Thrombophilia screening revisited: an issue of personalized medicine

Clinical thrombophilia is the consequence of multiple gene and/or environment interactions. Thrombophilia screening requires a targeted patient with specific indication, in which a finding would have implications. Carrying out a thrombophilia examination in the physician’s practice is often a cause of uncertainty and concern. The concerns begin in choosing the right patient to be examined, are associated with the time of investigation, with the choice of analysis, the test-material and with the correct interpretation of the results. Difficulties, which can influence the results, can occur with both organization and blood sampling. As common for any analysis, pre-analytical, analytical and post-analytical factors should be considered, as well as the possibility of false positive or false negative results. Finally, recommendation of correct therapeutic and prophylactic measures for the patient and his relatives is an additional focus. In this article we want to provide—on the basis of the evidence and personal experience—the theory of thrombophilia-investigation, the indications for testing, as well as practical recommendations for treatment options.


Introduction
Thrombophilia-θρομβοφιλία-originating from Greek is a term meaning both affinity (-philia: φιλία) and blood clot (θρομβο). Thrombophilia indicates an increased tendency to form pathological intravascular venous or arterial thrombosis, mainly as a consequence of interaction of multiple inherited and/or acquired predisposing factors [1]. The coagulation system-usually in a balance between pro-and anticoagulant influences-shifts towards a prothrombotic state which may become clinically manifest as a thromboembolic disease [2]. The clinical complexity to understand the in-vivo mechanism(s) shifting the coagulation into a pro-thrombotic state-i.e. a state with an excessive thrombin generation-is the result not only of numerous coagulation factors and their interactions, but also the result of their dynamic interactions with blood vessels, endothelial cells, platelets and other cells in the circulation. Venous stasis from impaired blood circulation was already described from Rudolf Virchow in 1856 as one of the main etiologic factors for venous thrombosis [3]. Although the role of venous stasis was accepted, the link between stasis and thrombosis for example after a long flight or immobilization remained for long time elusive [4][5][6]. A reduction of the fibrinolytic capacity is conceivable [7] but evidence in the human model is lacking [8]. The interactions between coagulation and other humoral systems such as complement [9] and immune system [10] are ongoing and complex. The risk of thrombosis increases ultimately with age as a consequence of punctual and constant influencing factors [11]. Venous thromboembolism (VTE) is therefore considered a multifactorial disease [12] and the final clinical sign of interaction of single or multiple genetic, epigenetic and/or acquired predisposing factors [1,13]. Therefore, focusing on and screening for hereditary thrombophilia should represent a comprehensive evaluation of the patient's pro-thrombotic state and not a purely laboratory testing.
Despite the association of genetic risk factors with VTE, screening for inherited thrombophilia has not shown a direct clinical benefit in the management of these patients [14]. The lack of preventive and therapeutic consequences after the first thrombotic event reduced the indications for thrombophilia screening [15,16]. Indeed, not testing blindly for thrombophilia in patients with VTE is on the Choosing Wisely list endorsed by many scientific societies [17][18][19]. But, it seems that the books are still not closed. In the era of big-data acquisition and genome-wide association studies (GWAS), it might be that newer aspects concerning risk evaluation for thrombosis come into consideration, based on the influence of newly identified genetic variations linked to thrombosis.
In this contest, in order to act against an indiscriminate, universal, population screening and to limit medical expenses we suggest to first carefully select the patient. In this review, based on evidence and personal clinical experience, we propose arguments favoring an individual decisionmaking regarding thrombophilia screening only in selected patients with VTE.

Venous thromboembolism
VTE includes the deep venous thrombosis (DVT) in typical or atypical localization and pulmonary embolism (PE). VTE may be divided between provoked (secondary) and unprovoked (idiopathic) ( Table 1). Risk factors for provoked VTE are major, minor, transitory (reversible) or persistent (irreversible) ( Table 2). Differentiation between idiopathic and secondary VTE is important because it affects the decision on the duration of antithrombotic therapy [20].

