Diagnostic Immunology Services – Immunochemistry & Related Testing

Regional Immunology Laboratory – Context & Scale

• Sole immunology reference laboratory for all of Northern Ireland
  • Serves 1.82\,\text{million} people
  • Receives ≈ 160\,000 primary samples per year
  • Generates \ge 540\,000 individual test results annually
• Most investigations performed in-house; only rare / ultra-specialist investigations sent to external centres
• Physical visit for students/trainees anticipated

Personnel & Organisation

• Karen Goodall (Senior BMS, Band 7)
  • Section lead: Cellular Immunology / Allergy
  • Responsible for training & competency assurance in the laboratory
• Staffing matrix
  • Specimen reception (now centralised NI-wide)
  • Cells & Allergy section
  • Autoimmune Serology section
  • Workforce: 2 MLA, 8 BMS laboratory staff, 2 BMS managerial staff, 1 Clinical Scientist
  • Clinical advice supplied by Immunology Day Centre

Learning Objectives for Students

• Be able to list serum proteins routinely measured & their physiological roles
• Explain analytical principles of nephelometry, turbidimetry, radial immunodiffusion (RID) & electrophoretic techniques
• Interpret result patterns & link to differential diagnoses / disease mechanisms

Immunochemistry – Portfolio of Tests

• Serum immunoglobulins: \text{IgG, IgM, IgA}
• \text{IgG} subclasses (1 → 4)
• Functional (vaccine-specific) antibodies
• Complement split products: \text{C3, C4} (quantitative)
• Rheumatoid factor (RF)
• Cryoglobulin detection & characterisation
• Cerebrospinal fluid (CSF) oligoclonal band analysis

Core Analytical Platforms

• Roche Cobas Modular
  • Quantitation of IgG/A/M, C3, C4, RF
• Helena Nexus & SAS
  • Serum Protein Electrophoresis (SPE)
  • Immunofixation electrophoresis (IFE)
  • Immunotyping (IT)
  • CSF oligoclonal band analysis
• Binding Site SPA Plus
  • Dedicated for IgG subclass determination

Nephelometry & Turbidimetry – Technical Principles

• Shared workflow
  • Patient serum diluted → addition of analyte-specific antibody
  • Formation of insoluble antigen–antibody immune complexes
  • Instrument measures optical change; performs antigen excess safeguard by adding extra antibody & reassessing
  • Latex enhancement may be used for very low-concentration targets
• Nephelometry
  • Detector placed off-axis; signal proportional to light scattered by complexes
• Turbidimetry
  • Detector in-line with light source; signal inversely proportional to light transmitted

Rheumatoid Factor (RF)

• Predominantly IgM auto-antibody against Fc region of IgG
  • Other isotypes possible (IgG, IgA, etc.)
• Pathogenic potential: immune-complex formation → complement activation → chronic joint inflammation
• Elevated in multiple conditions:
  • Rheumatoid arthritis (RA)
  • Systemic lupus erythematosus (SLE)
  • Chronic infections
  • Autoimmune liver disease
  • Glandular fever
  • Component(s) of mixed cryoglobulins
• Clinical staging terminology highlighted (Early, Intermediate, Severe RA per ACR) but numeric table omitted in transcript (only “1.5” shown)

Anti-CCP: The Successor Biomarker?

• Anti–cyclic citrullinated peptide (CCP) antibodies increasingly utilised
  • Higher specificity than RF for RA
  • Prognostic for progression from undifferentiated arthritis / early synovitis → established RA
  • Aids selection of patient-tailored, aggressive DMARD / biologic therapy early in disease

Complement System – Quantitative & Functional Assessment

• Pathways: Classical, Lectin, Alternative → converge to form C3/C5 convertases → Membrane Attack Complex (MAC =\text{C5b–C9})
• Quantitative assays
  • \text{C3} & \text{C4} measured by nephelometry (Cobas)
  • Clinical correlation
  – Elevated (acute-phase): infection, inflammation
  – Decreased/paradoxical: SLE, MPGN, partial lipodystrophy, angioedema, cryoglobulinaemia
• Functional assays
  • Detect qualitative defects even with normal protein levels
  • Two gold-standard tests
  – CH100 (a.k.a. CH50): classical haemolytic capacity
  – AH100 (AH50): alternative pathway haemolysis
  • C1-INH quantity/function for hereditary & acquired angio-oedema

Radial Immunodiffusion (RID) for Haemolytic Complement

• Agarose gel embedded with sensitised sheep (or chicken) red cells + rabbit anti-sheep hemolysin
  • Patient serum diffuses radially → if complement functional, MAC forms → RBC lysis → clear ring
• Measurement
  • Ring diameter \propto complement activity
  • Linear-regression vs. calibrator curves to calculate % lysis (target =100\%)
• Interpretation
  • Low CH100 only → defect upstream of C3 in classical pathway
  • Low AH100 only → defect unique to alternative factors (B, D, properdin)
  • Both low → terminal pathway defect (C5–C9) → impaired bacterial lysis
• Pre-analytical pitfalls: improper transport temperature → consumption & falsely low results

