Pre-analytical Variables in Coagulation Testing and Their Impact on Diagnostic Accuracy in Hemostasis

Pre-analytical Variables in Coagulation Testing and Their Impact on Diagnostic Accuracy in Hemostasis

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  Pre-analytical Variables inCoagulation Testing Associated With Diagnostic Errors in Hemostasis. Emmanuel J. Favaloro, PhD, MAIMS, FFSc (RCPA),1 Dorothy M. (Adcock) Funk, MD,2 Giuseppe Lippi, MD3 (1Department of Haematology, ICPMR, Westmead Hospital, Westmead, NSW, Australia, 2Esoterix Inc., Englewood, CO, 3Clinical Chemistry and Hematology Laboratory, Academic Hospital of Parma, Parma, Italy) Presented by ~ Dr Ravina Lalchandani MBBS, MD Lab Medicine PG1 MGUMST
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  Journal – LaboratoryMedicine Impact Factor – ~1.0–1.2 Conducted – (International review; Australia, USA, Italy)
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  INTRODUCTION • Modern instrumentationprovides highly accurate results; with internal quality control and external quality assurance, analytical errors in hemostasis testing are minimal. • Incorrect or inappropriate test results still occur, often due to circumstances beyond laboratory control. • Diagnostic errors impact patient care in ~9–15% cases, with inappropriate care in ~2–7%. • Most errors arise from inappropriate collection, handling, processing, wrong patient, or wrong timepoint—these are termed pre- analytical variables.
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  CONSEQUENCES OF PRE-ANALYTICALERRORS • Difficult to directly link spurious test results to patient outcomes; however, incorrect results can still cause clinically meaningful and economic consequences, and pose risks to laboratories. • Severity depends on test type, magnitude of error (reported vs true result), and ability of clinicians/lab personnel to recognize the issue. • In specialized hemostasis tests, errors may lead to false-positive or false-negative diagnoses, causing misdiagnosis and inappropriate or missed treatment. • Examples: false-negative APS → no anticoagulation (risk of thrombosis); false VWD diagnosis → unnecessary treatment and lifelong disease labeling. • Routine coagulation errors may cause unnecessary investigations/delays, missed diagnoses (e.g., hemophilia), or incorrect anticoagulant dosing → risk of bleeding or thrombosis.
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  Overview of Hemostasis,Laboratory Testing, and Pre-analytical Issues • Hemostasis is more complex than “coagulation” and includes primary hemostasis (platelets/VWF), secondary hemostasis (clotting factors), and fibrinolysis, often explained by Virchow’s Triad. • Routine laboratory tests include PT/INR, APTT, fibrinogen, TT, and D-dimer, used to assess secondary hemostasis disorders (congenital or acquired). • PT/INR and APTT detect deficiencies of clotting factors; used in diagnosing bleeding disorders (e.g., hemophilia) and monitoring anticoagulant therapy (warfarin, heparin). • Large number of tests and methodologies increase risk of errors, especially when unsuitable samples are submitted. • Pre-analytical errors can occur during collection, handling, transport, processing, or storage, often outside lab control; hence strict adherence to guidelines is essential.
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  Appropriate Sample Collection,Processing, and Storage ● These are critical to the attainment of appropriate test results but are often neglected, overlooked, or poorly applied. ● 1.Positive Patient and Sample Identification ● 2.Sample Collection ● 3.Sample Transport ● 4.Sample Processing and Storage ● 5.Controlled Thawing of Frozen Plasma Samples
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  1.) Positive Patientand Sample Identification • Proper patient identification is critical to avoid errors • Use “double identifiers” in outpatient settings: • Ask patient to state their name • Verify with an ID proof • In hospital settings: • Follow institutional protocols • Use electronic/barcode systems to reduce errors • Labeling guidelines: • Ensure labels match patient’s full name • Include a second identifier: • Date of birth (DOB) OR • Medical record number (MRN) • Sample details must include: • Date of collection • Time of collection
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  2.) Sample Collection Usesodium citrate tubes (3.2% preferred as per CLSI); standardize one concentration to avoid variation in PT, APTT, fibrinogen, and INR results • Follow proper order of draw; collect coagulation samples before EDTA, heparin, or clot activator tubes to prevent contamination • Ensure tubes are adequately filled (≥90%); under-filling causes excess citrate → false prolonged clotting times & dilution errors • Do not transfer blood between tubes; mixing tubes can alter anticoagulant ratio and compromise results • Mix samples gently (3–6 inversions); avoid under-mixing (clotting) and over-mixing (hemolysis, false results) • Avoid collection errors (line contamination, wrong sample type, improper needle size); ensure correct sample matrix (citrate plasma vs serum)
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  3.) Sample Transport Transportat 15–22°C (ambient) • Avoid heat & refrigeration • Test within 4 hrs (PT, APTT) • APTT (heparin): within 1 hr • Delay → ↓ FV, FVIII → ↑ clotting time • If delay: centrifuge → freeze → transport
