Laboratory Evaluation of Coagulopathies

Etiology and Epidemiology

Hemostasis is a complex biological process that involves a delicate balance between procoagulant and anticoagulant forces. Conditions that disrupt this balance can lead to coagulopathies, characterized by either excessive bleeding or thrombotic events. Generally, reduced procoagulant factors or impaired fibrinolysis, thrombocytopenia, or impaired platelet function increase the bleeding risk. Inherited coagulopathies with bleeding tendency most commonly arise from genetic mutations affecting clotting factor production or function, such as hemophilia A and hemophilia B, or von Willebrand disease, and rarely affect platelet function.[8][9] Von Willebrand disease is the most common, affecting up to 1% of the general population.[10] Platelet function disorders, such as Bernard-Soulier syndrome and Glanzmann thrombasthenia, are rare but may be underestimated.[11]

Acquired coagulopathies may result commonly from underlying medical conditions such as liver disease, vitamin K deficiency, or hematological malignancies.[12][13][14] Acquired von Willebrand disease and acquired hemophilia are uncommon and are associated with rheumatological disorders or hematological malignancies and cancer.[15] In addition, medications such as anticoagulants, antiplatelet medications, other commonly prescribed pharmaceuticals, over-the-counter products, and herbs can disrupt the coagulation cascade.[16][17] More importantly, trauma and the associated bleeding coagulopathy are responsible for more than 10% of the global health burden, whereas bleeding is responsible for 30% to 40% of trauma mortality.[18]

On the other side of the scale, thromboembolic diseases such as arterial thromboembolism, including myocardial infarction and stroke, and venous thromboembolism account for at least 1 in 4 deaths worldwide. Hypercoagulability may arise from multiple inherited and acquired factors associated with increased procoagulant factors or decreased anticoagulant or fibrinolytic factors. Venous thromboembolism affects 1 to 3 per 1000 adults and is one of the main mortality causes worldwide.[19] The inherited thrombophilias, such as natural anticoagulant deficiencies or gain-of-function mutations, including G20210AFII, increase venous thrombotic risk. The most common inherited thrombophilia is factor V Leiden. Acquired predisposing factors for venous thromboembolism include age, cancer, pregnancy, obesity, trauma, hospitalizations, and surgeries, among others.[20][21] Myeloproliferative diseases and paroxysmal nocturnal hemoglobinuria increase the risk for unusual site thrombosis.[22] Other rare immune-based hematological diseases, such as thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia, may present with severe thrombocytopenia and paradoxical thrombosis.[23] 

Pathophysiology

Three main components contribute to coagulation—the vasculature, the cellular compartment, and the plasma. Following vascular damage, tissue factor, and collagen exposure trigger the coagulation cascade. Platelets are central in the cellular compartment, working alongside monocytes, neutrophils, and potentially abnormal cells such as cancer cells. The plasma contains coagulation factors, natural anticoagulants, and microparticles, contributing to thrombin generation. Hemostasis is traditionally divided into primary and secondary stages.[24]

Primary Hemostasis

Primary hemostasis involves vasoconstriction, platelet adhesion, activation, secretion, and aggregation to form a temporary plug. Platelets adhere to the subendothelium through the von Willebrand factor (vWF) receptor, platelet glycoprotein GPIb, and collagen receptor GPIa through IIa. Several physiological factors activate platelets, including adenosine diphosphate (ADP) through P2Y1 and P2Y12 receptors, epinephrine, thrombin (through PAR1 and PAR4), and collagen (through GPVI). ADP and epinephrine are relatively weak platelet activators, whereas collagen and thrombin are the most potent. As platelets are activated, they change shape and degranulate crucial factors contributing to the coagulation cascade, such as vWF and fibrinogen from α-granules and serotonin, ADP, and Ca++ from δ-granules. As already mentioned, platelets possess numerous receptors, including glycoproteins GPIb-IX-V and GP IIbIIIa, to name a few. GPIb is involved in platelet adhesion to subendothelium by binding to vWF, whereas GPIIbIIIa is crucial in platelet aggregation by binding fibrinogen and vWF.[25][26]

Secondary Hemostasis

The platelet plug is stabilized through secondary hemostasis, a complex cascade of sequential activation of proenzymes, converging in thrombin generation, subsequently converting fibrinogen to fibrin. Factor XIII (FXIII) stabilizes the fibrin clot.[27] The coagulation cascade is traditionally divided into the extrinsic, intrinsic, and common pathways. Tissue factor activates FVII in the extrinsic pathway, which in turn activates FX (FXa). The intrinsic pathway involves the activation of FXII, FXI, FIX, and FVIII, ending up with FXa. Ultimately, in the common pathway, FXa combines with FV, platelet phospholipids, and calcium ions to convert prothrombin into thrombin, which cleaves fibrinogen into fibrin.[28]

These pathways primarily reflect ex vivo mechanisms observed in laboratory testing but do not fully capture in vivo processes. Emerging understanding favors a cellular model of hemostasis over the traditional cascade paradigm, where all interactions start on cellular membranes of platelets and endothelial cells, in 3 overlapping stages—(1) initiation, initiated by tissue factor on cellular membranes; (2) amplification, involving activation of platelets and cofactors to prepare for extensive thrombin production; and (3) propagation, characterized by significant thrombin generation on the platelet surface. The final step is fibrinolysis, with enzymatic degradation of the fibrin clot to balance the uncontrolled fibrin clot formation and prevent catastrophic thrombosis. Plasminogen activators, such as tissue plasminogen activators, activate plasminogen to plasmin, cleaving fibrin at specific lysine residues and dissolving the clot. Opposing forces guided by various fibrinolysis inhibitors, such as plasminogen activator inhibitors (PAI-1 and PAI-2), α2 antiplasmin (α2-AP), and thrombin activatable fibrinolysis inhibitors counterbalance the excessive lysis to prevent from bleeding, by impeding binding of fibrinolytic enzymes to fibrin.[29][30]

Specimen Requirements and Procedure

Coagulation tests are sensitive to preanalytical errors, underscoring the importance of meticulous attention to detail throughout the testing process. Preanalytical errors occur at various stages, including specimen collection, handling, transportation, and processing. Minor deviations from standard protocols can lead to inaccurate test results, impacting patient diagnosis and management.

Patient Preparation

Fasting is generally not recommended for most coagulation tests, such as homocysteine levels in thrombophilia screening may be affected by caffeine intake). Avoiding high-fat meals immediately before sampling is recommended, as lipemic plasma may interfere with coagulation tests and analyzer performance. For platelet function analysis, avoiding certain medications, foods, or herbal supplements that affect platelet function may be necessary for several days before testing. Other important information to gather before blood sampling includes:

• Current or recent pregnancies, oral contraceptive pills, and hormonal replacement in women are associated with decreased natural anticoagulants and increased coagulation factors.

• History of recent thrombosis, inflammation, or infections, including several coagulation factors, such as fibrinogen, FVIII, vWF antigen, PAI-1, protein C, S, antithrombin, and FXII, as acute phase reactants. 

• Blood group affects vWF levels.

• Treatment with anticoagulants, antiplatelets, or heparin contamination of the sample affects most coagulation studies.[31][32][33][34]

Sample Collection and Handling

• For most coagulation tests, a suggested needle size range is typically 19 G to 22 G. A larger gauge needle, such as 19 G, may be preferable for coagulation factor testing to minimize potential hemolysis and maintain sample integrity for platelet function and coagulation factor testing. In addition, atraumatic venipuncture should be performed as soon as possible, and tourniquet time should be minimized during blood collection to avoid potential artifacts in coagulation test results such as hemolysis. 

• Most coagulation tests require plasma. Plasma is typically collected in sodium citrate 3.2% tubes in a plasma-to-anticoagulant ratio of 9:1. Inadequate tube filling or high hematocrit levels in the sample (Ht>55%) increase citrate levels and affect recalcification performed during analysis with CaCl₂ and test results. In cases of high hematocrit levels, anticoagulant volume may need adjustment.

• Gently mixing is required by inverting the tube 3 to 6 times immediately after drawing the sample into the anticoagulated vial. Vigorous shaking or agitation should be avoided, as this may cause frothing or hemolysis, which can affect the accuracy of coagulation test results.

• Initial discard tubing is not required (except for the thrombin generation assay).

• Adherence to a standardized sequence during blood sampling is essential to prevent contamination between tubes. According to national and international guidelines, such as those from the World Health Organization (WHO) and Clinical and Laboratory Standards Institute (CLSI), the recommended order of draw during phlebotomy is as follows—blood culture/sterile tubes should be drawn first, followed by coagulation tubes, then plain tubes/gel tubes, and finally tubes containing additives.

• Freezing should be avoided before processing.

