3 Laboratory diagnosis of hemophilia
1 Introduction
- A diagnosis of hemophilia is made with the support of laboratory service. This includes plasma-based coagulation tests and genetic testing in whole-blood.
1.1 RECOMMENDATION
Testing for diagnosis of hemophilia and monitoring of therapy needs experience in coagulation laboratory testing using equipment and reagents that have been validated for this specific purpose.
Coagulation laboratories must implement quality assurance procedures e.g. participation in external quality assessment programs to ensure the reliability of laboratory testing.
2 Coagulation laboratory testing
2.1 Pre-analytical aspects of hemophilia testing
The pre-analytical phase is the time from the blood collection to the point when the sample is analyzed in the laboratory. Errors in this phase are often explained by incorrect patient identification or incorrect collection, handling, transportation, centrifugation or storage of the sample. In order to reduce the pre-analytical error rate, it is important to understand the sources of variability and mechanisms that may lead to false assay results.
Coagulation tests are exceptionally susceptible to suboptimal sample quality as the sample collection itself will initiate a hemostatic response. Thus, improper sample collection technique and/or incorrect handling prior to analysis will increase the risk of coagulation activation and clot formation in the tube. Furthermore, coagulation factor VIII (FVIII) is one of the most labile coagulation factors and is rapidly degraded in vitro. In worst case scenario, erroneous results of screening and specific factor assays may be reported and lead to mismanagement of the patient. There are guidelines/recommendations, how to assure sample integrity during the pre-analytical phase [1,2].
2.2 RECOMMENDATION
For plasma-based coagulation assays the recommendations for sample collection are:
Direct venipuncture: atraumatic phlebotomy with minimal tourniquet use.
Collection tube and order of draw: 3.2 % (109 mmol/L sodium citrate, light blue stopper) tube first, or only after a non-additive tube.
Fill the tube correctly to the mark (line).
Mix blood with anticoagulant in the tube (reverse the tube immediately 5-10 times).
Transport the whole-blood promptly at room temperature.
Centrifuge preferentially within 1 hour after phlebotomy at a minimum of 1700 g for 15 min to obtain platelet poor plasma (<10 x 109/L).
If laboratory testing cannot be performed within 4 hours, the plasma should be transferred by pipetting to another tube and frozen at -70 ºC for later analysis.
Underfilling (<90%) of tubes or presence of clots lead to rejection of samples.
The presence of anticoagulants in the sample, for example heparin contamination from a vascular access device, may interfere in the assay and give false test results.
2.3 Initial testing for detection of hemophilia
Global screening tests of coagulation, i.e. APTT and PT (INR), in addition to fibrinogen, are important for the initial laboratory evaluation of patients with bleeding disorders, and are available in most hospital laboratories. If congenital or acquired hemophilia A or B is present, the APTT will usually be prolonged and the PT (INR) remains within reference ranges. Deficiency of FXI and FXII might also cause an isolated prolonged APTT.
APTT is a global plasma assay that measures the collective functional activity of 10 different coagulation factors including FVIII or FIX. If one or several of the coagulation factors are increased, a deficiency of one coagulation factor may be masked. Furthermore, during certain conditions such as an acute phase reaction, FVIII is increased and APTT may be within reference ranges.
A normal APTT does not rule out mild hemophilia A or B and does not exclude a clinically relevant bleeding disorder. There are several commercially available APTT reagents that vary in their sensitivity for detecting coagulation factor deficiencies. To be accepted as a screening test for factor deficiency, it is recommended that the APTT reagent should give a prolonged APTT at FVIII and FIX activities ≤ 30% [3]. The presence of lupus anticoagulant may prolong the APTT and APTT reagents insensitive to lupus anticoagulant are advantageous.