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  4.) Sample Processingand Storage 🧪 Sample Processing (Coagulation) • Follow CLSI guidelines • Most tests (PT, APTT, factor assays) → once-centrifuged plasma • Lupus anticoagulant (LA) → double centrifugation (platelet-free plasma) • Double-spin preferred if plasma to be frozen ⚙️Centrifugation Conditions • Temperature: 15–22°C (ambient) • Speed: ~1500 g for 10–15 min • Avoid: • ❌ High speed (>1500 g) → platelet activation, hemolysis • ❌ Centrifuge brakes → remixing of cells • Non-refrigerated centrifuge: acceptable • Refrigerated centrifuge: • Maintain ambient temp (NOT cold) • Low temp → platelet activation
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  🔬 Special ProcessingNotes • Double-spun plasma → suitable for all hemostasis tests • ❌ Filtered plasma NOT recommended (LA testing errors) • Some tests need special handling (e.g., platelet function tests) ⸻ ️ ⏱️Sample Stability Factors • Depends on: • Collection system • Whole blood vs plasma • Storage temperature • Instrument/reagents • Specific test parameter
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  🩸 Whole BloodStorage • Acceptable (some tests): 24–48 hrs before centrifugation • NOT suitable for: • Factor V • Factor VIII • Protein S • ❌ Refrigerated whole blood → activates: • Factor VII, VIII, Von Willebrand factor ⸻ ⏳ Ideal Processing Timeline • Process within 1 hour of collection • Perform testing within 4 hours • If delayed: • Centrifuge → separate plasma → freeze
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  ⸻ ️ 🌡️Short-Term Storage • Wholeblood: • Keep capped at room temperature • Plasma: • Stable for few hours at room temp or refrigerated ⸻ ❄️Freezing & Storage • Separate plasma without disturbing cell layer • Storage recommendations: • -20°C → test within 2–4 weeks • -80°C → stable for months–years • ❌ Avoid frost-free freezers (freeze-thaw cycles)
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  5.) Controlled Thawingof Frozen Plasma Samples • Rapid thawing: 37°C water bath for 5–10 min (until fully thawed); avoid under/over-thawing • Strict temperature control: Prevent overheating → preserves coagulation factor activity & avoids false results • Post-thaw handling: Mix thoroughly before testing to ensure sample uniformity
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  Typical Issues Relatedto Inappropriate Sample Collection, Processing, and Storage ● 1.Incorrect Patient Collected or Wrong Label Attached ● 2.Incorrect Anticoagulant Matrix Collected or Provided to the Laboratory ● 3.Serum or Clotted Samples ● 4.EDTA Plasma ● 5.Heparin ● 6.Processing Issues ● 7.Hemolysis ● 8.Hematocrit ● 9.Lipemia ● 10.Freeze-thawing Events
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  1) Incorrect PatientCollected / Wrong Label Attached • Patient misidentification → high-risk error leading to misdiagnosis & inappropriate treatment • If suspected → recollect sample and repeat testing (safest approach)
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  2) Incorrect AnticoagulantMatrix Collected / Provided • Wrong anticoagulant (serum, heparin, EDTA) → unsuitable sample • Errors may be missed in secondary tubes / transferred samples • Routine tests (PT, APTT) → no clot or prolonged clotting time • Subtle effects in specialized tests: • Serum in VWF → may mimic Type 2 VWD pattern • Heparin/EDTA → misleading results (e.g., D-dimer, VWF) • ❌ False low/absent FVIII in clot-based assays • Detection depends on test type & lab vigilance
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  3) Serum orClotted Samples • Clotting occurs due to poor collection (slow fill, prolonged tourniquet, vein trauma, inadequate mixing, under-filled tubes) • Suspect in prolonged clotting tests; confirm visually or with applicator sticks → reject if clot present • Serum causes loss of fibrinogen and key factors (FII, FV, FVIII, VWF) with possible ↑ FVII due to activation • Leads to no clotting in PT/APTT/TT and may cause false diagnosis of factor deficiency, VWD, or LA errors • TT and mixing study help differentiate serum vs heparin contamination; anti-FXa confirms heparin
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  4) EDTA Plasma •Causes prolonged PT/APTT and ↓ FV, FVIII → may mimic factor deficiency or inhibitors • Mixing studies show no correction, falsely suggesting presence of inhibitor • May lead to false weak LA detection, while some tests (VWF:Ag, D-dimer) may appear normal • Recognition depends on test pattern; results can be misleading • Very high potassium and very low/absent calcium help confirm EDTA contamination
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  5) Heparin Contamination •Common in hospital settings (therapy, line flushes, heparinized needles); often more frequent than EDTA/serum errors • Causes prolonged APTT (marked) and TT, with ↓ fibrinogen and clotting factors (FVIII, FIX, FXI, FXII) • May lead to false diagnosis of dysfibrinogenemia, factor deficiencies, LA, inhibitors, and ↓ antithrombin • Variable results: PT may be normal or abnormal depending on heparin level and presence of heparin neutralizers; some tests (D-dimer, VWF:Ag) may remain normal • Detection: suspect with APTT/TT → confirm by mixing studies, anti-FXa assay, or correction after heparin neutralizer
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  6) Processing Issues •Improper processing may cause hemolysis or platelet activation, leading to falsely prolonged or shortened clotting times • Inadequate mixing can result in clot/partial clot formation with altered coagulation results • Freezing plasma with cellular contamination → hemolysis and activation effects • May also lead to false-negative LA results