• Blood samples should be kept at room temperature and processed within 24 hours for prothrombin time and 4 hours for activated partial thromboplastin time (aPTT). Ideally, processing must be performed within 2 to 4 hours of collection. For patients under heparin treatment, aPTT measurements should be performed within 90 min from collection to avoid the risk of heparin inactivation by platelet-derived factor PF4.[35][36][37][38][39][40]

Sample Processing

• Most coagulation tests require platelet-poor plasma. Rigorous centrifugation in 2500 g for 15 min or 4500 g for 5 min is required. Each laboratory should adjust the time and centrifugation force to achieve a residual platelet number of <10×10⁹/L.

• Double sample centrifugation is recommended for certain coagulation tests such as lupus anticoagulant, anti-Xa activity, protein S, and mixing studies.[41][42]

Sample Storage

• The platelet-poor plasma may be stored at −70 °C for 12 months. Freezers should not have automatic defrost cycles to avoid coagulation factor activation and unstable specimens.

• The sample must be thawed at 37 °C for 5 min before analysis. Thawing for longer periods may lead to factor V and FVIII consumption.[43]

Diagnostic Tests

Several laboratory methods are available to investigate primary coagulopathy disorders, such as von Willebrand disease and platelet dysfunction, and secondary coagulopathy, such as clotting factor deficiencies. The initial approach involves basic coagulation screening, and based on the patient's medical history and associated symptoms, the clinician proceeds to more specialized tests. A standardized questionnaire approach using bleeding assessment tools such as ISTH-BAT offers a more complete and structured approach.[44]

Primary Hemostasis Investigation

Screening tests

• Complete blood cell count with peripheral blood smear: In addition to automated analyzers, platelet assessment in the complete blood cell count should include examination of a peripheral blood smear under a microscope, providing a comprehensive evaluation of platelet number, size, the presence of platelet aggregates, and the identification of any abnormal cells. Abnormal morphology in other peripheral blood cells may identify conditions, such as Dohle bodies in neutrophils in the May-Hegglin anomaly. A mean platelet volume higher than 12.4 fL signifies large platelets.

• Bleeding time: Historically, bleeding time has been a laboratory method to assess in vivo platelet function and vessel integrity. This parameter measures the time needed for bleeding to stop after a standardized incision on the patient's skin. However, most laboratories tend to replace the test with other, more automated processes, such as platelet function analyzer, due to the lack of standardization, reproducibility, low sensitivity and specificity, and low positive predictive value.

• Semi-automated platelet function analysis in whole blood: Point-of-care testing of platelet function, such as PFA-100 or PFA-200® system, is easy and reliable for assessing platelet function in whole blood. The sample is applied to cartridges coated with specific agonists, such as collagen and ADP (COL/ADP) or collagen and epinephrine (COL/EPI). Subsequently, a simulation of high shear flow, akin to that in blood vessels, is initiated through a capillary, activating platelets. The closure time until clot formation is measured and expressed in seconds for both combinations (COL/EPI and COL/ADP). An additional cartridge, P2Y12, containing ADP, calcium, and prostaglandin E1, was developed to study resistance to antiplatelets inhibiting P2Y receptors, such as clopidogrel. VerifyNow is a similar point-of-care testing that uses cartridges coated with platelet agonists to study aspirin and clopidogrel resistance.[45][46][47][48][49][50]

Specialized tests

• Light transmission aggregometry: Light transmission aggregometry is considered the reference and gold standard for assessing platelet function. This test involves photometric measurement of the ability of various agonists to induce in vitro platelet activation and aggregation. Platelet-rich plasma obtained through low-speed centrifugation is placed in a cuvette, maintained at 37 °C, and stirred with a small magnet between a light source and a photodetector. The signal of the platelet-rich plasma in the absence of any agonists is considered to have 0% permeability, corresponding to 0% aggregation. Autologous platelet-rich plasma is used as a calibrator at 100% permeability, corresponding to 100% aggregation. After adding an agonist, such as epinephrine, ADP, collagen, arachidonic acid, ristocetin, or thrombin analog, platelets aggregate, reducing the optical density of the signal and increasing light transmission detected by the photodetector. The change in optical density is depicted against time over a graph. Significant drawbacks of this method are its time-consuming process, the requirement for large sample volumes, dependence on operator expertise, numerous pre-analytical variables leading to methodological issues and significant fluctuations, lack of standardization, and the inability to mimic physiological blood flow processes at high-shear rates.

• Flow cytometry: Flow cytometry is a powerful technique for assessing platelet function in disorders such as Bernard-Soulier syndrome and Glanzmann thrombasthenia. The test uses monoclonal antibodies against platelet membrane glycoproteins and fluorescent dyes. The measurement can also be used to diagnose paroxysmal nocturnal hemoglobinuria. The sample is introduced into a fluidic system, where a laser beam is directed at the cells, and the light scattered from the cells is measured. In addition, fluorescence emitted by fluorochrome-labeled cells or particles is detected.

• Electron microscopy: Some rare platelet function disorders require visualization of ultrastructural changes with electron microscopy, such as δ-storage pool deficiency and grey platelet syndrome.

• von Willebrand factor panel: von Willebrand disease is initially diagnosed by a basic panel that includes measurement of FVIII, vWF antigen (VWF: Ag), and activity assays (vWF: Act). More specialized studies, such as multimer electrophoresis and vWF collagen binding capacity, may be required to subclassify von Willebrand disease into various subtypes.[10][51][52][53][54][55][56]
  • vWF: Ag: Measured using enzyme-linked immunosorbent assay (ELISA), latex particle-enhanced immunoturbidometric assay (LIA), and chemiluminescence. The ELISA method is based on the sandwich technique, using microplates with anti-human VWF antibodies and a second antibody labeled with an enzyme such as alkaline phosphatase or peroxidase. Recently, the LIA method with polystyrene microparticles was covered with mouse anti-human vWF antibodies that bind to the vWF in the patient's plasma. Both methods are relatively fast and reliable at low antigen levels. 
  • vWF: Act: Traditionally, vWF activity (vWF: Act) was studied using the ristocetin cofactor (vWF: Rcof). Ristocetin is an antibiotic that never reached the level of a commercial drug but has been used for decades to estimate the binding of platelet glycoprotein Gp1bα to vWF qualitatively. The ristocetin method has a high coefficient of variability (CV>20% to 30%) and can detect polymorphisms without clinical significance, such as D1472H. Recently, automated alternative ELISA and LIA methods have been developed to determine the functional binding of platelet glycoprotein GPIb to VWF (VWF: GPIbM), which has greater sensitivity and lower variability.
  • FVIII activity: The vWF antigen is a carrier of FVIII. The percentage of FVIII activity (VIII:c) is calculated with a one-stage clotting assay, as described below in the section on secondary hemostasis investigation. FVIII markedly decreases to near absence in type III vWF, distinguishing it from hemophilia A.
  • vWF multimers electrophoresis: High-molecular-weight vWF multimers have the highest hemostatic effectiveness. To distinguish between type 2 and 3 von Willebrand disease, multimer analysis is conducted using electrophoresis on sodium dodecyl sulfate agarose gels. This method is time-consuming, influenced by many pre-analytical factors, and only performed in specialized centers.
  • vWF binding capacity to collagen (vWF: CB): Various methods study the binding of high molecular weight vWF multimers to collagen, including ELISA, immunological methods with magnetic beads, and chemiluminescence. As the examination is sensitive to a high molecular weight multimer, this test may replace the vWF: Act assay. Most methods use collagen types 1 and 3, although collagen type 6 has recently identified mutations that reduce vWF binding to different types of collagen. However, this method does not measure the binding capacity of the factor to the GPIb receptor and, therefore, cannot replace the vWF: RCo assay. Using both methods increases the diagnostic value of the type 2 vWF. 
  • vWF propeptide (vWFpp): vWF propeptide is synthesized and released in a 1:1 ratio with monomers. Measurement of the propeptide helps identify pathological clearance of the antigen that affects only vWF: Ag and not vWFpp, such as vWF antibodies, and for type 1C vWF identification. This assay is performed only in a few specialized laboratories. 
  • vWF: FVIII binding capacity: VWF: FVIII binding capacity detects the binding capacity of the patient's vWF to recombinant FVIII in vitro using ELISA. The method identifies patients with disorders of VIII binding capacity, such as type 2N VWD (differential diagnosis with mild hemophilia A).[57][58][59][60][61][62][63][64]

Secondary Hemostasis Investigation

Screening tests

• Prothrombin time: Prothrombin time measures the activity of coagulation factors of the extrinsic and common pathways, such as FII, FX, FV, and fibrinogen. Prothrombin time is based on the time (measured in seconds) required for a fibrin clot to form after adding thromboplastin reagent, a mixture of tissue factor, phospholipids, and calcium, to citrated platelet-poor plasma. Prothrombin time results exhibit significant variation between laboratories that use different commercially available thromboplastins. To enable inter-laboratory comparison, the WHO has proposed an International Normalized Ratio (INR) and calibration with a reference thromboplastin with an International Sensitivity Index (ISI) of 1. Each manufacturer calibrates their thromboplastin based on WHO standards and reports the ISI for each reagent batch. The INR can be calculated by logarithmic conversion of measured prothrombin times to those of normal donors and patients under anticoagulant therapy using the reference thromboplastin and the specific thromboplastin in use. Ideally, manufacturers choose thromboplastins with ISI values close to 1. Thromboplastins with high ISI values are less sensitive to small changes in prothrombin time. The INR is used to monitor patients under anticoagulant therapy with coumarin anticoagulants.

