Diagnostic testing for muscle disease Flashcards
Non-specific markers of muscle damage
- Creatine Kinase (CK) and Aspartate Aminotransferase (AST)
- These enzymes are normally found intracellularly within muscle, their appearance in the systemic circulation represents rupture of cells→damaged tissues
- Measured in terms of ACTIVITY not CONCENTRATION (same applies to hepatic enzymes)
When does CK activity peak and fall?
- peak early (6 hours)
- return to normal quickly (24 hours)
When does AST activity peak and fall?
- increase more slowly (18-24 hours)
- return to normal slowly (20 days)
Dynamic creatine kinase activities
- In some horses with clinical myopathy, resting enzyme activities might be within reference ranges
-> systemic enzyme response to a bout of sub- maximal exercise (15-20 minutes of trotting on the lunge is sufficient for most horses) - Take Sample 1 before exercise
- Exercise the horse for 15-20 mins
- Leave to rest
- Take Sample 2 6h after the initial sample
Significant finding:
= Sample 2 ≥ 2 x Sample 1
- (even if both are within the reference range)
Why is pigmenturia a (non-specific) sign of muscle damage?
Cell rupture→myoglobin into circulation→filtration by kidney→PIGMENTURIA
How is urine a useful muscle marker?
- The presence of dark red urine should alert the clinician to potential myopathy but needs to be differentiated from diseases leading to haematuria (blood contaminated urine)
– Usually by simply measuring muscle enzyme activities - Myoglobin is highly nephrotoxic – indication for fluid therapy
Indications for muscle biopsy
- Repeated bouts of clinical disease, poorly managed
- Cases with doubling of baseline CK activity
- Suspicion of underlying storage myopathy
- If seeking a definitive diagnosis
- Poor performance work-up with suspicion of muscle disease
Contraindications for muscle biopsy
- The diagnosis is clearly evident (atypical myopathy)
- Condition is successfully managed with husbandry changes and symptomatic treatment (equine rhabdomyolysis)
Muscle biopsy procedure for exertion myopathies
(biopsy of the semimembranosus muscle [part of the hamstring group])
- Contact your laboratory (RVC) with at least 48 hours notice
- Sedate horse, restrain in stocks if possible
- Inject local anaesthetic subcutaneously – avoid injecting muscle!
- Aseptically prepare the skins with chlorhexidine and surgical spirit
- Make a linear incision (4cm) and retract skin with gelpi retractors
- Make two parallel incisions (3cm) into the muscle tissue 1cm apart
and 1cm deep, following the direction of muscle fibres - Join your two incisions at the proximal end and then slowly
undermine the length of your incision, eventually transecting the
distal end - Place the sample on damp gauze while you close dead space and
suture the skin - Divide the sample in half – one part should be stretched onto a piece
of card and preserved in formalin, the other half should be sent fresh.
Lab testing from biopsies
- Periodic acid Schiff (PAS) staining for PSSM
- H&E staining suggestive of RER
- Desmin stain for myofibrillar myopathy
Periodic acid Schiff (PAS) staining for PSSM
- Amylase digestion first removes all normal accumulations of
glycogen - Dark staining cells from affected horse denoting
abnormal accumulations of resistant glycogen
*PAS stains glycogen and other sugars very dark
* But as amylase has first been added, it will only stain those resistant to amylase
* Amylase resistance is permanent (for life)
Haematoxylin and eosin (H&E) staining suggestive of RER
- Internalised nuclei (should be peripheral), marked variation in fiber
sizes, infiltration of macrophages - Caffeine sensitivity is also a feature – performed on fresh samples
– Muscle that is hyperreactive to caffeine (i.e. it contracts quite dramatically when exposed to caffeine) is a finding consistent with RER
Desmin stain for myofibrillar myopathy
- Abnormal accumulations of desmin in an affected horse
Genetic testing for muscle diseases
There is a genetic basis to many myopathies, but many multiple genetic foci, and few tests are validated for clinical use
PSSM1 – validated genetic testing available
* mutation in glycogen synthase (GYS-1) gene
* Widely available, samples include blood and hair plucks (including
follicles)
PSSM2 – many unvalidated tests available
* There is no evidence for the use of genetic testing with other storage
myopathies
Atypical myopathy toxin testing
Toxin testing can be performed on blood samples or samples of vegetation
Hypoglycin-A (HGA) and its toxic metabolite methylenecyclopropylacetic acid (MCPA)
* Principle metabolite causing AM
* Submit whole blood or spun serum sample (best sample = serum, 2nd best = spun heparinised plasma)
* Send next day postage