If APTT and/or PT (INR) is prolonged, a mixing test should be performed to distinguish between deficiency of coagulation factor(s) or presence of inhibitors/interfering anticoagulants. In congenital hemophilia the APTT will be corrected when patient plasma is mixed 1:1 with pooled normal plasma [3]. If mixing does not correct the prolongation of APTT, it may indicate the presence of an inhibitor (against coagulation factor VIII /IX in particular or lupus anticoagulant) or presence of other anticoagulants in the plasma (e.g. heparins).
2.4 RECOMMENDATION
For screening purposes, it is recommended that the APTT reagent gives a prolonged clotting time at FVIII or FIX activities ≤ 30%.
A normal APTT result should not be used to rule out the presence of mild hemophilia A or B.
It is important that the treating physicians are familiar with the local screening methodologies and reference intervals and understand the limitations of screening tests.
2.5 Specific Factor VIII and IX assays
FVIII:C or FIX:C in plasma represents the functional (coagulation) activity of the factors and can be measured with either clotting or chromogenic substrate assays [3,4]. These analyses are important in the diagnostic setting, during monitoring after administration of replacement products (measurement of trough and peak activity of the coagulation factor), and also for detecting the presence of inhibitory antibodies against endogenous and exogenous FVIII and FIX. When a family history is present, umbilical cord blood is tested in male infants at birth to determine FVIII:C or FIX:C. For prenatal diagnosis, see chapter “Carriers of hemophilia”.
The FVIII:C and FIX:C assays should be calibrated with material that has traceability to the current international standard for FVIII or FIX in plasma [3]. In this way the unit is given in international units (IU) and one IU is the factor activity present in one mL of pooled normal plasma. The results should be reported in IU/mL or IU/dL, which indicates traceability to the international standard (IU/dL is the same as percentage in absolute numbers; i.e. 5 IU/dL= 5%).
FVIII:C and FIX:C functional activity assays can be measured with both a one-stage clotting assay (OSA) or a chromogenic substrate assay (CSA).
2.6 RECOMMENDATION
- FVIII:C and FIX:C can be measured with either one-stage clotting assays (OSAs) or chromogenic substrate assays (CSAs) and are used in the diagnostic setting, for monitoring of replacement therapy and detection of inhibitor development.
2.7 Differential diagnosis
- Once a decreased FVIII activity has been confirmed, the differential diagnosis includes congenital hemophilia A, acquired hemophilia A and von Willebrand disease (VWD), type 2N VWD (Normandy) in particular. To determine the cause to low FVIII:C information about the case history, inheritance pattern and bleeding score is important, and the presence of lupus anticoagulant or interfering anticoagulants in plasma needs to be excluded. VWF antigen and activity should be measured and in some cases an inhibitor against VWF needs to be excluded. If the APTT is not corrected after mixing with pooled normal plasma measurement of antibodies against FVIII may be appropriate. If type 2N VWD is suspected a VWF:FVIII binding activity is recommended which determines the capacity of the patient’s VWF to bind FVIII. Definite diagnosis may be dependent on exon sequencing of the F8/9 and VWF genes.
2.8 RECOMMENDATION
Low FVIII:C may be caused by congenital hemophilia, acquired hemophilia or von Willebrand disease (VWD).
After measurement of von Willebrand factor (VWF) antigen and activity, extended investigation to detect antibodies against FVIII and VWF or measurements of VWF:FVIII binding activity may lead to a final diagnosis.
In some cases, definite diagnosis may be dependent on exon sequencing of the F8/9 and VWD genes.
2.9 Factor VIII:C assays
The OSAis the most frequently used assay principle in the world [3,4]. The main feature of the OSA is that it is based on the APTT test with the difference that the sample is pre-diluted in FVIII-deficient plasma before analysis. In this way, a test system is created that works with the simplicity of the APTT reaction but the pre-dilution procedure makes the FVIII activity in the sample the limiting factor, and thus determines the final clotting time. The ability of the sample to correct the APTT of a FVIII-deficient plasma can be expressed as the FVIII:C activity if the assay is calibrated with a plasma with known concentrations of FVIII:C.