• Activated partial thromboplastin time: The aPTT depends on the activity of intrinsic pathway coagulation factors, such as XII, XI, IX, and VIII; common pathway coagulation factors, such as V, X, II, and fibrinogen; contact system factors, such as prekallikrein and high molecular weight kininogen; and inhibitors and anticoagulant proteins. Plasma is incubated at 37 °C in phospholipids, such as cephalin, and an activator, such as kaolin, ellagic acid, or silica, leading to the activation of FXI. Subsequently, calcium chloride is then added, and the time (in seconds) for fibrin clot formation is measured.

• Fibrinogen: Fibrinogen is converted to fibrin through thrombin to produce the final clot. For the quantitative determination of fibrinogen, the Clauss functional method is considered a reference method. The patient's plasma is diluted to minimize the effect of any inhibitors present, such as heparin. A high thrombin concentration ensures that clotting time is independent of thrombin concentration over a wide range of fibrinogen levels. Reference plasma with a known fibrinogen concentration, standardized against a known international reference standard, is used in various dilutions to create a calibration curve. The clotting time measured after diluting the plasma and incubating at 37 °C, followed by the addition of thrombin, is compared to the calibration curve, and the fibrinogen concentration is determined and measured in mg/dL or g/L. Other immunoassay methods such as ELISA, immunodiffusion, and electrophoresis are used to measure antigen concentration and have diagnostic value in cases of dysfibrinogenemia.[65][66][67][68]

Specialized tests

• Thrombin time: Thrombin time is a test used to assess functional and quantitative fibrinogen disorders. This test measures the time required for a fibrin clot to form after a thrombin reagent is added to platelet-poor plasma at an incubation temperature of 37 °C to mimic physiological conditions.

• Reptilase time: Reptilase time is used in addition to thrombin time to exclude sample heparin contamination in cases of prolonged thrombin time. Reptilase is a serine protease from the venom of the Bothrops atrox snake (batroxobin), mimicking thrombin and cleaving fibrinogen's fibrinopeptide A. In contrast, thrombin cleaves both fibrinopeptides A and B. Adding reptilase to platelet-poor plasma activates thrombus formation, which is detected using optical or electro-mechanical methods.

• Mixing studies: Mixing studies are performed in cases of prolonged coagulation times to investigate the cause, whether due to a clotting factor deficiency or the presence of lupus anticoagulant. These studies can be performed for aPTT or prothrombin time. If prolongation occurs both times, mixing is performed at the time when the greatest prolongation is observed. A mixing test is recommended when at least a 5-second prolongation occurs to draw safe conclusions. Pooled normal plasma from healthy donors or commercially available normal plasma is used as a control for mixing. If the patient is prescribed heparin or novel oral anticoagulants, it is important to clarify this, as their presence affects most coagulation tests. If the patient's medical history is unavailable, thrombin time and anti-Xa activity can exclude the presence of anticoagulants. An equal amount of patient and control plasma is mixed, and the clotting time is measured directly and after incubation at 37 °C.

• One-stage clotting assays: One-stage clotting assays are coagulation factor assays performed when prothrombin time or aPTT is prolonged, typically after mixing studies. Factor assays determine the percentage (%) activity of individual clotting factors in patient samples. The method is based on the ability of the patient's plasma to correct a prolonged prothrombin time or aPTT of a reference plasma deficient in the factor under examination. Extrinsic pathway factors, such as FVII, and common pathway factors, such as FII, FV, and FX, are measured using one-stage prothrombin time-based methods. Intrinsic pathway factors, such as FVIII, FIX, FXI, and FXII, are measured using one-stage aPTT-based methods. The clot formation time is measured in the mixture of the test plasma (patient or control sample) with plasma deficient in the specific factor, typically in a 1:1 ratio. Calibration curves with successive dilutions of a reference plasma are used to calculate the concentration of the factor.

• Two-stage clotting assays (chromogenic): Commercially available two-stage chromogenic methods for FVIII, FIX, and FX that use chromogenic amidolytic substrates are available. Upon activating the inert zymogens in active serine proteases, a chromophore cleaves from the C-terminal end of a chromogenic substance, and the color change is measured at 405 nm. The change in optical density is proportional to the protein concentration. Measuring FVIII with chromogenic methods has the advantage of being able to distinguish falsely low concentrations of FVIII in the presence of inhibitors; monitor treatment with long-acting recombinant factor replacement therapy, such as pegylated recombinant FVIII; and identify mutations in FVIII in the A1-A2-A3 regions.

• FXIII assays: Hereditary and acquired FXIII deficiency is relatively rare, characterized by normal clotting times for prothrombin time and aPTT and delayed bleeding. FXIII consists of 2 subunits, an enzymatic subunit A and a non-enzymatic subunit B. Traditionally, the thrombin clot solubility test with urea was used. However, the test is abandoned due to the poor sensitivity of the method to detect only severe FXIII deficiencies <5%. Nowadays, quantitative methods are used to measure FXIII antigen A and B levels (ELISA) and FXIII function (ammonia release from FXIIIa transglutaminase and chromogenic methods). Severe FXIII deficiency can be additionally revealed as hyperfibrinolysis with hemostasis global assays.

• Prekallikrein: In prekallikrein deficiency, a marked prolongation of aPTT is observed, typically in a non-bleeding patient, which is corrected after incubation of the patient's plasma with aPTT reagents for 10 min before adding calcium chloride. Prolonged incubation time increases the activation of FXII in the absence of prekallikrein. Kaolin or ellagic acid are activators, not silica, as incubation with the latter reagent may not correct aPTT in prekallikrein deficiency. Chromogenic determination is required for confirmation.[27][69][70][71][72][73][74]

Screening for Acquired Coagulation Inhibitors

• Lupus anticoagulants: Lupus anticoagulants are a heterogeneous group of autoantibodies, typically immunoglobulin G (IgG) type, against protein-phospholipid complexes associated with increased thrombotic risk and antiphospholipid syndrome. Diagnostic guidelines have been proposed by various scientific societies, such as the International Society of Thrombosis and Haemostasis Scientific Standardization Committee (ISTH SSC), the British Committee for Standards in Haematology (BCSH), and the CLSI, without consensus. Several aPTT-based methods are available for detecting lupus anticoagulants, such as dilute Russell viper venom time, silica clotting time, hexagonal phase phospholipid neutralization (STACLOT-LA), Kaolin clotting time, dilute prothrombin time, and platelet neutralization procedure. Due to the heterogeneity of lupus anticoagulants, all guidelines recommend using at least 2 different detection methods, namely aPTT and dilute Russell viper venom time. Laboratory detection includes 3 stages—initial screening with dilute Russell viper venom time to reveal the presence of an antiphospholipid antibody; mixing test to demonstrate the presence of an inhibitor; and confirmatory test with APTT-based assay containing a high amount of phospholipids and silica as an activator to confirm phospholipid dependence of the antibody.

• Antiphospholipid antibodies: Laboratory criteria for antiphospholipid syndrome include 2 immunoassays (solid-phase ELISA) or chemiluminescence methods for detecting antiphospholipid antibodies, anticardiolipin antibodies, and anti-β2-glycoprotein I antibodies (anti-β2GPI) IgG and IgM.