Inherited thrombophilia
Genetic risk factors for VTE are deficiencies of natural anticoagulant proteins (antithrombin deficiency [21], protein C deficiency [22], protein S deficiency [23]), genetic dysfibrinogenemia [24], hyperhomocysteinemia [25] or mutation of factor II (F2, G20210A mutation [26]) or factor V-Leiden (F5, G1691A mutation [27]). GWAS revealed several additional genetic polymorphisms with borderline but measurable statistical association with VTE [13,28]. The non O-blood group status is in this sense the most common mild predisposition [29][30][31]. Two VTE associated loci,  [32]. A comprehensive review about the genetics of VTE describing the allele prevalence and influence on VTE of various genetic variances was recently published [33]. The ThromboGenomics group in the United Kingdom using next generation sequencing and a high-throughput screening panel for genetic analysis of patients with coagulation, platelet or thrombotic disorders was able to identify a genetic diagnosis in 48.9% of patients with thrombotic disease [34].
Hereditary thrombophilia predisposes to an imbalance of the coagulation mechanisms. The cumulative prevalence of hereditary thrombophilic defects in the general population is not rare [35,36] (Table 3). Still, these defects should be excluded only in patients with a specific indication. Inherited thrombophilia in combination with acquired thrombophilic risk factors-which may be transient or persistent [37]may lead to VTE at young age. Inherited thrombophilia has a different impact on the relative risk of first VTE or recurrence: hereditary thrombophilia increases the relative risk of    the first thrombosis, while the risk of recurrence is marginally but still not negligibly affected [38] (Table 4).

Inherited thrombophilia screening in children
The risk of VTE in children is low with two peaks in neonates and adolescents [39]. Risk factors for VTE in these two categories of patients are sepsis, dehydration, congenital heart failure, congenital anomalies of vena cava inferior and the use of catheter interventions [40]. In addition, cancer, polychemotherapy, immobilization after surgery or plaster casts, obesity, rheumatic disease, infection and use of oral contraceptive pills in girls were the leading triggers in adolescents [41]. Although one or more of these risk factors are often present in the majority of children with VTE, inherited thrombophilia is significantly associated with the first VTE [41,42]. Furthermore, a significant association with recurrent VTE in children was found for protein C, protein S, and antithrombin deficiency; the factor II mutation and the combination of two or more genetic traits [41]. In neonates with purpura fulminans, skin necrosis or idiopathic VTE, as well as adolescents with idiopathic VTE, thrombophilia screening is strongly recommended [43].

Thrombophilia screening in adult patients
Thrombophilia screening-generally performed after a thromboembolic event-is not only a laboratory test for inherited diseases. Thrombophilia screening in adults should be a comprehensive evaluation of the patient's pro-thrombotic state, allowing to determine the etiology of VTE, to estimate the risk of recurrence, to recommend therapy or prophylactic measures for patient or descendants. These objectives cannot be achieved by purely performing laboratory tests. The clinical evaluation of thrombophilia is based on personal and family history, clinical examination and basic laboratory diagnostics. It is a physician's task to advance step by step in the evaluation and then to decide if indicated to perform-or not-genetic tests. We suggest to split-up the thrombophilia screening into clinical thrombophilia evaluation and laboratory testing, in particular genetic ( Fig. 1).

Clinical thrombophilia evaluation
All patients with first VTE-i.e. Index patient-should be evaluated clinically for thrombophilia. This starts with a careful personal and family history for venous (or arterial) thrombosis, past clinical history, associated diseases and their prognosis, evaluation of acquired risk factors for VTE and the therapy's adverse effects. Full physical examination is indicated with particular attention to habitus, skin, cardiovascular i.e. peripheral venous, arterial and lymphatic, lung, abdominal, and neurological systems. Based on information such as age, thrombus localization, i.e. arterial, venous or paradoxical embolism and acquired predisposing risk factors the treating physician decides on the next steps. A basic laboratory test is indicated in all patients (Table 5). An important issue at this point is to carefully select the patient who should be screened with supplementary thrombophiliaspecific laboratory tests. Clinical VTE is multifactorial and the sum of gene-gene and/or gene-environment interactions which lead to overcome a threshold and trigger the event [44]. In this framework of predisposing factors inherited thrombophilia interacts dynamically with environmental factors, which are partly modifiable. Therefore, the single contribution and finally the risk of VTE may change over time [12]. Acquired predisposing factors for VTE-that may be classified in major/minor and persistent/transient (Table 2)-play a different role in the presence of an inherited thrombophilia. Indeed, a minor risk factor may be sufficient to trigger an episode of VTE in a young patient typically in association with a genetic risk factor. On the other hand, most patients over 60 years may develop a VTE in the presence of one or more acquired risk factors, even without an inherited thrombophilia. All predisposing factors that-alone or in combination-increase the risk of VTE should be screened or excluded ( Table 2). The combination of two or more factors increases the risk of thrombosis multiplicatively.
In the case of a patient aged > 60 years and/or one or more robust causes of acquired thrombophilia the physician should omit thrombophilia testing, particularly genetic tests. It is important to determine if VTE was triggered by a strong risk factor, i.e. antiphospholipid syndrome, malignancy and so forth. Age and acquired thrombophilia are the most important factors influencing the decision to exclude or not an inherited thrombophilia, including genetic screening.

