Thrombophilia Testing: Methodology, Instrument Limitations, and Principles of New Test/Equipment Selection

Overview

• Thrombophilia testing encompasses functional, immunological, and molecular assays designed to identify heritable or acquired conditions predisposing to venous (and, in the context of APS, arterial) thrombosis.
• Assay performance is influenced by pre-analytical variables, the patient's biological state at sampling, anticoagulant therapy, and inherent platform limitations.
• For the haematologist, a comprehensive understanding of test methodology and instrument characteristics is essential for accurate result interpretation and for informed decisions about adopting new assays or technologies into clinical practice.

Classification of Thrombophilia and Associated Testing

Pathophysiology Relevant to Testing Strategy

• Heritable thrombophilias disrupt natural anticoagulant pathways (AT, protein C/S system, APC resistance) or introduce gain-of-function in procoagulant proteins.
• Most confer incomplete penetrance and interact with acquired and environmental risk factors.
• Acquired thrombophilia, principally antiphospholipid syndrome, involves antibody-mediated interference with phospholipid-dependent coagulation.
• This biological heterogeneity must be reflected in assay selection, validation, and interpretation.
• Testing should be reserved for patients in whom a positive result would materially affect anticoagulant choice, intensity or duration of therapy, family counselling, or reproductive decision-making.
• Testing "just to know" is neither cost-effective nor clinically appropriate; testing uncovers an abnormality in >50% of patients presenting with an initial VTE but has minimal actual impact on management in most.

Test Methodology in Detail

Functional Assays for Natural Anticoagulants

• Antithrombin is measured by chromogenic substrate assay using heparin cofactor activity (anti-Xa or anti-IIa principle).
• Chromogenic assays are preferred over clot-based methods because they avoid interference from heparin, lupus anticoagulant, or elevated FVIII.
• Type I (quantitative) and type II (qualitative/functional) deficiency are distinguished by parallel immunological quantification.
• Unfractionated heparin (UFH) reduces AT levels substantially (up to ~30%); LMWH has a lesser but real effect.
• Testing on heparin therapy is therefore unreliable.
• Protein C is preferentially measured by functional chromogenic assay, which detects both type I and type II (functional) deficiencies.
• Clot-based protein C assays are susceptible to false-normal results in type II variants due to co-inheritance of FVL or elevated FVIII.
• Vitamin K antagonists (VKAs, e.g. warfarin) suppress protein C synthesis, rendering interpretation impossible during therapeutic anticoagulation.
• Acute thrombosis also transiently depresses protein C levels.
• Protein S is measured as free protein S by immunoassay (ELISA or latex-based immunoturbidimetric methods).
• Total protein S has low clinical utility because only the free fraction (~40%) is biologically active.
• Protein S behaves as an acute-phase reactant, levels fall during inflammation, pregnancy, and with oestrogen-containing contraceptives or HRT.
• Functional protein S assays are notoriously difficult to standardise and are not recommended as the primary screen.

Activated Protein C Resistance and Factor V Leiden

The modified APC sensitivity ratio uses patient plasma diluted in factor V-depleted plasma to normalise for co-existing anticoagulant deficiencies, FVIII elevation, or LA. The ratio of clotting times with and without added APC is calculated:

$$\text{APC sensitivity ratio} = \frac{\text{aPTT}_{\text{+APC}}}{\text{aPTT}_{\text{−APC}}}$$

• A ratio below the locally validated cut-off (platform-dependent) is considered positive.
• Reflexive molecular testing for the F5 G1691A (Factor V Leiden) mutation by allele-specific PCR confirms positivity and distinguishes heterozygous from homozygous carriage.
• Molecular testing is unaffected by anticoagulation status or acute illness.

Prothrombin Gene Variant F2 G20210A

• Detection is exclusively by allele-specific PCR or equivalent molecular method.
• No functional assay exists.
• Like FVL testing, this is unaffected by anticoagulant therapy or acute thrombosis, the most temporally flexible tests in the panel.

