Heamophilia and VWD Dr Deepankar ipgmer and sskm kolkata.pptx

discussion of heamophilia , inhibitors and vwd

1. HEAMOPHILIA AND VON WILLEBRAND DISEASES HEAMOPHILIA AND VON WILLEBRAND DISEASE Presented by : Dr. Deepankar shriwas, PGT Department of pathology Moderator : Dr. Mou Das , Associate professor, Department of pathology IPGME&R AND SSKM HOSPITAL , KOLKATA.

2. INTRODUCTION Deficiency of procoagulant clotting factors results in a reduction in the amount of thrombin generated, leading to impaired clot formation and increased bleeding tendency. Haemophilia is an inherited bleeding disorder due to deficiency of plasma coagulation factors. People with haemophilia have low level of either factor VIII or factor IX or factor XI.

3. INCIDENCE It is primarily found in males but transmitted by female carriers. Hereditary haemophilia account about 80% of cases as sex linked chromosomes. About 20% of cases may account as acquired haemophilia due to autoimmune disorder/ spontaneous mutation.

4. History of haemophilia Hemophilia is sometimes referred to as " the royal disease ," because it affected the royal families of England, Germany, Russia and Spain in the 19th and 20th centuries. Queen Victoria of England , is believed to have been the carrier of hemophilia B, or factor IX deficiency. She passed the trait on to three of her nine children. Her son Leopold died of a hemorrhage after a fall when he was 30. Her daughters Alice and Beatrice passed it on to several of their children. Hemophilia was carried through various royal family members for three generations after Victoria, then disappeared.

5. CLASSIFICATION OF COAGULATION DISORDER

6. More common :- Haemophilia A (Classic Hemophilia) :- This type is caused by a lack or decrease of clotting factor VIII, the antihemophilic factor (AHF). It accounts for 80-85% of all cases. 1 in 5,000 male births. Haemophilia B (Christmas Disease) :- This type is caused by a lack or decrease of clotting factor IX, the plasma thromboplastin component (PTC). It accounts for 15-20% cases.1 in 60,000 male births.

7. • Less common Hemophilia C :- This type is caused by a lack or decrease of factor XI, plasma thromboplastin antecedent (PTA). Hageman's disease :- Deficiency of factor XII. Von Willebrand's disease :- Reduced level of Von Willebrand's factor, a protein that binds with factor VIII & protects it from rapid breakdown within blood.

8. CAUSES Hemophilia is caused by a mutation or change in one of the genes, that provides instructions for making the clotting factor proteins needed to form a blood clot. This change or mutation can prevent the clotting protein from working properly or to be missing altogether. These genes are located on the X chromosome .

9. Haemophilia A (FVIII deficiency) Haemophilia is the most common, severe inherited bleeding disorder recognized in humans. Haemophilia A (HA) is due to deficiency or complete absence of FVIII . It is caused by mutations in F8 gene , located on the long arm of the X chromosome. For all severities of haemophilia , the estimated incidence is 1  in 5000 live male births. Inheritance is X-linked, with a third of patients having no family history

10. What is Factor VIII FVIII is a large 330 kDa glycoprotein synthesized in endothelial cells. It is organized into six domains denoted as A1-A2-B-A3-C1-C2. It circulates as a heterodimer, made of a heavy chain (A1-A2 domains, plus parts of the B domain) and a light chain (A3-C1-C2 domains) linked non-covalently via divalent metal cations.

11. 95–97% of FVIII circulates in complex with von Willebrand factor (VWF), which protects against its proteolytic degradation and clearance. Both proteins are lower in blood group O individuals due to higher endogenous clearance of VWF and of the FVIII attached to it. Without VWF, the half-life is about 1–2 hours. Continue…….. Factor VIII

12. Inheritance The mode of inheritance of haemophilia A is X-linked recessive . The abnormal gene (or the gene coding the synthesis of F VIII) is located on the X chromosome. The disease manifests only in males because they lack the complementary normal X chromosome . Females are carriers but do not manifest the disease as they have a normal allele on the complementary X chromosome .

