MSE-2015 L7 Case 6 3 Year Old Orphan

Haemophilia A

A genetic disorder caused by a deficiency in factor VIII, leading to increased bleeding.

• X-linked recessive disorder, therefore rare in females

• most common cause: inversion within FVIII gene where it breaks during meiosis at intron 22, causing exons 1-22 to reattach in wrong direction

• rare in females, but can be due to

  • Turner’s syndrome (where one X is absent)

  • homozygous FVIII deficiency (both X chromosomes carry the mutation)

  • inappropriate lyonisation (random X inactivation leading to a significant proportion of cells expressing the mutated X).

FVIII

Factor VIII, a blood-clotting protein that is deficient in Haemophilia A.

• gene located on long arm of X chromosome

• part of intrinsix tenase complex which activates FX

X-linked recessive disorder

A type of inheritance where the gene causing the condition is located on the X chromosome.

Haemophilia B

A genetic disorder caused by a deficiency in factor IX, also known as Christmas disease. Also an X-linked recessive disease.

FIX

Factor IX, a blood-clotting protein that is deficient in Haemophilia B.

• also located on the long arm of X chromosome, near FVIII gene

• plays a crucial role in the coagulation cascade, activating factor X in the presence of factor VIII and calcium ions

Haemophilia clinical presentation

• haematuria

• purpura

• bruising

• epistaxis (nose bleed)

• haemorrhage

• haemarthrosis (bleeding into joints)

Haemophilia Treatment

• Recombinant FVIII.FIX produced using tissue culture techniques

• Clotting factor concentrates e.g. cryoprecipitates

• Desmopressin

• Gene therapy

Von Willebrand Factor (vWF)

• Produced by endothelial cells, platelets and sub-vascular connective tissue

• has 2050 amino acids, found in plasma as large multimers

• binds and protects inactive FVIII

• binds to both exposed collagen and platelets

• binds to thrombin

Thrombocytopenia

A condition characterized by a reduction in platelet count, which can lead to bleeding.

Desmopressin

A treatment for von Willebrand disease that stimulates the release of factor VIII and von Willebrand factor. For minor Haemophilia A disease also.

Prothrombin Time (PT)

A blood test that measures the time it takes for blood to clot, used to assess coagulation factors.

Activated Partial Thromboplastin Time (APTT)

A blood test that assesses the intrinsic pathway of the coagulation cascade, prolonged in haemophilia.

Immune Thrombocytopenic Purpura (ITP)

A condition characterized by low platelet counts and purpuric rash, often due to autoimmune mechanisms.

Megakaryoblast

A large bone marrow cell from which platelets are derived.

Thrombopoiesis

The process of platelet production, including pre-endomitosis and post-endomitosis phases.

• Pre-endomitosis begins with initial differentiation of common myeloid progenitor, TPO (Thrombopoietin) is a early driver

• Endomitosis is mitosis without telophase and cytokinesis phase, RUNX1 key to suppress Rho signalling

• Terminal differentiation (post-endomitosis) = distinct morphological cells, appear within bone marrows e.g. megakaryoblast

Thrombocytopoiesis

aka platelet shedding, reduction in platelet count

• also regulated by TPO, with roles for IL-6 and Il-11

• Demarcation system = megakaryocyte ultrastructure feature

  • membrane lined channels that reach from cell membrane into cytoplasm

  • delineates final mature thrombocytes

• Megakaryocyte will produce proplatelet process which can cross the endothelium

  • sinusoidal capillaries within bone marrow facilitate this

• Minor/moderate reduction in platelets doesnt always = increase in haemorrhage risk as spontaneous bleeding risk is high when platelet count is ~20 x10⁹ cells/L.

• Acquired or hereditary

Cryoprecipitate

A blood product used to treat clotting disorders by providing fibrinogen and other clotting factors.

Adenovirus Mediated Gene-transfer - Gene Therapy

Involves delivering a functional copy of the FVIII to the patient's liver cells.

• By using an adenovirus vector to carry the normal FVIII gene, it is possible to enhance the production of factor VIII in the body, helping to restore proper blood clotting function.

• This approach aims to provide a long-term solution for managing the disorder, reducing the need for regular clotting factor injections.

Factor-poor plasma

Plasma that has been depleted of specific coagulation factors to create a baseline for testing so activity of specific coagulation factors can be assessed without interference from other factors.

Clotting Factor Test

Pt plasma mixed with factor-poor plasma and PT/APTT test undertaken. If pt has good quality/quantity of factor it will correct the clotting time of factor-poor plasma.

• monoclonal antibodies also used for quantitative factor count, may be preferred for specificity and sensitivity in quantifying coagulation factors, allowing for more precise measurements.