Serum Immunoglobulins – Quantitation & Meaning

• Adult reference intervals
  • \text{IgG: }6.0–16.0\,\text{g}\,\text{L}^{-1}
  • \text{IgA: }0.8–2.8\,\text{g}\,\text{L}^{-1}
  • \text{IgM: }0.5–2.0\,\text{g}\,\text{L}^{-1}
• Biology
  • IgG → long half-life (~3 weeks), secondary/specific responses
  • IgA → mucosal protection; secretory dimer
  • IgM → pentamer; earliest immune response
• Limitations of nephelometry
  • Cannot distinguish polyclonal vs. monoclonal immunoglobulin increase
  • Requires SPE + IFE to identify paraproteins → essential in B-cell malignancy work-up (further detail to be covered in Biochemistry lecture)
• Patterns
  • Low Ig: protein loss (nephrotic, gut), malnutrition, primary/secondary immunodeficiency
  • Selective IgA deficiency (~1:400 adults) → usually asymptomatic but watch for anaphylaxis risk to IgA-containing products
  • High Ig: autoimmune disease, infection, multiple myeloma (monoclonal IgG/IgA), Waldenström (monoclonal IgM)
  • High IgM polyclonal: early infection; Hyper-IgM syndrome (class-switch defect)

IgG Subclass Investigation

• Subclasses: \text{IgG1}, \text{IgG2}, \text{IgG3}, \text{IgG4}
• Technique
  • IgG1–3: nephelometry / turbidimetry (SPA Plus)
  • IgG4: referred to Supra-regional Protein Reference Lab (Sheffield)
• Clinical pearls
  • Deficiencies often paired (IgG1 + 3 or IgG2 + 4)
  • IgG2 deficiency commonest; linked to recurrent sinopulmonary infections
  • Normal total IgG may mask subclass deficiency because IgG1 is quantitatively dominant
  • Some patients asymptomatic due to compensatory class-switching

Functional Antibody Testing (Vaccine Response)

• Purpose: confirm adaptive humoral competence
• Protocol
  • Baseline serum sample
  • Administer vaccine (e.g. tetanus toxoid, \text{Hib}, pneumococcal polysaccharide \rightarrow \ge 23 serotypes, meningococcal C)
  • Post-vaccination sample at ~4 weeks
  • Fold‐rise (or protective threshold) assessed
• Blunted / absent rise → consider CVID, specific antibody deficiency, immunosuppression

Cryoglobulins – Pathobiology & Laboratory Handling

• Cryoglobulins: immunoglobulins (± complement) that precipitate at <37\,^{\circ}\text{C} and redissolve on warming
• Clinical picture
  • Raynaud’s phenomenon
  • Purpuric vasculitis
  • Arthralgia/arthritis
  • Glomerulonephritis
  • Hyper-viscosity syndrome
  • Hepatosplenomegaly
• Pre-analytical protocol
  • Collect blood into pre-warmed tubes, maintain 37\,^{\circ}\text{C} to clot & separate
  • Split serum: keep one aliquot warm (control) & cool the other to 4\,^{\circ}\text{C}
  • Observe for precipitate daily (up to 5 days)
  • If positive → wash precipitate & characterise by IFE
• Types
  • Type I: monoclonal Ig (often IgM κ)
  • Type II: monoclonal RF (IgM) + polyclonal IgG (mixed)
  • Type III: polyclonal IgM RF + polyclonal IgG (mixed)

CSF Oligoclonal Band Analysis – Isoelectric Focussing (IEF)

• Paired CSF & serum required to discriminate systemic vs. CNS-restricted IgG production
• Methodology
  • Agarose gel with pH gradient; proteins migrate until isoelectric point (pI) reached → sharply focused bands
  • Immunofixation overlay enhances IgG visualisation
• Interpretation patterns (Type 1 → 5)
  • Type 1: No bands (normal)
  • Type 2: Bands in CSF only (intrathecal synthesis)
  • Type 3: Bands in CSF + some extra unmatched bands
  • Type 4: Identical bands CSF & serum (systemic monoclonal)
  • Type 5: Matching bands with monoclonal gammopathy pattern
• Positive (Type 2/3) implies intrathecal IgG production
  • Infective (transient) vs. autoimmune (sustained, e.g. Multiple Sclerosis)
• Multiple Sclerosis overview
  • Immune-mediated demyelination → plaques/scarring
  • Symptoms: variable neurological deficits
  • OCB positivity supports but does not confirm diagnosis
  • Management: corticosteroids, IFN-β, natalizumab, ocrelizumab; tolerability issues common

Integrated Diagnostic Relevance

• Assay menu (IgG/A/M, subclasses, RF, C3/C4, functional C’ & Ig, cryoglobulins, CSF OCB) enables detection & monitoring of:
  • Autoimmune disorders: RA, SLE, autoimmune liver disease
  • Primary/secondary immunodeficiencies
  • Hematological malignancies: multiple myeloma, Waldenström’s
  • Complement deficiencies & hereditary angio-oedema
  • Neurological autoimmune disease: Multiple Sclerosis

Summary of Key Analytical Techniques

• Nephelometry / Turbidimetry: rapid, automated quantitation of soluble proteins
• Serum Protein Electrophoresis (SPE) & Immunofixation (IFE): separate & type monoclonal immunoglobulins
• Radial Immunodiffusion (RID): manual but informative for complement haemolysis
• Isoelectric Focussing (IEF): gold standard for CSF oligoclonal banding
• ELISA: mentioned but not detailed; usually for specialised antibodies (e.g. CCP, ANA subsets)

"Time for a well-deserved break… the coffee machine is broken – be back in \approx 15 minutes."