The performance of the OSA is affected by the type and quality of the APTT reagent and the FVIII-deficient plasma. The FVIII-deficient plasma can be obtained from a patient with severe hemophilia A (<1 IU/dL and without antibodies) or be immunodepleted. It is important to verify that new lots of FVIII-deficient plasmas are free from FVIII (<1 IU/dL) as this otherwise will compromise the test. Also, normal levels of VWF and other clotting factors of the FVIII-deficient plasma is recommended.
The choice of APTT reagent will also have an impact on the general assay characteristics. It is important that the laboratory choose reagents that have proven capacity to detect all hemophilia categories i.e. mild to severe hemophilia A. The results obtained by OSAs may be affected by the presence of lupus anticoagulant, heparins, etc.
Measurement of FVIII:C can also be performed by CSAs [5]. There are several commercial CSAsfor measurement of FVIII:C available. The assay procedure involves two separate reactions in a way that makes FVIII activity in the sample the rate-limiting factor. In the first step the diluted sample (or standard) is mixed with a reagent cocktail with purified FIXa, FX and phospholipids, leading to the formation of FXa. In the second step a specific chromogenic peptide substrate for FXa is added. Cleavage of the substrate yield a colour formation that is recorded spectrophotometrically. The amount of colour development is directly proportional to the FVIII:C in the sample. In general, the CSA has a lower detection limit than the APTT-based OSA and, due to the high dilution factor of the sample, the influence of interfering substances is less. CSA is common among the Nordic hemophilia centers. The CSA is also used by the pharmaceutical industry when the potencies of FVIII concentrates are assigned.
2.10 RECOMMENDATION
The OSA and CSA for FVIII:C should be performed with validated methods.
It is important that the laboratory choose reagents that have proven capacity to detect all hemophilia categories i.e. mild to severe hemophilia A.
Laboratories should ensure that new lots of FVIII-deficient plasmas are free from FVIII (<1 IU/dL).
Normal levels of VWF and other clotting factors of the FVIII-deficient plasma is recommended.
2.11 FVIII:C - Assay interpretation
The different FVIII:C assays should give similar results in cases with severe hemophilia A. However, in some cases of mild/moderate hemophilia A patients, a significant assay discrepancy between OSA and CSA has been reported, with OSA FVIII:C at least 2-fold higher than CSA FVIII:C [5,6]. Patients with certain mutations in the F8 gene causing mild/moderate hemophilia A may therefore be undiagnosed using the OSA FVIII:C. On the other hand, there are some genotypes causing inverse assay discrepancy in patients with mild hemophilia, where CSA FVIII:C is higher than OSA FVIII:C. Thus, mild hemophilia A may be challenging to identify correctly in the laboratory.
The recommendation in the current WFH guideline is to use both OSA and CSA in the initial diagnostic work-up [3]. For the management of hemophilia A, both methods should be performed to ensure detection of all mild/moderate cases and to correctly assess the severity.
Interpretation: Hemophilia A patients have low FVIII:C. FVIII:C < 1 IU/dL (<1%) are seen in severe hemophilia A. Moderate deficiency is characterized by a FVIII:C between 1-5 IU/dL, and patients with mild deficiency have FVIII:C > 5 IU/dL up to 40 IU/dL. Carriers of hemophilia A usually have approximately 50% of the normal FVIII activity, but occasionally they have FVIII:C consistent with mild haemophilia A. FVIII is an acute phase reactant and the levels of FVIII may increase several fold under certain conditions (e.g. trauma, infection, etc).
2.12 RECOMMENDATION
- Both methods, OSA and CSA are recommended to use in the initial diagnostic work-up. Assay discrepancies can be caused by different mutations but also due to analytical factors such as presence of lupus anticoagulant. OSAs are generally more prone to interferences from lupus anticoagulant, heparin etc.