• Specific coagulation inhibitors: Specific coagulation inhibitors are antibodies that target and inhibit certain coagulation factors, most commonly FVIII, leading to increased bleeding risk. This condition is often observed in older patients with acquired hemophilia or inhibitors against factor supplementation in hereditary hemophilia or in individuals with hereditary hemophilia who develop inhibitors against factor supplementation. Functional assays, either one-stage clot-based or chromogenic, can measure the residual coagulation factor after mixing the patient's sample with normal plasma. Because patients with acquired hemophilia typically present lupus anticoagulants together with anti-factor antibodies, if the chromogenic assay is not available, a lupus-insensitive aPTT reagent should be used in one-stage assays to avoid false-negative low-factor measurements. The Bethesda assay is used to measure the titer of FVIII inhibitors quantitatively. The assay measures the residual activity of FVIII after incubation with an equal amount of purine nucleoside phosphorylase for 2 hours at 37 °C in serial dilutions. One unit of Bethesda is defined as the amount of inhibitor that neutralizes 50% of 1 unit of FVIII:c in normal plasma after incubation at 37 °C. FVIII inhibitors are time-dependent, whereas other inhibitors, such as IX, do not require incubation. The Nijmegen modification method has improved specificity, detection of low-titer inhibitors, and greater FVIII stability during the 2-hour incubation with purine nucleoside phosphorylase.[75][76][77][78][79][80]

• Fibrinolysis investigation:
  • D-dimers: D-dimers are fragments resulting from the dissolution of the fibrin clot by plasmin after stabilization by FXIIIa and are widely used to investigate fibrinolysis. Many different assays are available, but they can be classified into 3 methods. Semi-quantitative methods detect macroscopic agglutination on polystyrene microparticles (latex). ELISA methodology is more sensitive and quantitative but time-consuming. The most widely used method is automated quantitative immunoassay with polystyrene microparticles that use antibodies against D-dimers. D-dimers have a high negative predictive value, and the negative result is commonly used in thrombosis exclusion algorithms.
  • The following assays are not routinely used in clinical practice to investigate fibrinolysis:
    • Plasminogen: Plasminogen is measured using a chromogenic method in excess streptokinase acting as an activator.
    • α2-AP: α2-AP measurement is performed using a chromogenic method in the presence of excess plasmin. The amount of plasmin inhibited is proportional to the amount of α2-AP in the patient's plasma.
    • PAI-1: PAI-1 measurement is performed using functional (chromogenic) and antigenic (ELISA and immunoassay) methods on automated analyzers. Normal daily variation is expected. 
    • Tissue plasminogen activator: The physiological role of tissue plasminogen activator is plasminogen activation into plasmin and, subsequently, degradation of fibrin into soluble degradation products. Functional measurement is performed using chromogenic assays and antigenic (ELISA) methods in excess PAI-1. Collection in citrate tubes in a slightly acidic environment is required for sample stabilization.[72][81][82][83][84]

• Global assays (viscoelastic) of hemostasis: Unlike traditional coagulation methods, global assays of hemostasis can provide rapid point-of-care testing and information on coagulation, clot stabilization, and fibrinolysis by studying the viscoelastic properties of whole blood. As whole blood is used, cellular interactions between platelets, red blood cells, white blood cells, and clotting factors can also be studied.
  • Thromboelastography (TEG®): Whole blood in citrated tubes is incubated at 37 °C in a special rotating cuvette, with a pin connected to a coiled wire. Calcium is added to initiate the clotting mechanism along with a specific activator. During thrombus formation, the fibrin produced mechanically binds the pin to the cup, and as the connection strengthens, the rotation of the cup is transmitted to the pin, and this torque is transferred from the wire and presented as a graphical representation. TEG-platelet mapping, a modification of TEG, is used to monitor antiplatelet therapy effectiveness in acute perioperative settings.
  • Thromboelastometry (ROTEM®): The methodology is similar to TEG, except in this case, the cuvette is stationary, and the pin oscillates and connects to a mirror that detects light reflection. A specialized detector records reflected light, processes samples on a computer, and converts the samples into a graphical representation. Five different channels are used with the addition of different activators—EXTEM with tissue factor controlling the extrinsic pathway, INTEM with ellagic acid for the intrinsic pathway, FIBTEM with cytochalasin D for platelet inhibition and fibrinogen estimation, APTEM with aprotinin inhibiting fibrinolysis, and HEPTEM with heparinase removing heparin from plasma. The main parameters used are clotting time affected by clotting factors and the time required to form a pre-determined level of clot strength, clot formation time affected by fibrinogen, FXIII, and, to a lesser extent, platelets. Similarly, the maximum clot firmness and the areas at specific times A10 and A20 represent interactions between fibrinogen, FXIII, and platelets. The lysis indices maximum lysis (ML), lysis at 30 min (LY30), and lysis at 60 min (LY60) reflect the presence of hyperfibrinolysis.[85][86][85][87]

Thrombin Generation Assay

Thrombin generation assay is a functional test on plasma with or without platelets that can monitor thrombin generation in real-time using a fluorogenic substrate that emits fluorescence when cleaved by thrombin. The resulting thrombin generation curve provides information on the kinetics and dynamics of thrombin formation, including parameters such as lag time (the time until thrombin generation begins), peak thrombin concentration, and endogenous thrombin potential.[88]

Thrombophilia Testing

• Antithrombin: Two-stage chromogenic assays using FXa or bovine FIIa are recommended for screening antithrombin levels. The residual activity of FXa or FIIa is measured through the enzymatic cleavage of a chromogenic substrate. Most methods use heparin and are referred to as heparin cofactor antithrombin tests (hc-anti-FIIs and hc-anti-Xa). In contrast, methods without heparin are progressive antithrombin activity measurement tests (p-anti-FIIa and p-anti-FXa). Many commercially available reagents have similar effectiveness. Further confirmation with antigenic assays (ELISA or LIA) or genetic analysis is needed in cases of confirmed low levels. The most common mutation detected is SERPINC1.

• Protein C: Commercially available methods for protein C include functional (coagulation-based) and chromogenic assays. Most laboratories use functional assays that rely on the ability of activated protein C to prolong the aPTT or Russell viper venom time. Chromogenic methods measure the ability of activated protein C to cleave the chromogenic substrate and are recommended for screening purposes.

• Protein S: Protein S circulates in plasma in 2 forms—free protein S and bound to protein C4b-BP. Only free protein is a cofactor of activated protein C. Three methods are widely used—functional assays, antigenic measurement of free protein S, and antigenic measurement of total protein S. Generally, antigenic measurement of free protein S through an immunoassay is recommended for screening. In doubtful cases, molecular testing and Sanger DNA sequencing are recommended.

• Activated protein C resistance: The V Leiden mutation causes FVa to be resistant to activated protein C, also known as APCR. The addition of activated protein C is expected to prolong aPTT due to the inactivation of Va and VIIIa in normal subjects. The method is based on measuring aPTT with and without the addition of exogenous activated protein C, and the ratio of clotting times is calculated (aPTT+APC/aPTT-APC).

• Genetic testing: The most common polymorphisms associated with inherited thrombophilia are factor V Leiden and the G20210A allele, increasing prothrombin levels. Factor V Leiden mutation renders factor V resistant to inactivation by activated protein C, leading to slowed inactivation and increased thrombin production. Testing for common polymorphisms of methylenetetrahydrofolate reductase (MTHFR), such as C677T and A1298C, is not recommended. Genetic testing is performed with polymerase chain reaction (PCR). Another useful genetic testing is the investigation of JAK2 and other mutations, such as JAK2 V617F, JAK2 exon 12, CALR, and MPL, in patients with unusual site thrombosis.

• Homocysteine: Homocysteine levels are measured using high-performance liquid chromatography and electrochemical or photometric methods. More recently, commercially available immunoassay methods have been utilized as they are more readily available and equally effective.[72][89][90][91]

Anticoagulant Monitoring

• Unfractionated heparin: Traditionally, unfractionated heparin anticoagulation monitoring is conducted using aPTT, but this method has many drawbacks due to a lack of standardized results between different laboratories, individual variations, and many confounding factors, such as high FVIII in the acute phase. Thus, a therapeutic target of aPTT 1.5 to 2.5 times the normal is no longer recommended, but rather, a measurement of anti-Xa activity is preferred. Chromogenic anti-Xa measurement is recommended.

• Low-molecular-weight heparin: Low-molecular-weight heparins (LMWHs) have more predictable pharmacokinetics and bioavailability compared to unfractionated heparin. Depending on the molecular weight of LMWHs, the ability to inhibit FIIa and FXa differs. aPTT is not a suitable test for monitoring LMWHs due to reduced anti-IIa activity. The effect of different LMWHs on aPTT is inversely proportional to the anti-Xa/anti-IIa ratio. Laboratory monitoring of anti-Xa activity using chromogenic methods at peak time, around 3 to 5 hours after administration, is recommended in pregnant women, patients with obesity, underweight patients, neonates, patients with increased bleeding risk or active bleeding, and patients with impaired renal function.

• Vitamin K antagonists: INR is used to monitor vitamin K antagonists, such as warfarin and acenocoumarol, as described above. 