Personalized thrombophilia evaluation
Personalized thrombophilia evaluation begins with consideration of patient's characteristic. Starting with age, i.e > 60 years, 50-60 years or < 50 years and sex the clinician may take a first decision. Because age is a major risk factor for VTE, frequently associated with other persistent or transient risk factors (Table 2), the probability of an underlying genetic cause is very low. Therefore, in patients over 60 years genetic screening should not be performed because not indicated (Table 7).
In patients younger than 60 years, risk factors for VTE should be excluded: in the presence of one major or multiple minor risk factors, no other diagnostic procedures are indicated.
In patients younger than 50 years without apparent risk factors, additionally cancer, PNH or an autoimmune disease should be considered [45]. VTE can be the first presentation of malignancy and symptomatic deep-vein thrombosis is associated with a risk of subsequent overt malignant disease [46]. We suggest personal, familial history, physical examination and starting from clinical symptoms an ageand gender-specific screening [47]. We suggest a limited and against an extended cancer screening [48].
In younger patients, especially under 40 years, additional risk factors like illicit drug consumption or therapy (including cocaine, estrogens, testosterone, erythopoietin) should be considered and excluded. Rare diseases (Klinefelter syndrome, Behcet disease, Hughes-Stovin syndrome, antiphospholipid syndrome, cancer and hematological diseases (i.e. sickle cell disease or neoplasm including leukemia) should be excluded based on clinical probabillity and Table 6 Proposed indications for venous thrombophilia screening Adapted from [18] and [19] Idiopathic VTE < 50 years Young patients with arterial ischemia caused by paradoxical embolism (right-to-left shunt) VTE in unusual sites Women with VTE during pregnancy or puerperium Women with VTE during use of oral contraceptive or hormonal replacement Women with VTE before prescribing hormonal replacement Women with multiple inexplicable pregnancy losses Young women with a strongly positive family history, before prescribing oral contraceptive First VTE and a positive family history for VTE first laboratory screening (Table 5). It should be kept in mind that a VTE (or an arterial TE by paradoxical embolism) can be the first manifestation many years before the diagnosis of a hematological disease, including the myeloproliferative neoplasms. Patients should be clinically followed and all transient or provoking risk factors avoided.
In younger patients with unprovoked VTE genetic thrombophilia testing should be considered (Table 6). This practical personalized approach can be summarized as follow: -> 60 years: no genetic thrombophilia screening (Table 7) -50-60 years: consider what risk factors for VTE are present (Tables 1 and 2) and-if possible-avoid -< 50 years: consider if thromboembolism (TE) may be the first cancer manifestation, including hematologic and myeloproliferative neoplasm and PNH -< 50 years, female patients: consider as well autoimmune disease (i.e. Lupus Anticoagulans, antiphospholipid syndrome, Table 2) -< 50 years, male, idiopathic: perform genetic thrombophilia screening -< 40 years: consider illicit drug consumption or therapy (including cocaine, testosterone, erythropoietin) -< 40 years female: exclude minor transient risk factors (

Genetic thrombophilia testing
Genetic thrombophilia screening-indicated only in selected patients [49]-include inherited deficiency of the natural anticoagulants protein C, protein S, antithrombin, and the mutation of factor V Leiden and the prothrombin gene. Blood group, hyperhomocysteinemia and dysfibrinogenemia are other inherited causes of thrombophilia (Table 4). In the search for a possible hereditary thrombophilia we recommend adherence to the above-mentioned main defects. The heterozygous or homozygous mutation of methylenetetrahydrofolate reductase (MTHFR)-677C → T, which in the past was considered, has not been confirmed as a risk factor for the first VTE or for relapse (either alone or in combination with the F V Leiden [50,51]) and its determination is redundant [52]. A genetic predisposition to VTE is supposed in the following cases: (1) in patients with VTE before 40 years of age, (2) VTE at a young age-before 50 years-with a weak risk factor, (3) considerably positive family history of VTE in two generations, (4) VTE in an unusual site. Table 6 highlights the proposed indications for a venous thrombophilia screening. Thrombophilia screening is also important in order to estimate the risk of recurrence according to baseline risk factor profiles by detection of acquired thrombophilia. Major risk factors like malignancy or antiphospholipid syndrome influences the thrombosis risk and have clinical consequences regarding the anticoagulation therapy.
In conclusion, a clinical thrombophilia evaluation is indicated in all patients with VTE. According to age, acquired predisposing risk factors, duration of therapy and the patient's requirement the physician determines the clinical likelihood and the indication-or not-to search for an inherited thrombophilia.

