L08 Blood Haemostasis 3 Coagulation tests _ Intro to bleeding disorders

Overview

This lecture covers the plasma-based coagulation screening tests (PT/INR, APTT, fibrinogen assay, factor assays) and how they map onto the extrinsic, intrinsic and common pathways, then applies this to the clinical assessment of bleeding disorders: history-taking, the causes of prolonged bleeding, haemophilia A/B, vitamin K deficiency and warfarin anticoagulation, and disseminated intravascular coagulation (DIC). Worked cases link abnormal test patterns to underlying diagnoses.

Plasma and sample handling

• Plasma (not serum) is needed for clotting assays: an anticoagulant keeps fibrinogen present, whereas in serum (a clotted sample with no anticoagulant) fibrinogen is fully converted to fibrin and pelleted with the clot.
• Sodium citrate (blue-top) tubes are used, with a precise ratio of citrate to blood of 1:9.
• To activate clotting in the assay, a precise amount of Ca++ is added back.

Prothrombin time (PT) and INR

• PT: Ca++, phospholipid and tissue factor are added to plasma to activate the extrinsic pathway (tissue factor/FVIIa converging with X→Xa, then the common pathway: prothrombin→thrombin→fibrinogen→fibrin).
• Reference interval ~9-13 sec.
• Prolonged by deficiency of FVII, FX, FV (prothrombin and fibrinogen deficiency have less effect on PT).
• INR (International Normalised Ratio) standardises PT across different tissue factor/thromboplastin reagents (rabbit, human or recombinant), which otherwise give different PT results between kits.
  • INR = ratio of the patient’s PT to a normal plasma PT, corrected (via the Sensitivity Index for Thromboplastin) to give the same result as the International Reference Thromboplastin (derived from human brain).
  • Reference interval: 0.8-1.2.
  • Target range for patients on warfarin: 2.0-3.0.

Activated partial thromboplastin time (APTT)

• Ca++, phospholipid and silica are added to plasma to activate the intrinsic pathway (XII→XI→IX/VIIIa→X→Xa, then the common pathway with prothrombin/thrombin/fibrinogen).
• Normal APTT ~22-34 sec.
• Prolonged by deficiency of VIII, IX, XII, XI, X, V (prothrombin and fibrinogen deficiency have less effect on APTT).

Fibrinogen assay

• Measured as an activity assay but reported as a mass concentration; reference interval 1.8-4.0 g/L.
• Thrombin is added to plasma and the rate of fibrin formation is used to calculate the fibrinogen concentration.

Sensitivity of the basic tests to factor deficiencies

• APTT clinically detects deficiency of VIII, IX, XI (also detects XII deficiency, but this is not clinically important).
• PT clinically detects deficiency of VII.
• Both APTT and PT detect X and V deficiency, but neither is very sensitive to prothrombin or fibrinogen deficiency.
• The fibrinogen assay detects fibrinogen deficiency directly.

Coagulation factor assays

• Individual factor levels (e.g. FVIII) are measured with clotting assays: the patient’s plasma is added to a synthetic plasma lacking only the factor of interest, and clotting time is read off a standard curve (time to clot vs % factor present) to give the factor level as a percentage of normal.
• “Normal” plasma = 100%; reference interval ~50-150%.
• APTT and PT are prolonged once factor concentration falls to roughly <40-50%.

Causes of prolonged bleeding

• Defective vessel wall disorders: e.g. fragile skin in the elderly, vasculitis, scurvy.
• Platelet disorders:
  • Thrombocytopenia (reference interval 150-400x10^9/L); bleeding/bruising increases progressively as platelet count falls below 100x10
  • Defective platelet function: drugs, some foods, or inherited conditions.
• Von Willebrand disease (~1:1,000 people): low levels or an abnormal vWF molecule, causing reduced platelet adhesion and prolonged bleeding.
• Coagulation disorders: a large number of conditions, acquired or inherited.

Assessing bleeding disorders

• History:
  • Location of bleeding: mucosal (nose/epistaxis, heavy periods, gut) suggests platelet dysfunction; joints/muscles suggest a coagulation defect such as haemophilia; skin bleeding can indicate either.
  • Pattern: frequency/severity, and whether bleeding occurs with surgery/trauma, menstruation, or spontaneously.
  • Drug history and diet (anti-platelet agents, anticoagulants).
  • Family history (for inherited disorders).
• Tests:
  1. Blood screen: platelet count and morphology.
  2. Coagulation screen: APTT, PT, fibrinogen.
  3. Neither of the above tests platelet function.
  4. If appropriate, second-stage specialist tests: von Willebrand factor assays, platelet function studies, coagulation factor assays.

Case: a 1-year-old boy with several large bruises, being investigated for child battering, had platelets 325 (ref 150-400), fibrinogen 2.2 (ref 1.8-4.0 g/L), APTT 65 (ref 22-36 s, prolonged), PT 10.0 (ref 9-13 s, normal). The slide left the differential diagnosis and further tests blank as a teaching/discussion prompt (not answered in the transcript). This test pattern (isolated prolonged APTT with normal PT and fibrinogen) is consistent with an intrinsic-pathway factor deficiency such as haemophilia.