• Novel oral anticoagulants: Novel oral anticoagulants have prefixed doses and do not require monitoring. Nevertheless, in cases of overdose, bleeding, need for anticoagulation reversal, or preoperatively, noting any residual therapeutic dose is important. Treatment with a direct thrombin inhibitor, such as dabigatran, typically leads to prolonged aPTT, although normal aPTT does not exclude the presence of clinically relevant drug levels. Thrombin time is a sensitive assay that detects low drug-level concentrations. In a few laboratories, measuring dabigatran levels is possible. On the other hand, treatment with anti-Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban, does not affect aPTT but prolongs prothrombin time in parallel with the drug concentration but variably according to the sensitivity of the prothrombin reagents. Normal prothrombin time does not exclude clinically relevant doses of anticoagulant. Chromogenic anti-Xa measurements using suitable calibrators can be used preoperatively or in situations where the reversal of anticoagulation should be decided.[92][93][92][94]

Antiplatelet Antibodies

Heparin-induced thrombocytopenia diagnosis is primarily based on clinical criteria and assessment of the preclinical probability with the 4T score. Laboratory testing should only be performed in cases of high clinical suspicion (high 4T score).[95]

• Immunological assays: Immunoassay methods detect the presence of antibodies against the complex of platelet factor 4 and heparin (specifically IgG or poly-specific IgG/IgM/IgA) but not their ability to activate platelets. The most commonly used method is ELISA in a solid phase. A high antibody titer is typically associated with heparin-induced thrombocytopenia. This method has high sensitivity, meaning a negative test indicates a low probability of heparin-induced thrombocytopenia, but low specificity, meaning a positive test cannot exclude the absence of heparin-induced thrombocytopenia.

• Functional assays: Functional methods can identify antibodies that activate the patient's platelets. They are performed only in a few specialized centers and used to confirm heparin-induced thrombocytopenia in cases where the result of immunoassay methods is doubtful or a discrepancy between clinical presentations. The immunoassay method is evident, for example, negative ELISA in a patient with high clinical suspicion and a high 4T score).
  • Serotonin release assay: The serotonin release assay is the gold standard for heparin-induced thrombocytopenia but is time-consuming and available only in a few specialized laboratories. This method is based on platelet activation of normal donors through serotonin release in the patient's serum and heparin. Platelets from normal donors are radio-labeled with 14C-serotonin and incubated with the patient's serum in therapeutic and higher doses of heparin. Positive results indicate the release of 14C-serotonin when therapeutic doses of heparin (0.1 units/mL) are used but not higher doses (100 units/mL). This discrepancy occurs because the binding of heparin-induced thrombocytopenia antibodies is specific to a particular ratio of heparin to PF4.
  • Heparin-induced platelet activation: In this method, serum or plasma from the patient with suspected heparin-induced thrombocytopenia is added to platelet-rich plasma from healthy donors, and the activation of platelets is measured through platelet aggregation in the absence and presence of low and high doses of heparin. The use of washed platelets increases the sensitivity of the method, making it the most widely used specialized confirmation method. A positive test shows minimal platelet activation in the absence of heparin and with high doses of heparin with pronounced activation in the presence of low-dose heparin.

von Willebrand Factor Protease Assays

The diagnosis of thrombotic thrombocytopenic purpura is based on a disintegrin and metalloproteinase with a thrombospondin type 1 motif, ADAMTS13 activity, and detection of autoantibodies. Commercially available chromogenic or fluorogenic ELISA estimates the ability of ADAMTS13 to cleave the synthetic molecule GST-VWF73. Inhibitory and non-inhibitory autoantibodies against ADAMTS13 can be detected using ELISA.[96][97][98][99][100]

Interfering Factors

Coagulation tests are subject to several interfering factors that may affect their accuracy and reliability, prolonging or shortening clotting times. The guidelines from the CLSI suggest discarding samples with excess hemolysis, lipemia, or icterus, as these abnormalities can interfere with clot formation or affect light transmission during analysis. As described above, careful patient preparation and sample preprocessing help mitigate sources of errors. Most errors stem from preanalytical factors, repeating abnormal results with a new sample. In addition, the effects of anticoagulant treatment should be considered, as these medications affect most coagulation assays.

In cases where medical history is unavailable, thrombin time can be useful for excluding the presence of heparin, LMWH, and direct thrombin inhibitors. Age is another significant parameter that affects coagulation studies, with infants and children having different reference ranges compared to adults—moreover, factors such as D-dimer, VIII, and VWF increase with age. D-dimer levels also rise in pregnancy, inflammation, sepsis, surgeries, hospitalizations, and cancer. In pregnancy, significant hemostatic changes occur to accommodate the increased demands of maternal physiology and prepare for childbirth. These changes prevent excessive bleeding during delivery, ensuring adequate blood flow to the placenta and fetus. Several coagulation factors such as FVII, FVIII, FIX, FX, and vWF increase, protein S and antithrombin decrease, whereas protein C slightly increases, fibrinolytic activity increases, and mild thrombocytopenia is common.[31][72][101][102] 

Common Factors That Affect Screening Tests

Polycythemia (Ht>55%) prolongs prothrombin time and aPTT unless the amount of anticoagulant in the tube is adjusted. C-reactive protein can prolong aPTT depending on the reagent. On the other hand, the acute phase reaction increases FVIII,  occasionally leading to shortened aPTT.[42] Similarly, patients with factor V Leiden may exhibit a shorter aPTT due to APCR, leading to a spurious reduction in protein S levels in functional studies. To avoid such pitfalls, measurement of the free protein S antigen is recommended for screening. Another interference is from lupus anticoagulants, which can artificially reduce coagulation factor levels in one-stage aPTT assays.[103] Lupus anticoagulant-insensitive aPTT reagent should be used to avoid this pitfall.

The blood group also affects vWF antigen levels. Blood group O shows 25% lower vWF: Ag levels compared to blood group A. Careful interpretation of results regarding vWF and multiple measurements are required as they are influenced by other factors such as pregnancy, oral contraceptive pills, hypothyroidism, menstruation, acute phase reactants, inflammation, and stress.[34][64] Platelet function assay closure time is prolonged by low hematocrit, low platelet number, diet, and several medications such as antidepressants and antibiotics.[48] In thrombocytopenia (PLT<150,000/μL), results in light transmission aggregometry may be affected. Smoking, exercise, and coffee consumption may also increase platelet aggregation. Circadian variation has been observed, with morning hours showing the highest results.[104]

Lupus Anticoagulants Interferences

Lupus anticoagulants are subject to various interfering variables in all stages of analysis. As platelets contain phospholipids, residual platelets in the plasma can neutralize lupus anticoagulant effects. Thus, double centrifugation of the sample is required to obtain platelet-free plasma. Different aPTT reagents vary in sensitivity due to differences in phospholipid concentrations and types of activators. False-positive or false-negative results in lupus anticoagulants may occur in patients under anticoagulant treatment with coumarin anticoagulants or novel oral anticoagulants and in the acute phase due to high FVIII and C-reactive protein levels. Medications such as antibiotics and antiarrhythmics have been associated with lupus anticoagulant positivity. Several manufacturers add heparin neutralizers in the reagent so that unfractionated heparin and LMWH do not interfere with lupus anticoagulants. In addition, different LMWHs affect aPTT-based lupus anticoagulant assays, depending on their anti-Xa/IIa ratio. Recently, methods have been studied to absorb in vitro novel oral anticoagulants before testing for lupus anticoagulants.[105]

Generally, obtaining lupus anticoagulants without any anticoagulation is recommended. However, in high-risk patients where stopping anticoagulation is not advisable, switching to LMWH may be more prudent, with testing conducted at least 12 to 24 hours after the last dose or before the next dose.[75][77][78] On the other hand, lupus anticoagulants may interfere with several coagulation tests. INR determination may be challenging, particularly by point-of-care testing devices in patients with antiphospholipid syndrome under vitamin K antagonists. In these cases, an alternative approach is measuring chromogenic factor Xa with a target range of 20% to 40% (corresponding to an INR 2-3). Unfortunately, this method is not readily available in most laboratories and is more expensive compared to INR. An alternative approach in high-risk patients with antiphospholipid syndrome under vitamin K antagonists is increasing the INR target between 3 and 4.[106][107] Antiphospholipid antibodies are not affected by the presence of novel oral anticoagulants. 

Thrombophilia Testing Interferences

Generally, thrombophilia testing should not be performed at the acute phase or under anticoagulation treatment. Only genetics studies with PCR testing can be safely performed without interfering. Functional studies are preferred as screening methods for antithrombin deficiency, as antigen levels may be normal in type II deficiency. No reagent can detect all types; for example, FXa-based methods are less sensitive in detecting specific mutations, such as Cambridge II, and hc-anti-Xa methods are more sensitive to other mutations, such as type II heparin-binding site). The origin of the thrombin reagent is also important, for example, bovine origin in hc-anti-FIIa methods. 

