Laboratory diagnosis of muscle diseases

LABORATORY DIAGNOSIS
OF MUSCLE DISEASES

• Muscle is a specialized type of C.T
• Three types of muscle tissue can be identified

on the basis of structure, contractile
properties, and control mechanisms into:
1. Skeletal M.
2. Smooth M.

3. Cardiac M.

1) Skeletal muscle:

• Attached to the skeleton.
• Muscle fibers are tubular, multinucleated, and

striated.
(Striation from thick myosin with thin actin myofibres)
• Voluntary in action.

2) Smooth muscle:

• An integral part of many internal organs

(stomach, intestines, U.B, uterus, B.V, and airways)
• Muscle fibers are spindle-shaped, uninucleated
and non-striated
•Involuntary in action, controlled by A.N.S &
hormones

3) Cardiac muscle:

• Forms the heart.
•Muscle fibers are tubular, branched,

uninucleated, and striated.
•Involuntary in action, determined by specialized
pace maker cells in the SAN

Physiology:
• All three muscle types use the movement of
actin against myosin to create contraction.
• All skeletal muscle and many smooth muscle

contractions
are
facilitated
neurotransmitter acetylcholine

by

the

• Proper muscle contraction needs contractile

myofibrils (actin & myosin) together with
calcium and ATP.

DISEASES OF THE MUSCLE
I) Cardiac muscle diseases: a separate entity.
II) Smooth muscle diseases:
A. GIT:
 Achalasia
 Gastroparesis
 Hypertrophic pyloric stenosis
 Diffuse Esophageal Spasm
 Gastro-esophageal Reflux Disease
 Chronic idiopathic intestinal pseudo-obstruction (CIIP)
 Hirschsprung disease
 Anal fissure
 Hemorrhoids
 Irritable bowel syndrome

B. Urinary bladder:
 Impaired detrusor contractility or detrusor areflexia
 Detrusor hyperflexia
C. Lung:
 Asthma
 Emphysema
 Bronchiactasis

D. Blood vessels:
 Aneurysms
E. Myometrium:
 Fibromyoma

III) Skeletal muscle diseases:
 Acquired myopathy
A) Inflammatory myopathy:
 Polymyositis
 Dermatomyositis
 Collagenic diseases
 Malignancy
 Viral, bacterial & parasitic infection
 Sarcoidosis
B) Metabolic and endocrine myopathy:
 Cushing’s syndrome
 Thyroid disease
 Calcium metabolism disorders
 Hypokalemia
 Ethanol
C) Myasthenic disorders:
 Myasthenia gravis
 Lambert-Eaton Myasthenic myopathic syndrome

 Genetically determined myopathies

A) Muscular dystrophies:
• Duchenne muscular dystrophy
• Facioscapulohumeral dystrophy
• Limb girdle dystrophy
B) Myotonias
C) Channelopathies (periodic paralysis)
Hypokalemic, hyperkalemic or normokalemic periodic paralysis
D) Specific metabolic myopathies:
• Glycogen storage diseases
• Fatty acid metabolism defects (Carnitine deficiency)
• Malignant hyperthermia
• Mitochondrial diseases (ragged red muscle fibres)

DIAGNOSIS OF MUSCLE DISEASES
 Clinical
 Electromyography (EMG)
 Imaging
 Laboratory investigations

 Muscle biopsy
 Special tests

LABORATORY INVESTIGATIONS OF MUSCLE DISEASES
Preliminary investigations
II.
Enzyme activities
III.
Proteins
IV.
Antibodies testing
V.
Other laboratory tests that might aid in the diagnosis
VI.
Immunohistochemistry
VII. Genetic studies
VIII. Skeletal muscle biopsy
IX.
Special tests
I.

I) Preliminary investigations
1) Complete blood picture:
 Anemia: associates systemic diseases, phosphofructokinase
deficiency (hemolytic),
 Leukocytosis: polymyositis and dermatomyositis
 Eosinophilia: polymyositis
2) ESR & CRP:
 Elevated in polymyositis, dermatomyositis and during activity
of collagenic diseases.
3) Creatine & Creatinine:
 Elevated serum creatinine: rhabdomyolysis, scleroderma,
sarcoidosis,
 Elevated urine creatine: Myopathy associated with alcoholism
 Elevated creatine/creatinine ratio: Muscular Dystrophies,
active muscle diseases.