Avoid universal screening
Universal screening of the general population to assess the venous thrombosis risk is unjustifiable [53], expensive and should be avoided [54,55]. Consensus exists that universal unselected screening of young women before prescribing estrogen containing oral contraceptives is clearly not indicated [56]. The low prevalence in the general population of thrombophilia genetic defects requires a high number of patients to find out a mutation and even more to avoid one VTE. A source of trouble in clinical practice is the incorrect prescription of the screening test: some tests are frequently omitted and a negative thrombophilia screening may give a false hope. We strongly suggest to avoid thrombophilia screening in this scenario, especially true partial screening, because it is clinically futile and expensive [57]. First, we advise to assess the personal and patient's family history of VTE. If a relative has had a VTE, it is important to ask at what age and in what context. A strong family history of VTE and/or relatives with VTE before 50 years make inherited thrombophilia more likely and laboratory testing should be certainly considered. We advise, whenever possible, to

Selected screening
Knowledge of inherited thrombophilia may be important for young patients to avoid supplementary risk factors for VTE. Particularly smoking, dehydration, immobilization, estrogen containing medication (combined oral contraceptives or postmenopausal hormone replacement) should be avoided [37]. If chemotherapy is necessary, the treating physician should evaluate drug prophylaxis against VTE. After stopping anticoagulant therapy, in risk situations like pregnancy, acute illness, surgery, immobilization or long flights over 4 h a thromboembolic prophylaxis should be evaluated and respectively given [20]. Knowledge of the relative risk of possible pregnancy complications associated with the thrombophilic trait help in the clinical decision and management [58]. Particularly, a prophylaxis with low molecular weight heparin, Aspirin, substitution of coagulation factors and/or other management procedures should be implemented according to anamnesis, genetic defect and thromboembolic risk [59]. For each patient assessment of the individual risk during pregnancy and puerperium and a comprehensive evaluation of potential pregnancy complications depending on the genetic defect may help in the clinical decision [60]. Moreover, diagnosis of antiphospholipid syndrome either isolated or associated to other autoimmune disease like systemic lupus erythematosus (SLE) in females of childbearing age is important to evaluate preeclampsia, fetal loss, and preterm birth that are well-known risks in such pregnancies [61]. Women with antiphospholipid antibodies or antiphospholipid syndrome and lupus nephritis represent a group with high risk for obstetric complications. Factors such as appropriate preconception counseling, medication adjustment, strict disease control prior to pregnancy, and intensive surveillance during and after pregnancy are essential to improve pregnancy outcome and ensure the best maternal and fetal prognosis.

Family screening
Detection of an inherited genetic defect induce a physician's reflection about family screening. Thrombophilia screening in relatives should be selective and performed after adolescence or before prescribing birth control pills, especially to young Index patient's daughters. All other young family female members should be additionally evaluated for possible screening. The relatives should be informed that a genetic mutation is only one risk factor predisposing to venous thromboembolic disease. Not all patients with the same mutation will develop a thrombosis or PE or will become ill.
Index patient's relatives undergo a targeted thrombophilia screening, i.e. just the detected defect should be ruled out. Degree of relationship, age, therapy, indication or contraindication for anticoagulation should be included in the evaluation before performing the screening. As indicated above, potential risk of false positive results and over-treatment should be kept as low as possible. Therefore, vitamin K-deficiency in young women, drugs with potential interactions with the tests, circadian variation of the coagulation factors should all be considered in the final evaluation [62]. All results of functional tests outside the normal reference ranges and suspect as a deficiency should be confirmed in a second blood sampling, at the earliest after one month.
First degree relatives should be informed-concerning the detected defect-about the relative increased risk of first VTE, transient additional risk factors, possible thromboembolic prophylaxis ( Table 5). Evaluation of VTE risk should be regularly repeated and adapted based on age, on new risk factors or illness. Members of families with strong positive VTE's history but negative thrombophilia screening require comprehensive information. Thus, transient risk factors should be avoided whenever possible and women should receive an alternative medication to estrogen-containing pills or devices.