Lupus Anticoagulant Testing

LA testing is among the most technically challenging procedures in haemostasis. ISTH guidelines mandate two phospholipid-dependent clotting tests from different principles:

1. Dilute Russell's viper venom time (dRVVT): activates factor X directly; highly sensitive and specific for LA. A screening ratio is calculated and positivity confirmed by repeating with excess phospholipid (confirmatory step). Normalisation of the ratio after phospholipid addition confirms LA.

$$\text{dRVVT screen ratio} = \frac{\text{clotting time (screen)}}{\text{mean of normal reference range}}$$

A confirmatory dRVVT ratio >1.2 (or locally validated cut-off ≥99th percentile of healthy donors) is considered significant.

2. Sensitive aPTT (silica-based, low phospholipid): provides orthogonal sensitivity. Mixing studies with normal plasma help distinguish inhibitor from factor deficiency, though interpretation requires care.

LA must be confirmed on repeat testing ≥12 weeks after the initial positive result to fulfil APS classification criteria.

Interfering factors for LA assays:

• Samples should be obtained in the absence of anticoagulant therapy whenever possible.
• The dRVVT is particularly sensitive to anti-Xa therapies.

Anticardiolipin and Anti-β2-Glycoprotein I Antibodies

• Measured by ELISA or chemiluminescence immunoassay (CLIA).
• IgG and IgM isotypes are reported in standardised units (GPL, MPL).
• Only persistent positivity at moderate-to-high titre (>40 GPL/MPL, or >99th percentile) confirmed at ≥12 weeks fulfils APS classification.
• CLIA platforms offer improved reproducibility and throughput but require platform-specific reference intervals.

Instrument Limitations Relevant to Thrombophilia Testing

Optical vs. Mechanical Clot Detection

Automated coagulation analysers detect clot formation by either optical turbidimetry/nephelometry or electromagnetic mechanical methods.

• Optical methods: susceptible to interference from lipaemia, icterus, haemolysis, and elevated bilirubin. Turbid samples require appropriate blanking; elevated baseline turbidity can shorten apparent clotting times.
• Mechanical (viscosity-based) methods: less affected by optical sample properties but may be more sensitive to temperature fluctuation and reagent lot variation.

For thrombophilia assays where precise endpoint detection is critical (dRVVT, protein C chromogenic), the detection principle of the local instrument must be understood when establishing reference intervals.

Platform-Specific Reference Intervals

• Reference intervals for thrombophilia assays are not transferable between platforms.
• Each laboratory must establish or formally validate local reference intervals using a minimum of 120 healthy adult donors (stratified by sex where relevant) per CLSI guidelines.
• The 99th percentile cut-off for LA assays is particularly sensitive to the composition of the reference population.
• Failure to establish platform-specific intervals is a common source of diagnostic error.

Reagent Lot Variability

• Phospholipid composition in dRVVT reagents, protein C activator (e.g. Protac™) activity, and thromboplastin sensitivity (ISI for PT) can vary between manufacturing lots.
• Each new reagent lot must be assessed with appropriate controls before clinical use, particularly important for protein C, APC resistance, and LA assays.

Timing of Testing: Pre-Analytical and Clinical Considerations

Molecular tests (FVL and F2 G20210A PCR) are unaffected by all of the above and represent the most robust components of the panel when timing is suboptimal.

Acquired vs. Inherited Deficiency: Differential Diagnosis

Before attributing a reduced natural anticoagulant level to an inherited deficiency, acquired causes must be excluded:

Confirmation of an inherited deficiency requires testing in a clinically stable state, off anticoagulants, and ideally supported by family studies or genetic testing.

Special Considerations in Paediatric Thrombophilia Testing

• Age-related physiological variation in coagulation protein levels is substantial in neonates, infants, and children.
• Protein C, protein S, and AT levels are significantly lower in neonates and rise progressively to adult reference ranges over the first years of life.
• Applying adult reference intervals to paediatric samples will generate false-positive diagnoses of inherited deficiency.
• An ISTH consensus panel recommends that paediatric patients with thrombosis be tested comprehensively for both inherited and acquired disorders; however, age- and sex-specific reference intervals are mandatory.
• The most common thrombotic risk factor in hospitalised children is the presence of a central venous catheter.
• Homozygous deficiencies of protein C or protein S are causal in neonatal purpura fulminans.