2.13 Factor IX:C assays
The principle of OSAs for FIX is similar as for FVIII described above, with the only difference that the sample is prediluted in FIX-deficient plasma before analysis. Thus, the main FIX:C assay principle is a test system based on the APTT with dilution of the sample (patient or standard plasma) in a plasma lacking FIX, which means that the activity of FIX in the sample is the limiting factor. The assay is calibrated with a standard that is traceable to the current international standard of FIX:C in plasma and results expressed as IU/mL or IU/dL (see FVIII:C above).
CSAs for measurement of FIX:C have become commercially available as an alternative to the OSAs. However, these assays are to this date only available at few laboratories, and measurement of FIX:C in combination with the WHO IS for FIX plasma standard (for potency labelling) have not yet been fully approved by regulatory bodies such as EMA. However, FIX:C measured by CSA displays analytical advantages e.g. high dilution of sample, and may complement the measurement of FIX:C by OSAs. Assay discrepancy caused by mutations in the F9 gene, has been described also in hemophilia B patients [7].
Interpretation: Congenital deficiency of FIX is the cause of hemophilia B. Acquired hemophilia B, caused by specific inhibitors exists but is even less frequent than the rare acquired hemophilia A. The degree of the deficiency defines the different forms:
Severe hemophilia B has FIX:C < 1 IU/dL (<1%); moderate deficiency has FIX:C between 1- 5 IU/dL, and mild deficiency has FIX:C > 5 up to 40 IU/dL. Carriers of hemophilia B often express about 50% of the expected normal FIX:C activity, but rarely FIX:C may be reduced into the range of mild hemophilia B.
2.14 RECOMMENDATION
- FIX:C can be measured with either OSA or CSA. OSAs are generally more prone to interferences due to the presence of lupus anticoagulant, heparin etc.
2.15 Antibodies against FVIII or FIX
The development of neutralising inhibitors and non-neutralising antibodies (NNAs) is a complication of factor replacement therapy in hemophilia. The diagnostic methods generally available are based on defining the neutralising capacity in functional assays, i.e. original or Nijmegen-modified Bethesda assays for antibodies considered to be inhibitors [8]. However, NNAs might also affect clearance of administered products, therefore immunological assays can be used to define all antibodies, including NNAs. In the Nordics, ELISA and xFLI methods for NNAs are available at the Coagulation laboratory in Malmö, Sweden.
Neutralizing antibodies may develop against factor concentrates in individuals with hemophilia A or B and are termed alloantibodies. Alloantibodies are most frequent and have a fast and dose-dependent antigen-antibody reaction. In acquired hemophilia A, time-dependent autoantibodies with a low binding affinity can be present. For this reason, it is recommended to incubate the samples for 2 hours at 37 ºC during a mixing experiment in order to allow the autoantibodies to have effect. Anti-FIX antibodies have faster kinetics and a shorter incubation time can be used for neutralising anti-FVIII antibodies. It is also important to use buffered pooled normal plasma (ie HEPES). This stabilizes the pH and thus the FVIII activity during the incubation and the risk of obtaining false positive results is reduced.
The recommended test procedure for quantitation of the inhibitor titer is the Bethesda-Nijmegen mixing test, which is an assay for inhibitory antibodies [3,8]. In brief, a test sample is prepared by mixing equal volumes of patient plasma with buffered pooled normal plasma and then measure the residual coagulation factor activity in the plasma mixture after 2 hours of incubation. A control sample is prepared in parallel with buffered pooled normal plasma mixed with FVIII-deficient plasma. Both test and control samples are incubated for 2 h at 37ºC and then the factor activities in both samples are measured. In a plasma sample with an unknown value for the inhibitor, a series of dilutions will need to be made. These samples are prediluted in FVIII-deficient plasma and handled as described above. There is usually a good correlation between the inhibitor results based on the OSA and CSA FVIII:C assays [9]. Any residual activity in the sample between 25 and 75% can be used for calculations of the inhibitor titer. By definition, one Bethesda unit (BU) is the inhibitor titer that neutralizes 50% of the factor activity in one mL plasma. If the residual activity is less than 25% it indicates an inhibitor titer above 2 BU/mL. Hence, these samples are prediluted in FVIII-deficient plasma until a residual activity within the 25-75% range is reached.