13. Positive family history is not obtained in about 20% of patients with haemophilia . These cases probably arise from spontaneous mutation. Therefore , negative family history does not rule out the possibility of haemophilia . However, haemophilia may develop in carrier females when there is lyonisation predominantly of normal X chromosomes during embryogenesis . Other mechanisms by which haemophilia can occur in females are homozygosity of haemophilic gene in the female offspring of carrier female and affected male. And also hemizygosity of haemophilic gene due to chromosome anomalies such as Turner’s (XO) syndrome . Continue…….. Inheritance

14. In vivo, extrinsic pathway controls bleeding after injury. Tissue factor-F VIIa complex activates F IX and F X and initiates a rapid but limited generation of thrombin on tissue injury. The initial formation of thrombin is subsequently amplified by a feedback mechanism in which thrombin activates F XI and F VIII of intrinsic pathway. Pathogenesis of Bleeding in F VIII Deficiency :-

15. In F VIII deficiency, inadequate amounts of thrombin are generated. Secondly, increased amount of thrombin is required for activation of TAFI (thrombin-activated fibrinolysis inhibitor). Therefore, F VIII deficiency is associated with insufficient clot formation as well as rapid clot removal . Continue…..Pathogenesis of Bleeding in F VIII Deficiency :-

16. Clinical Features:- The clinical presentation of haemophilia A and B is related to the severity of the disease. The International Society of Thrombosis and Haemostasis (ISTH) has proposed a classification for disease severity based on coagulation activity, which correlates strongly with the clinical picture.

17. Continue…..Clinical Features Patients with severe deficiency present in the first few years of life with excessive bruising, prolonged bleeding from minor trauma, such as cut lips or tongue, prolonged bleeding post-circumcision or decreased movement due to joint or muscle bleeds. The defining feature is spontaneous bleeding, i.e. bleeds with no apparent trauma. Patients also present with bleeds that are disproportionate to the tissue injury. Fatal bleeding is not uncommon due to intracerebral haemorrhage or bleeding into other internal organs. Before the advent of modern treatment, the average life span was around 11 years.

18. Continue…..Clinical Features Haemophilic pseudotumours are large encapsulated haematomas with progressive cystic swelling from repeated haemorrhage . They are best visualized by magnetic resonance imaging (MRI). They may occur in large muscle groups, long bones and any fascial plane. They arise from large haematomas that have been poorly treated.

19. Continue…..Clinical Features :- A pseudotumor may be misdiagnosed as a neoplasm (e.g., Ewing sarcoma or osteosarcoma) or as an infection (e.g., osteomyelitis or tuberculous abscess). Biopsy of such lesions is contraindicated because of the potential for significant bleeding or infection. Small pseudotumors , particularly distal ones or pseudotumors in patients with inhibitors, are often treated conservatively with aggressive clotting factor replacement along with immobilization of the affected limb.

20. Laboratory findings:- Investigations show:- Prolonged activated partial thromboplastin time (APTT). Normal PT and normal platelet function. FVIII or FIX clotting assay confirms the diagnosis. In patients with mild FVIII deficiency, evaluation should include an analysis of VWF parameters to exclude VWD.

21. Continue……Laboratory findings:- Thromboplastin generation test (TGT) is a second-line test and reveals “plasma defect”. The combination of normal PT, prolonged APTT, and plasma defect in TGT are highly suggestive of F VIII deficiency. A genetic diagnosis can be undertaken through mutation analysis . This provides information on the phenotype and risk of inhibitor development and helps screen carriers.

22. Mutation analysis:- Mutations reported in the F8 gene are heterogeneous and include nonsense, missense, splice site, frameshift mutations or large insertions/deletions. A characteristic intron 22 inversion (Inv22) of the F8 gene accounts for around half of severe HA cases. This involves recombination between homologous sequences located in intron 22 and upstream of the F8 gene , resulting in a characteristic ‘flip-tip’ inversion and disruption of  the F8 gene. A single nucleotide change causes responsible for mild or moderate haemophilia A.

23. Carrier detection and antenatal diagnosis :- There are four ways in which hemophilia carriers can be identified. First, in light of the X-linked transmission of the disease , pedigree analysis will determine the carrier status of some women. The second mode of identification can be through the manifestation of abnormal bleeding caused by a low FVIII or FIX level . Estimates of the percentage of hemophilia carriers who experience excessive bleeding (most often demonstrated through menorrhagia) vary but likely approximate 20% to 30%.

24. Continue….Carrier detection and antenatal diagnosis :- The third mode of carrier detection involves use of the laboratory phenotype , in which tests of the intrinsic pathway (APTT) may be abnormal and the plasma levels of FVIII or FIX may be reduced below the normal range. The fourth and most definitive approach to carrier diagnosis is to use molecular genetics to identify the causative FVIII or FIX mutation. Antenatal diagnosis can be confirmed on chorionic biopsies at 8–10 we eks gestation, providing sufficient foetal DNA is available for analysis. Free foetal DNA isolated from maternal peripheral blood can be used to identify the causative mutations.