• some labs prefer traditional testing methods, due to their established protocols and historical reliability, even if they may be less specific than monoclonal antibody methods.

Ristocetin

A test that causes platelet aggregation, used to evaluate von Willebrand factor activity.

• Requires vWF to cause platelet aggregation

Platelet aggregation

The clumping together of platelets in the blood, essential for the formation of a clot.

Thromboelastography (TEG)

A method to test platelet function and clot strength by measuring changes during coagulation.

• also detect aberrant fibrinolysis

• blood slowly agitated around a wire to initiate coagulation

• measured by…

  • R time - time to start forming a clot, 5-10mins, shows problem with coagulation factor. Treat with FFP

  • K time and A angle - time until clot reaches fixed strength, 1-3mins. Shows problem w fibrinogen, treat with cryoprecipitate

  • Maximum Amplitude (MA) - highest vertical amplitude of TEG, problem with platelets. Treat with platelets or DDAVP

  • LY30 - percentage of amplitude reduction 20mins after MA, problem with excess fibrinolysis

Turner’s Syndrome

A chromosomal disorder that can lead to haemophilia A in females due to abnormal X chromosome inheritance.

Normal range for FVIII activity

70-150% in adults, essential for appropriate clotting function.

Von Willebrand’s disease

Common inherited bleeding disorder, characterized by a deficiency of von Willebrand factor.

• Heterogenous, with different subtypes.

• Most common inherited bleeding disorder

vWD Subtypes

vWD

• Desmopressin/Vasopressin - stimulates FVIII release from intra-cellular stores

• stimulates vWF from platelet granules

• cryoprecipitate

• recombinant vWF

vWF Testing

• Platelet aggregation (thromboelastography)

• vW antigen

• Ristocetin

Platelets and Homeostasis

• Primary Platelet Plug: adhere to the site of vascular injury, becoming activated and aggregating to form a primary platelet plug, which is essential for initial hemostasis.

• Reservoir of Coagulation Factors, act as a reservoir for various coagulation factors, such as factor V and factor VIII, which are secreted upon activation to facilitate the coagulation cascade.

• Calcium in Coagulatio, ions released from platelets and surrounding tissue are crucial for several steps in the coagulation cascade, including the activation of clotting factors.

• Haemodynamic Altering Compounds, release substances like thromboxane A2 and ADP that alter blood flow and vascular tone, promoting further platelet recruitment and aggregation

Platelet Investigations

• FBC reveal number and size

  • important to keep in mind of EDTA mediated platelet clumping

  • dont inform whether platelets function

• Function testing can including screening or diagnostic tests

Pre-analytical Variables of Platelet Testing

• Medicines e.g. aspirin, ibuprofen and naproxen

• Diet e.g. garlic, turmeric, onions, ginger can reduce platelet function

• Supplementation e.g. Vit C, E and B6

• Others e.g. alcohol, smoking, caffeine

Platelet Screening Tests

• Bleeding time test, 1mm incision damages capillaries only so no secondary haemostasis

• TEG

• Platelet function analyser aka PFA-100

Platelet Diagnostic Tests

Introducing agonists to pt platelets and measuring response, done by in two methods

• PRP aka patient platelet rich plasma - relies on light transmission

• whole blood method - relies on electrical charge in resistance

Platelet Disorder Mechanisms

Thrombocytopenia

• impaired production

• increased consumption

• splenic sequestration

Platelet Function Disorders

• Defect in surface receptors

• Defect in granule function/number

• Defect in metabolism

• Phospholipid exposure

Immune Thrombocytopenic Purpura

Low platelet count and purpuric rash

• purple regions on skin

• do not blanche on pressure

• bruising, epistaxis, menorrhagia

IgG produced targets glycoprotein IIb/IIIa on the platelet surface.

• Integrin complex.

• Receptor for vWF and fibrinogen

• Platelets are opsinized by IgG.

• Renders them susceptible to phagocytosis by splenic macrophages.

Good prognosis for young children

• Spontaneous remission in 2months is common

ITP Haematology

• Thrombocytopenia ( <150 x10⁹ cells/L)

• RBC decrease after bleeding

• usually normal unless ITP is secondary to something else

• important to establish true ITP from pseudo (EDTA)

ITP Treatment

• For acute ITP no pro-coagulant needed, excessive bleeding very rare

• Treat underlying infection, prevents antibody secretion

• Monitoring platelet counts until stabilised

Platelets

• Treatment for transfusion needs e.g. thrombocytopenia

• Each unit contains ~250x10⁹ cells/L.

• More perishable than RBC

  • produced at 22 degrees, kept for 5 days

  • pooled or aphresis