It is recommended to perform the Bethesda assay when there has been a washout of the concentrate.
Reference interval: The cut-off for a positive result is by definition 0.4 BU/mL. Many laboratories instead use 0.6 BU/mL as the cut-off for positivity (reduced risk of false positive results) [10].
Interpretation: The presence of inhibitors may be suspected in patients with unexpected bleedings despite regular prophylaxis with replacement therapy. This is also strengthened if the patient displays reduced recovery and half-life of the substituted factor.Patients with acquired haemophilia have very different clinical symptoms, caused by autoantibodies against FVIII or FIX.
Note: The Bethesda assay is usually performed on patients with a severe type of hemophilia without measurable FVIII:C (or FIX:C) activity. If the patient has an activity of 5 IU/dL or higher this must be taken into consideration. Then it is possible to remove the endogenous coagulation factor activity by heat-inactivation of the plasma sample at 56˚C for at least 30 min. After centrifugation of plasma, FVIII:C or FIX:C is measured [8–10]. The FVIII-mimetic emicizumab interferes with inhibitor measurements using OSAs, but a CSA with bovine reagents can be used (see below).
2.16 RECOMMENDATION
Nijmegen-Bethesda assay is the recommended method for measurement of neutralizing FVIII or FIX antibodies. It is most reliable in patients without measurable coagulation factor activity (i.e. severe haemophilia).
It is recommended to perform the Nijmegen- Bethesda assay when there has been a washout of the coagulation factor concentrate.
If the patient has an activity of 5 IU/dL or higher it is possible to remove the endogenous coagulation factor activity by heat-inactivation of the plasma sample at 56˚C before analysis.
2.17 Coagulation factor activity assays for monitoring treatment with replacement therapy with focus on Extended half-life products
For monitoring of post-infusion levels of full length recombinant (r)FVIII standard products, CSA results are generally reported to be about 20% higher than OSA results. Well-documented discrepancies have also been described for B-domain deleted rFVIII (ReFacto®), with CSA results 30% higher than OSA results. Calibration with B-domain deleted FVIII has therefore been recommended for OSA. For rFIX products, CSA results are instead in general 30% lower than OSA results [4,11–13].
There are a number of products modified for an extended half-life (EHL), both rFVIII and rFIX, recently on the market or under development (i.e. pegylated, glycopegylated, and fusion proteins) (See Chapter Prophylaxis). Some of the modifications affect factor assays and can result in under- or overestimation of results in postinfusion patient samples, which might have a potential impact on patient management [4,11–13]. Ideally, similar recovery results should be obtained in post-infusion samples with the same method as used for potency labelling.
For more than one of the modified products, a difference of > 30% has been obtained for factor activity levels when different APTT-reagents have been used in different OSAs. The recovery can be more consistent in CSAs. All reagent-product combinations have not been tested, and all reagents that contain the same activator (ellagic acid/phenol, silica/kaolin type) do not give the same results. More adequate factor levels are obtained with the chromogenic methods for some of the products. Comparable data about systematic under- or overestimation of coagulation factor activity for different modified products are still scarce for many instrument-reagent combinations.
The laboratory need information about the type of product used in every patient. Product-by-product-based information must be verified with the methods used by the local laboratory to maintain patient safety. Both ECAT and UK NEQAS (European external quality control providers in haemostasis) are providing external quality control programmes for EHL products.
Other options for measurement that are now beginning to be used in clinical routine settings, are other global coagulation tests, such as viscoelastic coagulation methods (ROTEM®, TEG®) and thrombin generation (CAT®, Ceveron alpha®), that can, if available, be used as a complement for monitoring purposes of certain products. However, standardisation of these methods is very much needed.
