What is an in vitro (in a laboratory setting) contraction test?

What is the In Vitro Contracture Test (IVCT)?

The in vitro contracture test (IVCT) is the gold standard laboratory diagnostic test for malignant hyperthermia (MH) susceptibility, involving pharmacological challenge of freshly excised skeletal muscle with halothane and caffeine under controlled conditions to assess abnormal contracture responses. 1

Core Principle and Purpose

The IVCT exploits the fundamental pathophysiology of MH susceptibility: muscle samples from MH-susceptible individuals demonstrate heightened sensitivity to contracture-inducing properties of caffeine and halothane compared to normal muscle. 1 This test serves as either a first-line diagnostic tool or confirmatory test when DNA analysis fails to establish high-risk status in patients suspected of MH susceptibility. 1

Technical Procedure

Muscle Biopsy Requirements

• Minimum patient age: 4 years, though most laboratories require patients to be at least 10 years old due to limited control data in younger children. 1
• Biopsy site: Quadriceps muscle (vastus medialis or vastus lateralis) using local, regional, or trigger-free general anesthetic techniques, avoiding local anesthetic infiltration of muscle tissue itself. 1
• Specimen dimensions: 20-25 mm length between ties, 2-3 mm thickness, weighing 100-200 mg. 1

Laboratory Conditions

• Time constraint: Testing must be completed within 5 hours from biopsy to test completion. 1
• Temperature: All tests performed at 37°C in tissue baths. 1
• Solution: Muscle maintained in preoxygenated Krebs-Ringer solution with specific ion concentrations (pH 7.35-7.45). 1
• Test protocol: Minimum of four tests using fresh specimens—two static caffeine tests and two halothane tests (either one static plus one dynamic, or two static tests). 1

Pharmacological Challenge

• Caffeine testing: Incremental caffeine concentrations applied to assess contracture threshold. 1
• Halothane testing: Incremental halothane concentrations applied to assess contracture threshold. 1
• Viability criteria: Specimens must demonstrate twitches ≥10 mN at test beginning and response to 32 mmol/L caffeine ≥50 mN at end. 1

Diagnostic Classification

The IVCT classifies patients into MH-susceptible (MHS) or MH-normal (MHN) categories based on contracture responses to both agents. 1 Patients with abnormal responses to either caffeine or halothane should be considered at risk for developing MH under anesthesia, regardless of specific subtype classification. 1

Clinical Indications for Testing

The European Malignant Hyperthermia Group identifies specific referral criteria: 1

• Family history of MH
• Adverse reaction to general anesthesia with trigger agents showing signs of increased metabolism (unexplained CO₂ increase, tachycardia, temperature elevation), muscle rigidity, rhabdomyolysis, or DIC
• Family history of unexplained perioperative death
• Postoperative rhabdomyolysis after excluding other myopathies
• Idiopathic hyperCKemia (persistently elevated CK >500 U/L after complete neurological work-up) 2, 3
• Exertional heat stroke requiring hospitalization after excluding known predisposing factors 1, 3

Test Performance Characteristics

The IVCT demonstrates 99% sensitivity and 94% specificity for MH susceptibility. 1 This represents the most robust diagnostic tool available, though DNA analysis plays an increasingly important complementary role in family screening after index case identification. 1, 2

Critical Limitations and Pitfalls

• Invasiveness: Requires surgical muscle biopsy, making it more invasive than genetic testing. 1
• Age restrictions: Concerns exist about cosmetic and functional consequences in children below certain body weights (e.g., 30 kg). 1
• Time sensitivity: The 5-hour window from biopsy to test completion requires precise coordination. 1
• Specificity concerns: Some false-positive results may occur, particularly with atrophied muscle samples. 1
• DNA testing comparison: While DNA analysis is less invasive, it lacks the sensitivity of IVCT and should not replace it as the primary diagnostic tool when MH susceptibility confirmation is needed. 1, 2