Antithrombin levels are decreased during pregnancy and the early hours after delivery, during treatment with unfractionated heparin or LMWH, in the acute phase, and in liver and kidney diseases. Protein C is falsely elevated in warfarin treatment. Factor V Leiden, activated protein C resistance, elevated FVIII levels, and lupus anticoagulants can lead to falsely low protein S levels in functional clotting methods.[108][109] In any case, repeated measurements should exclude preanalytical errors or other acquired factors. The activated protein C resistance phenotype is observed not only in factor V Leiden. Other mutations, such as factor V Liverpool (R485K), FVCambridge (R306T), and R2 haplotype, may present.[110] Acquired activated protein C resistance can be associated with pregnancy, oral contraceptives, cancer, lupus anticoagulants, and elevated levels of FVIII.[111]

Results, Reporting, and Critical Findings

Screening Tests and Coagulation Factor Assays

The reference range of normal values in coagulation tests varies depending on the manufacturer of different reagents. Generally, each laboratory establishes the reference ranges for each reagent or analyzer, considering the specific population studied, as age and ethnicity may be significant sources of variance. Sex differences are identified in global assays of hemostasis, with women more hypercoagulable compared to men.[112] Reference values for newborns and children are different and should be determined from relevant literature. Nevertheless, most laboratories agree on the following critical values for screening tests—PT >37 s, INR >5, aPTT >100 s, and fibrinogen <100 md/dL, as these values may be clinically significant and flag an increased bleeding risk. Considering the patient's clinical presentation and medical history to guide the requests for the appropriate testing is important.[113] 

Hemophilias

The normal adult range for most coagulation factors is between 50% and 150%. However, each laboratory has to establish the reference ranges for each reagent or analyzer. Reference values for newborns and children are different and should be determined from relevant literature.[72] FVIII and FIX critical values are categorized as follows—<50% increased bleeding risk following severe trauma/surgery (mild hemophilia), 1% to 5% increased bleeding risk after mild trauma (moderate hemophilia), and <1% increased risk for spontaneous hematomas and joint bleeds (severe hemophilia). These critical values do not apply to hemophilia C, as some patients with considerably low FXI levels may not experience bleeds.[114]

von Willebrand Disease

In von Willebrand disease, normal vWF values are between 50% and 200%, although levels of 35% to 50% are considered borderline and evaluated only in the presence of a bleeding history. Blood groups should always be investigated as blood group O exhibits up to 30% lower vWF levels. PFA-100 is a sensitive and specific screening tool for von Willebrand disease. Cut-off values depend on the laboratory, but generally, Epi >180 s and ADP >120 s are considered abnormal and may correspond to von Willebrand disease or, more rarely, Bernard-Soulier syndrome and Glanzmann thrombasthenia.[53][54] Type 1 von Willebrand disease is the most common, and the diagnosis is clear-cut when Act/vWF: Ag ≥0.7. Type 1 is the only type of von Willebrand disease with an appropriate desmopressin response. Type 1C patients with vWF exhibit significantly diminished vWF plasma survival, with half-life as brief as 1 hour, compared to the 8- to 12-hour half-life observed in healthy individuals and other type 1 patients. This characteristic can be identified during a desmopressin test, where there is a marked decrease in vWF antigen 2 to 4 hours after desmopressin injection.

When the vWF: RCo/vWF: antigen ratio is <0.7, this indicates type 2 von Willebrand disease. Although the vWF: RCo assay is pathological in types 2A, 2B, and 2M, only types 2A and 2B have pathological vWF: binding capacity. In contranst, type 2M is normal. Low-dose ristocetin-induced platelet aggregation detects the hyperreactivity of vWF using a lower dose of ristocetin than that used for the vWF: RCo assay to reveal type 2B von Willebrand disease. Multimer electrophoresis can confirm the specific subtype 2 of the disease. Simultaneously, testing for vWFAg and vWFpp can detect decreased vWF survival, a feature observed in acquired type 1C, type 2B, and specific type 2A cases. Mild thrombocytopenia is common in type 2B. Finally, vWF: FVIII binding capacity helps identify type 2N von Willebrand disease.[64] FVIII levels are significantly decreased in type 3 von Willebrand disease (differential diagnosis with severe hemophilia A) and type 2N von Willebrand disease, where a mutation in the vWF antigen prevents binding to FVIII.[115]  

Mixing Studies

Prolonged coagulation times require mixing studies to distinguish factor deficiencies from inhibitors. Failure to correct the clotting time during mixing indicates the presence of an inhibitor. Some inhibitors, such as FVIII inhibitors, are time- and temperature-dependent. In such cases, initial correction of the clotting time may be observed, followed by prolongation after incubation. In case of a clotting factor deficiency, clotting time correction is observed immediately and after incubation. However, clotting times tend to prolong after incubation due to the consumption of FV and FVIII. Hence, comparing the mixture with the platelet neutralization procedure assay is necessary.

The results can be estimated using the Rosner index = (1:1 mix PTT − platelet neutralization procedure PTT/patient PTT) × 100 or Chang index = (PTT patient − 1:1 mix)/(PTT patient − PNP PTT) × 100. Rosner index <12 refers to correction, 12 to 15 is the gray zone, and >15 is no correction. Chang index >75% shows correction, and <75% no correction. If no immediate correction occurs, an investigation for lupus anticoagulants is indicated. False-negative results in mixing studies can occur in weak inhibitors, and other mixing ratios, such as 1:4 mix, may be helpful in these cases. An aPTT reagent sensitive to lupus anticoagulants is chosen in ambiguous cases, or dilution is performed 4:1, for example, 400 μL patient + 100 μL platelet neutralization procedure assay. In case of suspicion of FVIII inhibitor, a Bethesda assay is ordered to measure the antibody titer.[116][117][118] 

Antiphospholipid Syndrome

The diagnosis of antiphospholipid syndrome requires both clinical and laboratory criteria. Recent updates to clinical criteria have expanded beyond thrombosis and pregnancy complications to include thrombocytopenia, cardiac complications, and microvascular thrombosis.[119] To assess the thrombotic risk, lupus anticoagulants can be interpreted as positive or negative and related to anticardiolipid and anti-β2GPI antibodies. Repeat testing of a positive result after 12 weeks is obligatory to confirm antiphospholipid syndrome.[120] Each testing laboratory should establish the reference ranges using samples from at least 40 healthy donors. A lupus anticoagulant test is considered positive if clotting time in the initial and mixing tests is >99th percentile compared to the normal population. Results are preferably expressed as the ratio of the patient's clotting time to the clotting time of normal plasma (normalized ratio) to ensure consistency. Generally, a dilute Russell viper venom time confirm ratio ≥1.2 is considered positive. In cases of weak lupus anticoagulant, aPTT in mixing studies may not be prolonged.

The results of antiphospholipid antibodies differ based on the laboratory method, such as ELISA or chemiluminescence. Anticardiolipin IgG and IgM antibodies are expressed in GPL and MPL, respectively, whereas anti-β2GPI are typically expressed in arbitrary U/mL. Samples at medium or high titers (>40 GPL or MPL, or >99th percentile) are considered positive if persistent after 12 weeks (reference range negative <10 GPL or MPL, 10-40 GPL or MPL weak positive). Triple-positive patients have the highest thrombotic risk. IgG antibodies are detected more frequently, and they are clinically significant.[75] Discordant isotypes between antiphospholipid antibodies (for example, the presence of anticardiolipin antibodies, IgM, and anti-b2GPI IgG) may be caused by laboratory errors. Isolated low-titer IgM antibodies are typically transient and not associated with high thrombotic risk.[121] 

Low-Molecular-Weight Heparin Monitoring

The therapeutic range of anti-Xa measurement for heparin is 0.3 to 0.7 IU/mL, but it lacks clinical validation and has considerable discrepancies among different laboratories. The target anti-Xa range for LMWHs depends on the specific LMWH and dosing regimen (once or twice daily) if used for prophylactic or therapeutic anticoagulation.[122][123] Recent guidelines from the American Society of Hematology (ASH) recommend against routine anti-Xa monitoring in treatment, even for high-risk patients such as those with renal failure or obesity, because of low evidence (conditional recommendation based on very low certainty in the evidence about effects).[124]

Clinical Significance

The clinical presentation of bleeding disorders varies depending on several factors. Primary hemostasis disorders typically present with mucocutaneous bleeding, such as easy bruising, epistaxis, heavy menstruation, and immediate bleeding following trauma or surgeries. In contrast, secondary hemostasis presents with deep tissue hematomas in muscles and joints and intracranial hemorrhage in severe factor deficiencies such as hemophilia A and B, which may occur early in life. However, in other factor deficiencies, symptoms may manifest later in life, with a significant variation in presentation from mild epistaxis or menorrhagia to delayed bleeding after surgeries, typically 1 to 2 days later. Spontaneous bleeding in hemophilia A or B is expected in severe factor deficiencies (<1%).[8][9] C factor levels do not correlate with bleeding symptoms in other hemophilias.[125] Hyperfibrinolytic disorders typically present with delayed bleeding after trauma or surgery, occurring a few days later. Umbilical stump bleeding has been reported in FXIII deficiency and rare afibrinogenemia.[27] Hypofibrinogenemias and dysfibrinogenemias may occasionally present with symptoms of thrombosis.[126]