Regarding thrombotic risk
Goals of thrombophilia screening are the search for possible causes of thrombosis [37, [63] and the identification of the patients who could benefit from a continuous anticoagulation after the first event. Other aims of the investigation are the identification of family members, at which VTE can be prevented by avoiding risk factors and/or with drug prophylaxis, as well as the counseling of patients, relatives and supervising physicians. The following parameters are helpful in the estimation of the individual recurrence risk for VTE and for the decision of the duration of anticoagulation [

Regarding therapy of thrombosis
The thrombophilia investigation should not be performed with the individual goal of defining the duration of the anticoagulation therapy. In the comprehensive assessment and advice, in addition to the risk of recurrence, the risk of bleeding under anticoagulation therapy should be estimated and the individual preference of the patient included.
The evidence shows a subordinate role of thrombophilia in the prediction of a recurrence of thrombosis. Three prognostic models for individual VTE recurrence risk after discontinuation of anticoagulation after one idiopathic VTE-HERDOO2 score, "Vienna prediction model" and DASH score-do not take thrombophilia into account [78]. In the score of Franco Moreno et al. [79] the genetic thrombophilia is statistically significant, but only as a retrospective observation, no prospective validation was performed with this score. If evidence of hereditary thrombophilia is present with possible consequences for offsprings, especially women of childbearing age, a family status (targeted partial thrombophilia evaluation) is recommended. It helps in the decision in risk situations for preventive action only by means of conservative measures (e.g. compression stockings, hydration, abstinence from estrogens) or even to apply thromboprophylaxis with medication. In addition, hereditary thrombophilia is an important component of thrombotic risk scores, such as "Caprini" and "Rodgers" for the risk stratification of patients within the context of perioperative thromboprophylaxis [80].

Role of a positive diagnosis of thrombophilia regarding thrombosis prophylaxis
If a thrombophilic variance is detected in a family member, the question arises how to proceed concerning prophylaxis. The first risk stratification of patients is based on the personal history. In addition, physical habitus, age, type of coagulation anomaly, type of mutation (heterozygous or homozygous), underlying disease and other risk factors should be taken into consideration. In these patients, we generally recommend to avoid immobilization and/or dehydration. Especially with varicose compression stockings in risk situations (immobilization, travel longer than 4 h, pregnancy, postoperatively) are recommended. In these risk situations we recommend a careful drug prophylaxis and in women we favor against the prescription of estrogen-containing preparations (Table 8).

Role of a negative diagnosis of thrombophilia regarding thrombosis prophylaxis
If the family history is clearly strong positive and no laboratory thrombophilia is proved, the prophylactic measures are recommended as above. Even more, if the personal medical history is also positive for VTE.

Role of thrombophilia in the context of a pregnancy
The physiological adaptations of the body, the blood circulation and the coagulation during pregnancy increase the risk of thrombosis. Although the risk is influenced ante-partum especially by the BMI, age, number of births, varicosis and post-partum due to premature birth, cesarean section and hemorrhage, the VTE risk in women with hereditary thrombophilia and positive family history is especially high. Possible obstetric complications in the presence of thrombophilic defects are e.g. pre-eclampsia in antithrombin-or protein S deficiency or the fetal growth retardation in factor V Leiden and prothrombin gene G20210A mutation [81]. The assessment of the personal risk prior to the initiation of medication for thromboembolic event prophylaxis and regular checks during pregnancy are indicated for these patients. Start (24th week of gestation or earlier), dose and duration of prophylaxis are individual to decide. We recommend the prophylaxis with low molecular weight Heparins (LMWH), risk and weight-adapted (usually 75-100 IU/kg body weight/day), until the onset of labor pains. An interval of 12 h from the last LMWH low-dose is enough to carry out a spinal anesthesia. An interdisciplinary management of these patients with involvement of gynecologists, midwives, anesthesiologists and hematologists during pregnancy, at delivery and in the post-partum period is strongly recommended.

Future diagnostic perspectives
High-throughput sequencing methodology is now available and affordable for every-day genetics [82]. In the era of the GWAS it is possible, that genetic cohort analysis of pre-specified patients or healthy persons can reveal single polymorphisms, which alone or in combination are associated with thrombotic risk. Genetic risk scores or clustered panels of thrombotic genes with respect to this have already been published [34,83,84]. The benefit of such a wide genetic analysis, though, remains uncertain. Many of such gene candidates and their biological influence are at present unknown. The identification of variants of unknown significance (VUS) can be at present disturbing than helping. Moreover, the identification of variants, known to be associated to other diseases than thrombosis, such as the RUNX1 variation for leukemia or the aneuploidies can cause more problems than answer questions [84]. Should they be reported within the context of thrombophilia investigation or should they be silenced? Is there a risk for misinterpretation of the genetic results? How can we risk-stratify a VUS? These questions are still open, there is an urgent need for redefining the indications and dimensions of extensive genetic testing.
Author contributions All authors have substantially contributed to conception, design of the article, interpretation of data, drafting of the article and have approved the final version to be published.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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