For the management of hemophilia patients with EHL-products, it is important that the laboratory has access to more than one method for FVIII and FIX respectively, preferably one CSA and one OSA. With the information available to this date, CSA is a more reliable laboratory method that is able to adequately measure the levels of most EHL-FVIII products. Most importantly, WFH recommends laboratories to use assays that has been validated with the specific concentrates used for treatment.
2.18 RECOMMENDATION
It is important that the laboratory has access to more than one method for monitoring of FVIII and FIX in post infusion samples, preferably one CSA and one OSA method for each factor.
It is recommended to communicate to the laboratory the specific factor replacement product used by each patient, and if the sample is drawn at a through or peak level.
2.19 Factor activity assays for monitoring treatment with non-replacement therapy
The bispecific FVIII mimetic Emicizumab affects all APTT-based laboratory methods and for determining FVIII:C levels in a patient on treatment, it is recommended to use a CSA FVIII activity assay containing bovine FX [14]. This assay is not able to detect emicizumab and is used for measurement of endogenous/exogenous FVIII in the presence of emicizumab. Also, for measurements of FVIII inhibitors a CSA containing bovine FX should be used. Anti-emicizumab antibodies may be indicated by a prolongation of the APTT.
The concentration of Emicizumab can be measured and reported in µg/mL using a modified OSA calibrated with emicizumab.
Discrepancies between OSA and CSA results have been reported after both FVIII and FIX gene therapy. Results of OSA FVIII:C have been approximately 1.5-fold higher than CSA results. Results of OSA FIX:C have varied with different reagents, but were higher with OSAs than with CSAs.
2.20 RECOMMENDATION
FVIII:C and inhibitor levels in patients receiving emicizumab can be measured using a CSA FVIII:C assay containing bovine FX.
Discrepancies between OSA and CSA results have been reported after both FVIII and FIX gene therapy, with generally higher results with OSAs compared to CSAs.
3 Genetic diagnosis
3.1 Indications for testing
For people with hemophilia, obligate carriers, females with low FVIII:C or FIX:C, or individuals with suspected hemophilia and potential carriers, measurement of FVIII:C and FIX:C activity l and VWF antigen and activity testing is recommended prior to genetic testing.
Genotyping is clinically useful to predict the risk of developing an inhibitor against coagulation factor, response to immune tolerance induction (ITI), phenotype severity and for carrier- and prenatal diagnosis. For a detailed description of the clinical interpretation of genetic variants, we refer to the 2019 UK Haemophilia Centre Doctors’ Organisation Good Practice Paper) [14]. Depending on the experience and competence of the hemophilia team and the local organisation of genetic services, a clinical geneticist or counsellor can be part of the hemophilia care team.
Genetic information should include a discussion of the experimental limits and possibility of incidental findings.
3.2 RECOMMENDATION
Measurement of coagulation factor activity is generally recommended prior to genetic testing.
Genotyping is clinically useful to predict the risk of developing an inhibitor and for carrier- and prenatal diagnosis.
3.3 Strategy and techniques for genetic testing
Genetic diagnosis of severe hemophilia A starts with screening for the intron 22 inversion of the F8 gene which is caused by homologous recombination involving intron 22 and related sequences outside the F8 gene [15]. Approximately 40% of cases of severe hemophilia A is caused by intron 22 inversion. Similarly, an inversion involving intron 1 found in 1-2% of severe cases can also be screened for with a PCR technique. In the remaining cases of severe hemophilia A as well as all moderate and mild cases, the whole F8 gene, 26 exons, must be sequenced usually through Sanger or next-generation sequencing (NGS) methodologies.
Most patients have their own unique mutation/pathogenic variant and today a broad spectrum of more than 3000 variants are known to cause hemophilia A and almost 2000 causing hemophilia B. Variants such as nonsense and deletions, “null-mutations”, will obviously cause severe hemophilia since the DNA reading frame will be altered, mRNA aberrant and the FVIII protein will not be synthesized. A missense variant will usually produce a dysfunctional protein with reduced clotting activity but may also result in a ‛neutral/silent variant’ or a benign variant/polymorphism. In such cases it is important to know if the same variant has been reported previously in patients with hemophilia and exists in databases such as the European EAHAD Coagulation Factor Variant Databases (http://dbs.eahad.org) or the American CDC Hemophilia Mutation Project databases CHAMP/CHBM (https://www.cdc.gov/ncbddd/hemophilia/champs.html) [16,17].