Prolonged coagulation times do not always predict bleeding risk. A common clinical scenario involves a prolonged aPTT in a non-bleeding patient, typically due to lupus anticoagulants, which are found in 2% to 4% of the general population and more commonly in pediatric or geriatric populations, pregnant women, and patients with rheumatological disorders. In rheumatological disorders, lupus anticoagulants are frequently associated with antiphospholipid syndrome and increased thrombotic risk.[75] Patients with a history of thrombosis and persistent lupus anticoagulants with or without antiphospholipid antibodies in 2 separate measurements, 12 weeks apart, suffer from antiphospholipid syndrome.[76] Prolonged aPTT without bleeding or thrombosis may be less commonly associated with FXII or contact pathway factor deficiencies.[74][127]

Inherited and acquired thrombophilia testing includes screening for natural anticoagulant deficiencies, such as protein C, protein S,  and antithrombin factor; activated protein C resistance; factor  V Leiden; and FII G20210A mutations. Testing for antiphospholipid syndrome is reserved for patients with unprovoked venous thromboembolism, unexplained arterial thrombosis, obstetric complications, and other clinical criteria of microvascular thrombosis.[119] Quantitative disorders of FVIII, FIX, FXI, and FXII, and mutations in fibrinogen genes (FGG, FGA, FGB), PAI-1 (4G/5G), and the protective gene of factor XIII (Val24Leu) are not currently indicated for investigation. In addition, polymorphisms of the MTHFR gene (677C→T, 1298A→C) are not associated with thrombosis and are therefore not recommended for inclusion in the panel. Previous guidelines proposed homocysteine testing, but recent changes suggest this is not a strong risk factor.[128][129]

Whether thrombophilia testing assists in the therapeutic management of patients and medical decisions regarding the duration of anticoagulant therapy is unclear. Unfortunately, no randomized studies exist to examine whether thrombophilia testing contributes to improved patient outcomes, such as reducing venous thromboembolism recurrences, minimizing major bleeding during anticoagulant therapy, and enhancing patients' quality of life. Studies provide conflicting evidence regarding the risk of venous thromboembolism recurrence in cases of thrombophilia, except in the cases of antiphospholipid syndrome or natural anticoagulant deficiencies, where indications for an increased recurrence risk are strong. Additional testing for thrombosis in unusual locations is recommended for paroxysmal nocturnal hemoglobinuria and myeloproliferative neoplasms.[90] No consensus is available among these guidelines, and their recommendations are weak. Most physicians follow an individualized approach in their clinical decision-making, though a complete blood count with a peripheral blood smear is recommended in most, if not all, cases.

Clinical Correlations

• Thrombocytosis: Essential thrombocythemia
• Thrombocytopenia with large platelets: Immune thrombocytopenic purpura and other rare inherited thrombocytopenias with giant platelets
• Rare inherited thrombocytopenia with giant platelets: Bernard-Soulier syndrome, May-Hegglin anomaly, Alport syndrome, and platelet storage disorders
• Thrombocytopenia with tiny platelets: Wiskott-Aldrich syndrome and X-linked thrombocytopenia
• Platelet aggregates and platelet satellites: Pseudo-thrombocytopenia (repeat complete blood count in citrate vials)
• Leukemias: Presence of abnormal blasts
• Microangiopathic hemolytic anemias (disseminated intravascular coagulation and thrombotic thrombocytopenic purpura): Thrombocytopenia and schistocytes [45][53][130][131][132][133][134][135]

Prolonged Bleeding Time

• von Willebrand disease
• Platelet dysfunction: Bernard-Soulier syndrome and Glanzmann thrombasthenia
• Connective tissue disease: Hereditary hemorrhagic telangiectasia, Ehlers-Danlos syndrome, and Chediak-Higashi syndrome
• Medications: Nonsteroidal anti-inflammatory drugs, anticoagulants, antiplatelets, tricyclic antidepressants, and antibiotics
• Uremia
• Liver failure
• Diseases that cause thrombocytopenia <50,000/μL [47] 

PFA-100

• Interpretation
  • Prolonged EPI/COL (>180 s), normal ADP/COL closure time (<120 s)
    • Recommended medications: Aspirin, nonsteroidal anti-inflammatory drugs, anticoagulants, tricyclic antidepressants, and antibiotics
  • Prolonged both EPI/COL (>180 s) and ADP/COL closure time (>120 s)
    • von Willebrand disease
    • Platelet dysfunction: Bernard-Soulier syndrome and Glanzmann thrombasthenia
  • P2Y12 closure time <106 s
    • Clopidogrel resistance 
• Possible uses
  • Excellent screening tool for von Willebrand disease and some of the platelet dysfunction disorders (Bernard-Soulier syndrome and Glanzmann thrombasthenia)
  • Assessment of desmopressin response in von Willebrand disease
  • Assessment of presurgical bleeding risk [48][49]

Prolonged Prothrombin Time

• Vitamin K antagonists
• Xa inhibitors (rivaroxaban, apixaban, and edoxaban)
• Vitamin K deficiency
• Liver disease
• Disseminated intravascular coagulation
• Hypofibrinogenemia (acquired and inherited)
• Rare factor deficiencies: FVII, FV, FX, and FII (the most common deficiency is FVII)
• Lupus anticoagulants (rarely antibodies against prothrombin)
• Recreational drugs (synthetic marijuana) [65][134][136]

Prolonged Activated Partial Thromboplastin Time

• Heparin and LMWH (medical history or thrombin time)
• Direct thrombin inhibitors (dabigatran) and other anticoagulants (medical history)
• Liver disease: Ascites and alcohol drinking
• Disseminated intravascular coagulation: Critically ill patient or patient with cancer
• von Willebrand disease
• Hemophilia A, B, and C: Joint bleeds, deep hematomas, typically early in life in A and B
• Lupus anticoagulants: general population, children, women, pregnancy, rheumatological disorders, antiphospholipid syndrome, and history of thrombosis
• Specific factor inhibitors: Most commonly FVIII, abrupt onset of bleeding, mucocutaneous, gastrointestinal, retroperitoneal, intramuscular, genitourinary, and intracranial hemorrhage-ICH
• Other factor deficiencies without bleeding symptoms: FXII, high-molecular-weight kininogen, prekallikrein, and common pathway factors [66]

Low Fibrinogen

• Liver disease: Ascites and alcohol drinking
• Disseminated intravascular coagulation
• Hypofibrinogenemia: Acquired, rare, and inherited [67]

Prolonged Thrombin Time

• Heparin, LMWH, and direct thrombin inhibitors
• Hypofibrinogenemia: Acquired and inherited
• Rare cases of dysfibrinogenemia
• Paraproteinemias [69]

Reptilase Time

• Prolonged
  • Dysfibrinogenemia
  • Fibrin degradation products
  • Fibrinolysis products
  • Paraproteinemia
• Normal
  • Heparin contamination [70]

Light Transmission Aggregometry

• Glanzmann thrombasthenia: Absence or reduced response to all agonists (ADP, epinephrine, arachidonic acid, and thrombin) except ristocetin
• Bernard-Soulier syndrome: Absence or reduced response to ristocetin, large platelets in peripheral blood smear with thrombocytopenia
• von Willebrand disease: Reduced response to ristocetin
  • Type 2B: Increased response to low-dose ristocetin
• Storage pool disorder: Reduced response to all agonists (ADP, epinephrine, arachidonic acid, and collagen)
• Aspirin: Reduced or absent response to arachidonic acid
• Clopidogrel: Reduced or absent response to ADP [137]

von Willebrand Disease 

The disease typically presents with nose bleeds, menorrhagia, easy bruising, gastrointestinal bleeding, and excessive bleeding following procedures such as dental, tonsillectomy, and adenoidectomy, except the rare type III, which presents with joint bleeds such as hemophilia A or B.