4) Electrolytes:
 Serum calcium:
 When decreases causes muscle rigidity and tetany.
 When increases causes muscular hypotonia.
 Serum potassium:
 Increases: malignant hyperthermia (> 7 mEq/L), hyperkalemic

familial periodic paralysis (may reach values > 8 mEq/L).
 Decreases: hypokalemic familial periodic paralysis (< 2 mEq/L)
5) Arterial blood gases:
1) Combined metabolic and respiratory acidosis
 Malignant Hyperthermia
(Diagnostic in the presence of muscle rigidity or rising temperature)
2) Increased pCo2:
 Indicates progressive respiratory failure in Myasthenia Gravis
3) Early respiratory alkalosis then respiratory acidosis follows:
Bronchial asthma

II) Enzyme activities
A)

Primary muscle enzymes:

1. Creatine kinase (mainly CK-MM): test of choice
•Increased in:
- Prolonged exercise
-Polymyositis
{the most useful diagnostic test. Increased in 70% of patients. Levels may vary greatly,
up to 50 X ULN. The level frequently becomes normal with steroid therapy}
-Duchenne muscular dystrophy
{always increased in affected children (5–100X ULN of adults) to peak by 2 years of age;
then begin to fall as disease becomes manifest. In fact, persistent normal CK virtually
rules out this diagnosis. Not affected by steroids}
-Facioscapulohumeral Dystrophy
{ mildly increased in 75% of patients to average 3X ULN}
-Limb-Girdle Dystrophy
{increased in 70% of patients to average of 10X ULN}
-Myotonic dystrophy
{ mildly increased in 50% of patients to average 3X ULN}

- Acute myopathy associated with alcoholism
{increased in 80% of patients; rises in 1 to 2 days; reaches peak in 4 to 5 days; lasts ~2
weeks}
- Chronic myopathy associated with alcoholism
{ increased in 60% of patients to average of 2X ULN}
- Malignant hyperthermia
{up to 20,000 - 100,000 U/L}
- Rhabdomyolysis
{ up to 20 X ULN which rises and falls rapidly}

• Normal in:
- Scleroderma
- Familial periodic paralysis
- Discoid lupus
- Muscle atrophy of neurologic origin (e.g., old poliomyelitis, polyneuritis)
- Hyperthyroid myopathy
•

Decreased in:
- Rheumatoid arthritis (~2/3 of patients)

2. CK-MB:
Skeletal Muscle Disorders That May Cause Increased Serum CK-MB:
(Reflecting its production by regenerating muscle)
 Malignant hyperthermia
 Alcoholic myopathy
 Dermatomyositis/polymyositis
 Duchenne muscular dystrophy
 Exercise myopathy
 Familial hypokalemic periodic paralysis
 Endocrine (e.g., hypoparathyroidism, acromegaly)
 Rhabdomyolysis
 Infections
 Severe skeletal muscle trauma

3) Aldolase:
 Marked increase in Rhabdomyolysis
 Elevated in 75% of cases of polymyositis and 20% of cases of
muscular dystrophy.
4. Lactate Dehydrogenase:
 LD-5 is the iso-enzyme that is elevated in skeletal muscle
diseases.
 Marked increase in Rhabdomyolysis
muscular dystrophy.
5. AST:
 Marked increase in Rhabdomyolysis (> 3000 IU/L)
muscular dystrophy.