The clinical interpretation of a new or an unpublished genetic variant in the F8 or F9 genes, as well as other genes, should be based on guidelines published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [17]; as pathogenic – likely pathogenic – variant of unknown significance (VUS) – likely benign - benign. One may also use various in silico variant prediction programs to evaluate the deleterious effect of a variant. In a small percentage, disease causing variants/alterations will not be found despite sequencing of the whole gene, some of these cases having a more complex genetic background. The MLPA technique (Multiplex Ligation-dependent Probe Amplification) may show deletions or duplications not revealed on conventional Sanger sequencing.
In hemophilia B, the 8 exons of the F9 gene are sequenced and in almost all cases the disease-causing variant will be found. Inversions are not present in the F9 gene, but some patients have complete gene deletions, a strong predictor for development of inhibitors or anaphylactic reactions on FIX treatment.
3.4 RECOMMENDATION
Genetic diagnosis of severe hemophilia A starts with screening for intron 22 inversion (approximately 40% of cases of severe hemophilia A).
An inversion involving intron 1 is found in 1-2% of severe cases.
For remaining cases of severe hemophilia A as well as all moderate and mild cases, the whole F8 gene, 26 exons, must be sequenced usually through Sanger or NGS methodologies.
In hemophilia B, the 8 exons of the F9 gene are sequenced and in almost all cases the disease-causing variant will be found.
The clinical significance of a new or an unpublished genetic variant in the F8 or F9 genes should be evaluated according to ACMG criteria to be reported as: pathogenic – likely pathogenic –VUS – likely benign - benign.
Carrier diagnosis in sporadic case of hemophilia A or B, which encompasses around 50-60% of all newly diagnosed cases, may be a problem. In about 70-80% the mother of a sporadic case also carries the pathogenic variant and is thus a carrier. In the remaining 20-30% of cases a pathogenic variant cannot be detected and these women may be either true non-carriers or being somatic and/or gonadal mosaics, i.e. it is not possible with conventional less sensitive techniques to conclude if she is a non-carrier or a mosaic carrier.
Mosaicism may cause a problem when genotyping mothers ofa sporadic case of hemophilia A with conventional techniques but may be detected by more sensitive techniques such as ddPCR or NGS [18–20]. Studies indicate that, depending on the type of pathogenic variant, approximately 20% may be somatic or gonadal mosaics [21]. In hemophilia B this seems to be unusual but is not sufficiently studied with sensitive techniques [22].
Prenatal diagnosis (PND) can be achieved by chorionic villus sampling during the 11 to the 13th week of gestation and karyotype analysis can be performed to determine the sex of the fetus, and in cases of male fetus, if it carries a gene causing hemophilia. The reasons for PND may be to prevent the birth of an affected boy by termination of the pregnancy, to prepare the obstetrical procedures or, for the parents-to-be, to psychologically prepare for having a child with hemophilia. Later in pregnancy amniotic fluid can be used as source of fetal DNA. Fetal sex determination can also be made by Y-chromosome analysis in blood from the pregnant woman very early in pregnancy and thus avoiding invasive diagnostic procedures in pregnancies with a female fetus. Pre-implantation genetic diagnosis (PGD) enabling the implantation of female or unaffected male embryos has become possible [23,24]. PGD is a demanding procedure which however may be indicated in selected cases.
3.5 RECOMMENDATION
In about 70-80% of sporadic cases, the mother also carries the pathogenic variant and is thus a carrier. In the remaining 20-30% of cases a pathogenic variant is not found.
Mosaicism may cause a problem when genotyping mothers ofa sporadic case of hemophilia A with conventional techniques.