• Type 1: vWF (vWF): albumin/globulin (A/G) & vWF: Act <30 IU/dL (or <50 IU/dL with bleeding symptoms), vWF: Act/vWF: A/G ≥0.7, vWF: CB ↓, VIII ↓ or ↔, desmopressin response
• Type 1C: vWF: A/G & vWF: Act <30 IU/dL (or <50 IU/dL with bleeding symptoms), vWF: Act/vWF: A/G ≥0.7, vWF: CB ↓, VIII ↓, Desmopressin response fails at 2 to 4 hours, vWFpp/ vWF: A/G >3
• Type 2A: vWF: A/G normal, vWF: Act< 50 IU/dL, vWF: Act/vWF: A/G< 0.7, vWF: CB ↓, VIII ↓ or ↔, loss of high- and intermediate-molecular-weight multimers in electrophoresis
• Type 2B: vWF: A/G normal, vWF: Act< 50 IU/dL, vWF: Act/vWF: A/G< 0.7, vWF: CB ↓, VIII ↓ or ↔, loss of high-molecular-weight multimers in electrophoresis, mild thrombocytopenia, low-dose ristocetin-induced platelet aggregation enhanced
• Type 2M: vWF: A/G normal, vWF: Act< 50 IU/dL, vWF: Act/vWF: A/G < 0.7, vWF: CB ↔, normal multimers in electrophoresis
• Type 2N: vWF: A/G & vWF: Act <50 IU/dL, vWF: Act/vWF: Ag> 0.7, VIII ↓↓, Normal multimers in electrophoresis, vWF: FVIII binding capacity↓
• Type 3: vWF: A/G & vWF: Act <5 IU/dl, VIII↓↓↓ <10 IU/dl, absent multimers in electrophoresis [10][64]

Hemophilia A and B are clinically indistinguishable, presenting with joint bleeds, deep hematomas, or intracranial hypertension, and the severity of symptoms depends on factor levels:

• <1% factor levels: spontaneous bleedings
• 1-5%: bleeding after mild-to-moderate trauma
• >5%: bleeding after severe trauma [8][9]

Bethesda assay or Nijmegen modification: Typically performed in acquired hemophilia (dramatic unexplained bleeding, extensive bruising, and large hematomas) and patients with known hemophilia that develop inhibitors (newly appeared breakthrough bleedings or altered pharmacokinetics of the replacement factor) and for the follow-up during treatment. More than 5 BU is considered a high titer.[138]

FXIII deficiency: Symptoms depend on factor levels, with severe bleeding in factor levels <15%. Common bleeding symptoms include delayed postoperative bleeding, impaired wound healing, and bruises in a patient with normal clotting times.

• Inherited: Umbilical stump bleeding, soft tissue, delayed surgical bleeding, and intracranial bleeding
• Acquired: Spontaneous or delayed life-threatening bleeding, idiopathic or associated with comorbidities [27]

Abnormal fibrinolysis is routinely screened with D-dimers and can be identified with global hemostasis assays.

Increased D-dimers

• Venous thromboembolism: Incorporated in pretest probability clinical scores
• Disseminated intravascular coagulation
• Acute promyelocytic leukemia
• Other: Pregnancy, cancer, hospitalization, sepsis, and surgery [81][139]

Low Plasminogen

• Hereditary quantitative or qualitative deficiencies of plasminogen
• Acquired deficiency: Disseminated intravascular coagulation, liver disease, and leukemia [82]

Low a2AP

• Rare congenital deficiency is associated with bleeding tendency in the first hours after injury
• Acquired deficiency: Liver disease and disseminated intravascular coagulation [140]

t-PA: Decreased levels are reported in acute myocardial infarction, deep vein thrombosis, and cerebrovascular accidents. Increased levels are observed in the acute phase, cancer, postoperative period, pregnancy, metabolic syndrome, and sepsis.[141]

PAI-1: Increased PAI-1 levels are associated with acute myocardial infarction, deep vein thrombosis, pregnancy, sepsis, cerebrovascular accidents, type 2 diabetes, and disseminated intravascular coagulation. Decreased levels are observed in severe liver disease, amyloidosis, and fibrinolytic therapy.[142]

Thrombophilia testing: Several guidelines provide recommendations regarding thrombophilia testing. The consensus is that thrombophilia testing should be performed when the result could lead to a change in the patient's treatment. Testing should be considered in unprovoked venous thromboembolism, unexplained arterial thrombosis, and obstetric complications (see Table. Clinical Guidelines Regarding Indications for Thrombophilia Testing and Duration of Treatment Anticoagulation). In rare cases, JAK-2 genetic testing and paroxysmal nocturnal hemoglobinuria testing (CD55, CD59 testing with flow cytometry) are also considered. Testing is considered in cases of unexplained hemolysis or pancytopenia.[89][90][143]

Antiphospholipid Syndrome 

Indications for screening are as follows:

• Unprovoked venous thromboembolism in patients younger than 50.
• Unusual site thrombosis
• Arterial thrombosis otherwise unexplained in patients younger than 50 (myocardial infarction, ischemic stroke, transient ischemic attack, and other organ infarcts)
• Microvascular thrombosis
• Recurrent venous thromboembolism under anticoagulation unexplained by subtherapeutic anticoagulation, non-adherence, or malignancy
• Pregnancy-related complications, including fetal loss after 10 weeks of gestation, repeated early miscarriages, prematurity (before 34 weeks gestation) linked to severe pre-eclampsia, HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet levels), placental insufficiency resulting in fetal growth restriction, and stillbirth.
• Systemic lupus erythematosus
• Cardiac valve thickening or vegetation otherwise unexplained
• Unexplained thrombocytopenia (PLT >20,000/μL).[119]

Patients with the following characteristics are considered high-risk antiphospholipid syndrome:

• Triple positive
• Double positive (lupus anticoagulant negative, anticardiolipin antibodies, and anti-b2GPI positive)
• IgG AcL, anti-b2GPI (vs IgM)
• High IgG titer (versus low IgG titer) increases thrombotic risk. In obstetric antiphospholipid syndrome, low-titer antibodies may have clinical significance.[121]

Global hemostasis assays are considered in the following clinical settings:

• Perioperative bleeding
• Trauma
• Peripartum bleeding
• Guiding transfusion needs
• Conditions with hyperfibrinolysis [85][87]

Interpretation requires adequate training and expertise. Several diagnostic and management algorithms exist to support clinical decisions. Interdisciplinary communication is essential to improve patient outcomes. Some examples of abnormal global hemostasis assays are given below:

• Hyperfibrinolysis: EXTEM ML≥15% or FIBTEM ML≥10%, LI60≤85%
• Hypofibrinogenemia: FIBTEM A5< 5 mm or A10< 10 mm
• Fibrin polymerization disorder (low fibrinogen, low XIII): EXTEM A5 <35 mm and FIBTEM A5 <9 mm
• Thrombocytopenia or severe platelet dysfunction: EXTEM A5 <35 mm and FIBTEM A5 ≥9 mm
• Hypercoagulability: EXTEM CT <45 s, EXTEM MCF >68 mm, FIBTEM MCF >22 mm, EXTEM LI60 ≤3%
• Vitamin K factor deficiency: EXTEM CT >80 s [144][145]

Thrombin generation assay: The assay is not widely available in all laboratories and has primarily been used in research settings, such as evaluating new anticoagulant drugs. Interest in monitoring hemophilia A treatment is increasing with the new VIII mimetics drugs such as emicizumab. The International Society on Thrombosis and Hematosis (ISTH) has recently proposed a standardized approach to improve the precision and reproducibility of the method.[146][147] 

Table 1. Clinical Guidelines Regarding Indications for Thrombophilia Testing and Duration of Treatment Anticoagulation

Quality Control and Lab Safety

Quality control in coagulation studies is essential to maintain accuracy and precision. Quality control encompasses procedures in clinical laboratories to oversee the functioning of testing processes, identify potential errors, and rectify issues before results are reported. Specifically, everyday internal quality control (IQC) and 3 to 6 monthly external quality assessment programs are utilized to assess and enhance quality standards in coagulation.[154]

Enhancing Healthcare Team Outcomes

Interprofessional communication is crucial in evaluating coagulopathies, given the expertise required for accurate assessment and management. Hemostasis disorders increasingly intersect with several medical disciplines, and close collaboration with the laboratory is essential for interpreting results, avoiding diagnostic pitfalls, and preventing medical misdiagnosis. Moreover, with our evolving understanding of complex hemostatic mechanisms and the emergence of new laboratory tests, education across all medical specialties is paramount for enhancing diagnostic accuracy and optimizing patient management. 

An ethical concern arises regarding the allocation of responsibility in such interprofessional cases and situations where clinical guidelines are weak, or consensus is lacking. Clinicians must rely on the best available evidence, expert consensus, and patient factors to guide decision-making, involving interdisciplinary discussions, consultation with hemostasis experts, consideration of patient preferences and values, and shared decision-making with patients and their families. Overall, ethical considerations in patients with hemostasis problems emphasize the importance of patient-centered care, interdisciplinary collaboration, evidence-based practice, and respect for patient autonomy and dignity. By upholding these principles, healthcare professionals can navigate complex ethical dilemmas and optimize outcomes for patients with hemostasis disorders.