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  7) Hemolysis • Causedby RBC destruction (in vitro handling errors or in vivo conditions like hemolytic anemia, infections, DIC, transfusion reactions) • Increases plasma absorbance → interferes with optical clot detection, affecting accuracy of test results • Releases tissue factors → may activate coagulation, causing ↓ fibrinogen, ↑ D-dimer, altered PT/APTT, ↓ antithrombin • Mechanical clot detection systems reduce interference but results may still be affected by activation • Grossly hemolyzed samples should be rejected; if unavoidable, use mechanical methods and interpret cautiously
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  8) Hematocrit • Lowhematocrit (anemia) → generally does not affect coagulation test results • High hematocrit (>55%) alters anticoagulant-to- plasma ratio → affects test accuracy • Adjustment required as per CLSI guidelines for correct anticoagulant volume • Practical method: remove 0.1 mL citrate from a 5 mL (3.2%) tube before collection
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  9) Lipemia • Causesboth biological and analytical interference in coagulation tests • High-fat meals → ↑ FVIIa activity, altered platelet function, and ↓ some clotting factors (FII, FIX, FX, FXII) • Interferes with optical clot detection methods; minimized by mechanical or dual-wavelength systems • Best approach → repeat sample in fasting state (if no underlying dyslipidemia)
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  10) Freeze–Thawing Event •Repeated freeze–thaw cycles → loss of labile factors (FV, FVIII) • Number of thaw cycles often unknown → may affect result reliability • Unexpected low factor levels → repeat testing with fresh sample
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  Under-recognized Pre-analytical Issues 1.1.Normal Reference Range Derivations and Related Issues 2. 2.Miscellaneous Variables 3. 3.International Normalized Ratio (INR) 4. 4.Filtered Plasma 5. 5.Physical Activity, Illness, and Stress 6. 6.Circadian and Diurnal Rhythms 7. 7.Patients on Anticoagulant Therapy 8. 8.Other Medications That May Interfere With Coagulation Testing 9. 9.Clinical Ordering and Inappropriate Requests as a Pre-analytical Issue 10.10.Test Methodology and Test Panel Selection
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  CONCLUSION 1. Normal ReferenceRange (NRR) limitations: Standard NRRs (mean ± 2 SD) include 95% of healthy individuals, meaning about 5% of normal people may show falsely abnormal results, especially problematic in rare disorders. 2. Influence of patient variables: Factors like age, gender, ethnicity, and blood group can affect test results (e.g., FVIII, VWF, platelet function), so these must be considered to avoid misinterpretation. 3.International Normalized Ratio (INR): INR is calculated as (patient PT / mean normal PT) ^ ISI. While patient PT is instrument-derived, MNPT and ISI are separate values and can act as pre-analytical variables, potentially affecting INR accuracy.
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  4. Filtered Plasma: ForLA testing, plasma must be platelet-free (<10 × 10⁹/L). Microfiltration is no longer recommended as it reduces FVIII and VWF levels and may cause false prolonged APTT or false-positive LA results. Double centrifugation is preferred to avoid misdiagnosis (e.g., hemophilia or VWD). 5. Physical Activity, Illness, and Stress: Exercise, illness, or stress can increase acute phase reactants (fibrinogen, VWF, FVIII), potentially masking mild hemophilia A or VWD (false negatives). Stress during blood collection can also alter results. 6. Circadian and Diurnal Rhythms: Some hemostatic factors vary with time of day (e.g., higher fibrinogen and PAI- 1 in the morning). These fluctuations, though subtle, may sometimes affect clinical interpretation.
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  7. Patients onAnticoagulant Therapy: Anticoagulants affect thrombophilia tests (e.g., LA, APCR, antithrombin, protein C/S), leading to both false-positive and false-negative results depending on the drug and test. 8. Other Medications: Various drugs can interfere with coagulation testing through different mechanisms, often producing unexpected or misleading results. 9. Clinical Ordering & Timing Issues: Inappropriate or poorly timed test requests (e.g., soon after thrombosis or during anticoagulant therapy) can cause diagnostic errors; many abnormal results may reflect such inappropriate testing. 10. Test Methodology & Panel Selection: Choice of test methods and panels influences accuracy; inadequate or unsuitable tests may lead to false diagnoses or missed conditions (e.g., VWD, APS).
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  Conclusion • Pre-analytical errorsin hemostasis testing are a significant source of diagnostic mistakes, though overall error rates remain relatively low. • Many errors are detected before reporting; goal is zero errors through early identification and correction. • Key prevention strategies: proper training, careful sample evaluation, accurate clinical information, and retesting when results are questionable. • Total quality management systems and strong clinician–laboratory collaboration are essential for minimizing errors. • Appropriate test selection, correct timing, and adherence to best practices improve diagnostic accuracy and patient outcomes. • New oral anticoagulants (e.g., Dabigatran, Rivaroxaban) can affect coagulation tests and should be considered in pre-analytical evaluation.
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  REFERENCES
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