Principles of New Test and Equipment Selection

When evaluating a new assay or analytical platform for thrombophilia testing, a systematic evaluation process aligned with accreditation requirements (NATA/RCPA in Australia; ISO 15189) is mandatory.

Evaluation Framework

Precision and Accuracy Standards

• For coagulation assays, acceptable within-run imprecision is generally ≤5% CV for clot-based tests and ≤3% CV for chromogenic assays.
• Between-laboratory reproducibility is assessed through EQA participation.
• For molecular thrombophilia assays (allele-specific PCR; and emerging next-generation sequencing panels), analytical validation must address allele call accuracy, sensitivity for mosaic or rare variants, and bioinformatic pipeline performance.

Evaluating Diagnostic Utility

The prior probability of detecting a clinically actionable result must justify the cost and complexity of testing. A formal framework using sensitivity, specificity, PPV, and NPV, derived from the 2×2 contingency table, should be applied:

$$\text{PPV} = \frac{\text{TP}}{\text{TP} + \text{FP}}$$

$$\text{NPV} = \frac{\text{TN}}{\text{TN} + \text{FN}}$$

$$\text{Likelihood ratio (+)} = \frac{\text{Sensitivity}}{1 - \text{Specificity}}$$

• In thrombophilia testing, where the population prevalence of most individual defects is low (e.g. AT deficiency ~0.1%), even tests with high specificity generate substantial false-positive results in unselected populations.
• This reinforces restriction of testing to appropriately selected patients.

Internal Quality Control and External Quality Assessment

Internal Quality Control (IQC)

• For each thrombophilia assay, at least two levels of control material (normal and abnormal) must be run with each analytical batch.
• Control limits are set using Westgard rules appropriate to the assay.
• Chromogenic assays require controls spanning the clinically relevant range.
• For LA testing, a LA-positive plasma control is essential alongside a normal control.

External Quality Assessment (EQA)

Participation in recognised EQA programmes is mandatory for accredited laboratories. Relevant schemes include:

• RCPA Quality Assurance Programs (RCPA QAP), Australia
• UK NEQAS for Blood Coagulation
• European ECAT Foundation
• EQA exercises cover natural anticoagulant surveys (AT, protein C, protein S), LA and antiphospholipid antibody surveys, and molecular testing for FVL and F2 G20210A.
• Poor EQA performance triggers formal root-cause analysis; persistent failure may require assay suspension pending remediation.
• Accuracy can be checked using reference materials assayed by reference methods against standards of known concentration (e.g. WHO reference plasma).

Summary: Principles for Selecting and Implementing New Thrombophilia Tests

1. Clinical need: Does the new test address an unmet diagnostic or prognostic need? Will it change patient management beyond currently available tests?
2. Analytical validation: Precision, accuracy, linearity, interferences, and comparison to the reference method must be formally documented before clinical deployment.
3. Reference interval establishment: Platform-specific, population-appropriate (including age- and sex-stratified) reference intervals are non-negotiable.
4. Quality systems integration: IQC and EQA must be in place before first clinical use.
5. Pre-analytical standardisation: Clear protocols for sample type, collection timing relative to anticoagulation, and transport must be documented in standard operating procedures.
6. Staff competency: Training and ongoing competency assessment for all staff performing and interpreting the assay.
7. Cost-benefit evaluation: Reagent, instrument, and labour costs assessed against the expected clinical benefit in the intended testing population.
8. Regulatory compliance: TGA registration for IVD devices in Australia; laboratory-developed tests require internal validation to ISO 15189 and NATA accreditation standards.

• The overarching principle, applicable equally to test selection and equipment evaluation, is that technical capability must be matched to clinical utility.
• A methodologically excellent assay that does not alter patient management offers limited value; conversely, a test with genuine clinical impact requires rigorous analytical underpinning to be trustworthy.