B) Specific enzymes:
1. Angiotensin-converting enzyme:
- Elevated in sarcoidosis
2. Carnitine palmitoyltransferase deficiency (Lipid storage diseases):

 CPT I and CPT II are essential for the transport of long-chain










fatty acids from the cytosol to the mitochondria.
It is the most commonly identified metabolic cause of recurrent
myoglobinuria (dark urine) in adults.
Patients often have episodes of muscle pain, stiffness, and
tenderness, usually without frank cramps. The attacks are
triggered by prolonged exercise, especially in fasting
conditions.
May cause a severe and fatal disease in the neonatal period
and during early infancy characterized by hypoketotic
hypoglycemia.
Plasma carnitine level may be increased in CPT I deficiency,
but it is usually normal in CPT-II deficiency.
Final diagnosis usually is established through biochemical
demonstration of CPT deficiency in the muscle.
CPT deficiency should be differentiated from glycogen storage
diseases, particularly McArdle disease.

3) Glycogen storage diseases (glycogenoses):
Deficiency of the following enzymes is biochemically detected in muscles:
 Glycogenosis type II - Acid maltase deficiency (AMD); Pompe disease.
 Glycogenosis type V - Muscle phosphorylase deficiency; McArdle disease.
 Glycogenosis type VII - Phosphofructokinase deficiency; Tarui disease.
 Glycogenosis type IX - Phosphoglycerate kinase deficiency.
 Glycogenosis type X - Phosphoglycerate mutase deficiency.

III) Proteins:
A. Myoglobin:
Urinary and serum Myoglobin is elevated in:
 Rhabdomyolysis
 Carnitine palmitoyltransferase deficiency
 Trauma
 Malignant hyperthermia
 Polymyositis / Dermatomyositis
 Duchenne muscular dystrophy & Facioscapulohumeral dystrophy
 Myopathy associated with alcohol intake

Myoglobin clearance:
 A value below 4 ml / min suggests high incidence for developing
ARF in patients with rhabdomyolysis. (Earlier than CCl)

B) Troponins:
Skeletal Troponin T and Skeletal troponin I, with its two distinct
isoforms (fast and slow), are elevated in:
 Exercise induced muscle injury.
 Myositis (suggested by some authors)

C) Dystrophin:
 Dystrophin protein quantitated by Western blot on biopsy of muscle
is <3% of normal in Duchenne muscular dystrophy.
 Immunofluorescence performed on biopsy of muscle is used to
confirm WB results in patients with suspected dystrophinopathy.
D) Alpha1-antitrypsin:
Its serum level decreases in emphysema patients.

IV) Antibodies testing:
A) Antibodies in Myasthenia Gravis (MG): (RIA & Radioreceptor assay)
1. Acetylcholine Nicotinic Receptor-Binding Antibodies
 The standard assay and should be ordered first.
 Has a sensitivity of 80 – 96 % and a specificity > 99 %.
 Negative in about 50 % of cases with ocular MG>
 Correlates with severity and ameliorated by treatment.
2. Acetylcholine Nicotinic Receptor-Blocking Antibodies
 More often associated with more severe forms of disease.
3. Acetylcholine Nicotinic Receptor-Modulating Antibodies
 Positive in 7% of MG patients when AChR-binding antibodies are not detected.
4. Antibodies to skeletal muscle cross-striations
 Found in 30% of adult MG patients.
5. Antibodies against Muscle Specific Kinase
 Present in about 50 % of sero-negative patients.
6. Other antibodies
 Anti-DNA, ANA, anti-parietal cell, anti-smooth muscle, antimitochondrial,
antithyroid antibodies, rheumatoid factor.

B) Antibodies against P/Q voltage-gated calcium channel:
- In Lambert-Eaton syndrome.
- D.D from MG.

C) Myositis specific auto antibodies: (ELISA, IF & immunoprecipitation)
Anti Ro-52, anti Jo-1, anti Mi-2, anti SRP,
anti PM / Scl-100, anti PM / Scl-75
D) Antibodies in scleroderma:
Anti-DNA, Anti-La/SS-B, Antinuclear antibody, Anti-RNP, Anti-Sm test.

V) Other laboratory tests that might aid in the diagnosis:

1) Urine cytology and toxicology screening: rhabdomyolysis
2) Fecal leukocytes and occult blood: diverticulosis
3) HLA B 27: MG
4) D-xylose test in serum or urine: scleroderma
5) CSF Immunoglobulin G ratios and immunoglobulin G index:
MS
6) Carnitine level: decreases in muscles and plasma on
carnitine deficiency myopathy.