25. Preimplantation Diagnosis :- Preimplantation diagnosis offers the genetic diagnosis in preimplantation embryos and thus eliminates the problem of termination of pregnancy. The advent of polymerase chain reaction and in vitro fertilisation techniques has made preimplantation diagnosis feasible. In this technique, a single cell is removed from the embryo (6–10 cell stage) following in vitro fertilisation and its DNA is amplifie d using polymerase chain reaction . Techniques for detection of mutation are applied on the amplified DNA . The unaffected embryo is implanted into the uterus. Preimplantation diagnosis has been applied to haemophilia A, β thalassaemia , sickle-cell anaemia , cystic fibrosis, Lesch-Nyhan syndrome, Tay-Sachs disease, and some other monogenic diseases.

26. Non-invasive Prenatal Diagnosis:- It is known that foetal cells (such as erythroblasts and lymphocytes ) are present in maternal circulation during pregnancy. These foetal cells though small in number, can be isolated from maternal blood by a method utilising antigenic differences between foetal and maternal cells. DNA is extracted from these foetal cells and analysed by a sensitive technique (polymerase chain reaction). If a mutation is detected, then it can be confirmed by doing chorionic villus biopsy in early pregnancy. The technique, at present, is in investigational stage.

27. Therapeutic options and comprehensive care :- The mainstay of treatment is the correction of factor deficiency and therefore, bleeding tendency. There are two main therapeutic aims: (1) Management of bleeds (spontaneous or trauma) and haemostatic insults for example trauma and surgery . (2) Prophylaxis aimed at prevention of spontaneous bleeds and excessive bleeding with trauma thus altering the clinical phenotype of the disorder.

28. Comprehensive care:- Two innovations improved outcomes even before the advent of definitive treatment regimens. The first was home treatment; the second was comprehensive care. At the earliest indication of bleeding, a patient can be treated at home reducing the need for hospitlization . Most patients in developed countries attend specialized haemophilia centres with a multidisciplinary team. Comprehensive care is now offered to almost all patients with inherited bleeding disorders.

29. What Are Inhibitors? Inhibitors are antibodies that the immune system develops when it reacts to the proteins in factor concentrates (hemophilia treatment) as if they were harmful substances. Inhibitors fight against the proteins, as if they are foreign substances, thereby preventing the treatment from effectively managing a patient’s bleeding risk. Most patients develop inhibitors within the first 75 exposures to factor concentrates, with the greatest risk occurring within the first 10 to 20 rounds of treatment.

30. Continue…….Inhibitors:- Inhibitors are often discovered during a routine laboratory test for activated partial thromboplastin time (APTT), which measures how long it takes for blood to clot. When inhibitors are present , the blood takes longer to clot and does not coagulate fully. To confirm the diagnosis, a Bethesda assay is performed (measured in Bethesda units [BU]). It determines the titer (strength) of the inhibitor, either high titer (>5 BU) or low titer (<5 BU).

31. Treatment Options of Inhibitors:- High-dose factor concentrates : administering factor concentrates at higher doses and/or at more frequent intervals. Bypassing agents : activated prothrombin complex concentrates and recombinant factor VIIIa . Tranexamic acid : an antifibrinolytic drug given as an additional therapy to help stop blood clots from breaking down. Epsilon aminocaproic acid : an antifibrinolytic drug given as an additional therapy to help hold clots in place in certain parts of the body, such as the mouth, bladder, and uterus. Plasmapheresis : a procedure that removes inhibitors from the patient’s bloodstream, usually performed when the inhibitor titer needs to be brought down quickly. Immune tolerance induction therapy : administering frequent doses of factor concentrates over several months to train the body to recognize the treatment without adversely reacting to it .

32.  HAEMOPHILIA B:- Haemophilia B (also known as Christmas disease, after the first patient described) is a hereditary F IX deficiency state with X-linked recessive mode of inheritance. The incidence is about 1:60,000 populations. Clinical features and inheritance pattern are similar to haemophilia A. It is essential to distinguish between haemophilia A and B in the laboratory because of different therapeutic products required . F IX assay should be done in all cases; its principle is similar to that of F VIII assay. About 1 to 2% of patients with severe hamophilia B develop inhibitor antibodies against F IX.

33. Continue……… HAEMOPHILIA B The therapeutic products for haemophilia B are recombinant F IX (treatment of choice), fresh frozen plasma (for mild/moderate cases) and prothrombin complex concentrate (for severe cases). Mutations are similar to HA . Haemophilia B Leiden : In this form of haemophila B, F IX levels in blood increase at the time of puberty followed by resolution of bleeding manifestations. It is seen in 3% of haemophilia B patients and results from specific F IX promoter mutations.

