernatant, and then freeze supernatant. The frozen samples should be shipped on dry ice by an overnight delivery service. Pyruvate is unstable unless plasma is collected by this methods.