VI) Immunohistochemistry:

1) Myeloid-related proteins MRP8 and MRP14:
Calcium binding proteins expressed in inflammatory myopathies
2) Ki-67 proliferation protein:
- Cellular marker of proliferation detected immunohistochemically
by a monoclonal antibody.
- Expressed in sarcoidosis.
3) Smooth muscle markers:
- Smooth muscle α-actin (α-SMA): in Chronic idiopathic intestinal
pseudo-obstruction
- Smooth muscle myosin heavy chain (SMMHC): in
gastrointestinal motility disorders
- Smoothelin (SM): in gastrointestinal motility disorders
- Histone deacetylase 8 (HDAC8): in gastrointestinal motility
disorders

VII) Genetic studies:
1) Malignant Hyperthermia:
- Autosomal dominant disorder.
- Defective Ryanodine receptor gene on chromosome 19q13.1
(low sensitivity)
2) Duchenne muscular dystrophy:
X-linked defect in locus Xp21
3) Facioscapulohumeral Dystrophy:
- Autosomal dominant
- Abnormality on chromosome 4q35
4) Carnitine palmitoyltransferase deficiency:
- Autosomal recessive trait
- Abnormality on chromosome 1p32

5) Glycogen storage diseases:
 Glycogenosis type II : autosomal recessive (17q23)
 Glycogenosis type V(McArdle disease): autosomal recessive (11q13)
 Glycogenosis type VII: autosomal recessive (12q13.3)

 Glycogenosis type IX: X-linked recessive (Xq13)
 Glycogenosis type X: autosomal recessive (11p15.4) (isozyme LDH-M on
chromosome 11/ LDH-H on chromosome 12)

6) Bronchial asthma:
- ADAM33 gene located on chromosome 20 and is expressed in lung and
muscle cells.
It is believed to be related to asthma as it causes the airways to over-respond
and constrict airway passage.
- Other genes: PHF11, DPP10, GRPA & SPINK5
(Their functions are still obscure)

VIII) Skeletal muscle biopsy: (Confirmatory)
- Muscular dystrophy: fibers necrosis, regeneration and
replacement by fat.
- Mitochondrial diseases: ragged red muscle fibers.
- Polymyositis: inflammatory changes with mononuclear

cells infiltration.
- Myopathic carnitine deficiency: increased number of lipid

droplets with minimal or no increase in mitochondria.
- Glycogenosis type V(McArdle disease): glycogen
accumulation in muscle fibers.

IX) Special tests:

1) Malignant Hyperthermia: (inhalation anesthesia, increase
calcium, M. rigidity)
- Gold standard for diagnosis by in vitro exposure of biopsied
skeletal muscle to caffeine-halothane contracture test; S/S =
99%/94%.
2) Edrophonium test:
- Its application is limited to the situation when other investigations
do not yield a conclusive diagnosis about MG.
- This test requires the intravenous administration of edrophonium
chloride (Tensilon), a drug that blocks the breakdown of
acetylcholine by cholinesterase and temporarily increases the
levels of acetylcholine at the neuromuscular junction.
- In people with myasthenia gravis involving the eye muscles,
edrophonium chloride will briefly relieve weakness.

3) Forearm ischemic exercise test:
- To D.D glycogen and lipid storage diseases.
Technique
- Draw venous blood for lactate as baseline samples.
- Apply a sphygmomanometer on the arm to be tested and raise its
pressure slightly above the systolic blood pressure.
- Ask the patient to exercise repetitively for 1 minute.
- Stop exercise, deflate the sphygmomanometer, and draw blood
samples at 1, 3, 6, and 10 minutes after 1 minute of exercise for
lactate and ammonia.
Findings
- In healthy subjects, lactate level should increase to 3-5 times the
basal level in the first 2 samples after exercise and then decrease
gradually to the baseline.
- In glycogen storage diseases, such as McArdle disease, serum
lactate levels do not increase after exercise (ie, flat lactate curve),
while in lipid storage diseases, lactate and level increase in a normal
fashion.

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