34. von Willebrand disease:- von Willebrand disease (VWD), due to reduced von Willebrand factor (VWF) function, either quantitative or qualitative , is the most common inherited bleeding disorder. The estimated prevalence varies from 1:100 to 1:10,000 depending on the assessment criteria. Symptomatic presentation requiring assessment and care is observed in about 1 in 1,000  individuals. The gene is located on the short arm of human chromosome 12 and inheritance depends on the type of VWD.

35. Continue……….von Willebrand disease The basic mature vWF molecule is a monomer composed of 2050 amino acids. vWF monomers associate with each other through disulphide bonds to form multimers of varying sizes. The large multimers of vWF are more effective in haemostasis as they have greater binding sites for mediating adhesion of platelets to subendothelium . Most of the vWF is synthesised by endothelial cells and stored in Weibel-Palade bodies for later secretion.

36. vWF Gene :- Encodes for a protein with multiple copies of homologous motifs with three A, six C and four D motifs.

37. Clinical features :-

38. Laboratory investigations:- The complex structure of VWF supports platelet adhesion and protects circulating FVIII from premature degradation . Investigations measure these two functions and aim to diagnose and classify subtypes. The assays used are listed below:- (1) VWF: Ag (antigen) levels are measured by ELISA assay, a measure of protein concentration.

39. Continue……Laboratory investigations (2) VWF: Rco ( ristocetin cofactor activity) assay is a functional assay and uses fixed platelets and ristocetin . Ristocetin alters VWF conformation, facilitating the interaction between the VWF A1 domain and platelet GP1b receptors; shear mediates this in vivo. This defines the platelet-dependent function of VWF. (3) VWF: Activity assays also measure the interaction between the VWF A1 domain and platelet GPIb α receptor .

40. Continue……Laboratory investigations (4 ) VWF: CB is measured by ELISA assay and measures VWF collagen-binding (CB) activity. (5) VWF multimers are evaluated by electrophoresis. Loss of HMWMs impacts VWF: RCo and other VWF activity assays. (6) Ristocetin -induced platelet aggregation (RIPA) uses low-dose ristocetin to induce platelet agglutination when a gain of function mutation in VWF increases affinity to platelets. (7) FVIII binding measures the ability of VWF to bind FVIII.

41. Treatment:- Therapeutic strategies a Local measures and antifibrinolytic agents can be used for mild bleeding. Desmopressin injection increases VWF levels by releasing endothelial stores 30  minutes after intravenous infusion. This is effective in VWD Type 1 and some Type 2M and 2A cases. A therapeutic trial is indicated before using it for a planned procedure or in an emergency. V WF concentrates are indicated in patients with low levels and major surgery. The concentrates include plasma derived, intermediate purity factor VIII/VWF concentrates and high-purity VWF-only concentrates.

42. Continue…..Treatment:- Recombinant VWF concentrate is available for the management of bleeds and surgery. When using VWF-only containing concentrates, a low FVIII at baseline necessitates additional FVIII replacement for management of a bleed. Otherwise, treatment should be started 8–12 hours pre-surgery to allow FVIII levels to rise to normal range

43. SUMMARY Severe haemophilia A, with FVIII activity < 1IU/dL, is the most common inherited severe bleeding disorder. Haemophilia B (FIX deficiency), five times less frequent, has identical clinical features. Patients with severe deficiency present with spontaneous bleeding, particularly joint and muscle bleeds. Patients with deficiencies of all severities have excess bleeding with trauma and surgery. Treatment advances in haemophilia A and B have resulted in a near-normal life span. Prevention of bleeding is the current standard of care for patients with severe deficiency or severe phenotype, thus decreasing long-term joint morbidity. Therapies for haemophilia A and B include replacement therapies, non-replacement therapies that restore thrombin generation, and gene therapy that restores endogenous expression of the deficient factor.

44. VWD is the most common inherited bleeding disorder. Mild quantitative deficiency is the most common subtype with multiple qualitative subtypes and severe deficiency recognized. Mucocutaneous haemorrhages are common presenting symptoms, and symptomatic presentation is more common in women. Desmopressin is a valuable treatment for mild VWD and mild haemophilia A by releasing endothelial stores of VWF and FVIII. VWF-containing plasma-derived and recombinant concentrates are appropriate for managing patients with severe VWD and qualitative defects. It is crucial to diagnose rare coagulation disorders as they are associated with increased mortality and morbidity. Most rare coagulation disorders can be treated with recombinant or plasma-derived concentrates. A few continue to require treatment with plasma.

45. REFERENCE  :-