Overview
MLPA uses hybridizing probe pairs that ligate only when adjacent sequences are present; ligated probes amplify proportionally to copy number and are sized by capillary electrophoresis.
Clinical Importance
Detect deletions/duplications in genes poorly covered by sequencing alone
Complement Sanger/NGS in germline disease workups
Specimen Requirements
- Specimen: whole blood or plasma as validated for the assay
- Tube: lavender-top K₂EDTA (preferred for many viral load and germline DNA assays)
- Handling: mix gently; separate plasma promptly when required; avoid heparin for many PCR assays (inhibition)
- Storage: refrigerate or freeze plasma per IFU; extract nucleic acid within validated windows
Analytical Method
Denatured DNA hybridizes to MLPA probes; ligation joins perfectly paired probes; PCR amplifies ligated products with universal primers; relative peak heights indicate copy number vs reference samples.
Instruments Used
Automated analyzers
- Applied Biosystems capillary electrophoresis systems
- Thermal cyclers for MLPA ligation/PCR
Manual methods
- Manual MLPA kit setup with capillary fragment analysis
Reference Values
Molecular cutoffs, units (IU/mL, copies/mL, Ct), reportable ranges, and variant classifications are assay- and laboratory-specific. Always interpret against the performing laboratory report, manufacturer IFU, and current CLSI / CAP / AMP / ACMG guidance as adopted locally. Limit of detection and inhibition controls are essential for negative results.
Dosage quotients near 1.0 = normal copy number; validated thresholds define deletion/duplication calls per kit.
Interpretation
Common Causes of Increased Results
- Elevated dosage peaks; duplication of probed exons
Common Causes of Decreased Results
- Reduced dosage peaks; deletion of probed exons
Clinical Significance
- DMD, hereditary cancer, and other genes where CNVs are common
- Confirmation of NGS-suggested copy-number events when validated
Factors Affecting Results
Pre-analytical
- Wrong specimen type or transport medium for the validated assay
- Delayed transport degrading RNA targets
- Contamination during collection (especially amplicon-rich environments)
- Inadequate volume or low cellularity/tumor content
Analytical
- PCR inhibition (heparin, humic acids, excess hemoglobin; assay-dependent)
- Extraction failure or pipetting/contamination in amplification areas
- Probe/primer mismatch with sequence variants causing false negatives
- Calibration/QC failure; expired reagents
Post-analytical
- Reporting Ct or raw copies without assay-specific interpretive rules
- Misapplying another laboratory’s LOD or units
- Variant classification without current ACMG/AMP framework when required
Advantages and Limitations
Advantages
- Efficient exon-level CNV detection
- Multiplex many exons
Limitations
- Does not sequence base-level variants
- Probe-site SNPs can mimic dosage changes
Clinical Pearls
MLPA counts exons . Sanger/NGS still own the point mutations.
References
Authoritative textbooks, guidelines, and reviews supporting this laboratory test reference. Verify reference intervals, critical limits, and method details against institutional protocols and current guideline editions.
Textbooks
- McPherson RA, Pincus MR. Henry's Clinical Diagnosis and Management by Laboratory Methods. 24th ed. Elsevier; 2021. (Molecular diagnostics chapters, verify current edition.)
- Buckingham L. Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications. 3rd ed. F.A. Davis; 2019. (Verify current edition and institutional adoption.)
CLSI and Professional Guidelines
- Clinical and Laboratory Standards Institute (CLSI). Consult current CLSI molecular method documents available through your laboratory, document numbers and editions change; do not treat any single CLSI title as universal.
- Institutional molecular laboratory standard operating procedures (SOPs), assay manufacturer instructions for use (IFU), and locally adopted accreditation requirements. Always verify current local practice.
- Clinical and Laboratory Standards Institute (CLSI). Molecular method evaluation and related documents (verify current CLSI editions applicable to the assay).
- College of American Pathologists. Molecular pathology accreditation and proficiency testing expectations (verify current CAP checklist requirements).
- Association for Molecular Pathology (AMP). Practice guidelines and position statements for molecular diagnostics (verify current AMP documents for the assay).
- American College of Medical Genetics and Genomics (ACMG). Standards and guidelines for interpretation of sequence variants and related genetic testing practice (verify current ACMG documents).
WHO Publications
- World Health Organization. Laboratory and diagnostic guidance relevant to molecular testing (verify current WHO publication for the assay/pathogen).
Frequently Asked Questions
Common questions about using this MLPA laboratory test Medical Laboratory Library reference in Medical Laboratory Science education.
What is the MLPA laboratory test reference used for?
This Medical Laboratory Library page summarizes the clinical importance of MLPA. Review the Clinical Importance section for why the test is ordered in laboratory medicine practice.
Where can I find MLPA specimen requirements?
See the Specimen Requirements section on this page for specimen information related to MLPA. Confirm collection details against your laboratory protocol.
Where are MLPA reference values listed?
See the Reference Values section on this Laboratory Library page. Reference ranges can vary by method and population, so verify intervals with your laboratory.
How do I interpret MLPA results?
Use the Interpretation section for laboratory interpretation framing, including increased and decreased result patterns when provided. Do not treat educational text as a patient-specific diagnosis.
What analytical method is used for MLPA?
See the Analytical Method section for principle and method notes. Method details may differ by instrument and institutional SOP.
Are related laboratory tests linked from MLPA?
Yes. The Related Laboratory Tests section lists connected references for continued lookup in the Medical Laboratory Library.
How can I continue learning after reading MLPA?
Use Continue learning links on this page for related Academy lessons, medical laboratory quizzes, or laboratory case studies when available for this Molecular Diagnostics topic.
What are the clinical pearls for MLPA?
See the Clinical Pearls section for concise laboratory practice notes associated with MLPA. Apply pearls alongside institutional protocols.
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