Laboratory diagnosis of muscle diseases


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A seminar in the Chemical pathology department
Presented by Ola Elgaddar

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Laboratory diagnosis of muscle diseases

  2. 2. • 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.
  3. 3. 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.
  4. 4. 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
  5. 5. 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
  6. 6. 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.
  7. 7. 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
  8. 8. B. Urinary bladder:  Impaired detrusor contractility or detrusor areflexia  Detrusor hyperflexia C. Lung:  Asthma  Emphysema  Bronchiactasis D. Blood vessels:  Aneurysms E. Myometrium:  Fibromyoma
  9. 9. 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
  10. 10.  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)
  11. 11. DIAGNOSIS OF MUSCLE DISEASES  Clinical  Electromyography (EMG)  Imaging  Laboratory investigations  Muscle biopsy  Special tests
  12. 12. 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.
  13. 13. 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.
  14. 14. 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
  15. 15. 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}
  16. 16. - 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)
  17. 17. 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
  18. 18. 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  Elevated in 25% of cases of polymyositis and 10% of cases of muscular dystrophy. 5. AST:  Marked increase in Rhabdomyolysis (> 3000 IU/L)  Elevated in 25% of cases of polymyositis and 15% of cases of muscular dystrophy.
  19. 19. B) Specific enzymes: 1. Angiotensin-converting enzyme: - Elevated in sarcoidosis 2. Carnitine palmitoyltransferase deficiency (Lipid storage diseases):
  20. 20.  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.
  21. 21. 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.
  22. 22. 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)
  23. 23. 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.
  24. 24. 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.
  25. 25. 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.
  26. 26. 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.
  27. 27. 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
  28. 28. 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
  29. 29. 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)
  30. 30. 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.
  31. 31. 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.
  32. 32. 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.
  33. 33. THANK YOU