Haemophilias

• Inherited X-linked disorders; males are affected because they have only one X chromosome.
• Reduced functional levels of FVIII (Haemophilia A) or FIX (Haemophilia B); both genes happen to be on the X chromosome. Female carriers are usually unaffected or have occasional mild bleeding.
• Pathogenic variants occur throughout the FVIII or FIX genes, reducing the amount or function of the protein. Factor level correlates with bleeding risk:
  • Severe: ≤1% factor level, spontaneous bleeding and bleeding after minor trauma.
  • Moderate: 2-5%, bleeding after minor/moderate trauma.
  • Mild: 5-50%, bleeding after moderate/major trauma.
• Clinical problem: reduced clot formation produces fibrin strands that are thinner and weaker than normal, easily disrupted physically and by fibrinolysis.
  • Mostly joint bleeding (haemarthrosis): joints and muscle have low tissue factor levels so rely on the intrinsic pathway for haemostasis. Crippling arthritis develops unless FVIII is kept above ~3-5% (reference interval 50-150%).
  • Muscle bleeding: can cause muscle fibrosis and calcification if not stopped early.
  • Other sites (kidney, brain, etc.): uncommon but can be severe.
  • Skin: large bruises after trauma.
• Joint disease consequence: recurrent joint bleeding causes chronic inflammation, cartilage damage and wear; synovium becomes stained brown from haemosiderin-laden macrophages; end-stage disease often needs reconstructive joint surgery (e.g. synovectomy) by middle age if effective treatment has not been provided.
• Treatment: intravenous infusion of recombinant FVIII (haemophilia A) or FIX (haemophilia B).
  • Emicizumab (monoclonal antibody) for haemophilia A: bridges activated FIX and FX in a manner similar to activated FVIIIa, restoring the FVIIIa-like bridging function on the phospholipid membrane to generate FXa. Given as a subcutaneous injection every 1-4 weeks; now available for severe haemophilia A in New Zealand.

Case: an elderly patient after major abdominal surgery, on antibiotics, with easy bruising around cannulation sites, had APTT 54 (ref 22-34 s, prolonged), PT 18 (ref 9.0-13.0 s, prolonged), fibrinogen 1.9 (ref 1.8-4.0 g/L, normal). No differential diagnosis or answer is given on the slide. Both APTT and PT are prolonged with normal fibrinogen, a pattern consistent with vitamin K deficiency (low intake plus antibiotics, post-surgery), which affects multiple factors in both pathways.

Vitamin K deficiency and warfarin mechanism

• Vitamin K is needed for the final activation step (carboxylation) of factors II, VII, IX and X (“1972”), and also of the coagulation inhibitors Protein C and Protein S.
• Vitamin K cycle: vitamin K (reduced) is converted by carboxylase (using CO2/O2, releasing H2O) into vitamin K epoxide, while simultaneously converting glutamic acid residues on the target factors into γ-carboxyglutamic acid residues (the active, calcium-binding form). Vitamin K epoxide is then converted back to vitamin K (reduced) by epoxide reductase (VKOR), completing the cycle.
• Without vitamin K, factors II, VII, IX and X are present but not functional: they cannot bind calcium, so bleeding risk rises.
• Causes of vitamin K deficiency: low intake, antibiotics, and small bowel malabsorption disorders; also occurs physiologically in the neonate and premature infant.
• In adults: vitamin K is fat-soluble, from vegetable sources and bacterial synthesis in the colon.
  • Causes: low intake (uncommon alone), low intake plus antibiotics (important in some patients), and malabsorption disorders (bile salts emulsify fats/fat-soluble vitamins, so biliary tract obstruction, e.g. gallstones, slows absorption).
  • Liver stores of vitamin K usually last 1-3+ weeks; deficiency causes prolonged PT and APTT and a bleeding tendency. Watch for the abdominal surgery patient with a wound infection, antibiotic therapy, and 1-2 weeks without eating.
• In the neonate: limited vitamin K crosses the placenta, and levels fall further after birth from negligible oral intake in the first 24 hours; vitamin K-dependent factor levels are lowest at 48 hours with normal feeding, risking Vitamin K Deficiency Bleeding (especially gastrointestinal and brain), occurring in about 1:10,000 births in high-income countries.
  • Vitamin K is always given after birth: 0.5 mg IM at birth, or 3 oral doses (poorly absorbed even in a full-term normal infant).
  • Premature infants have an increased risk of intracerebral bleeding and cannot absorb sufficient vitamin K orally, so must receive the intramuscular dose.
• Warfarin inhibits vitamin K recycling by blocking epoxide reductase (VKOR), which reduces carboxylase activity and so reduces production of functional (γ-carboxylated) factors II, VII, IX, X and Proteins C/S.

Warfarin anticoagulation

• Benefits: useful for treating venous clots and preventing clots on heart valves and in atrial fibrillation; an oral medicine, widely used for decades.
• Potential problems: risk of bleeding if over-anticoagulated, risk of clotting if under-anticoagulated; a narrow therapeutic window requires careful clinical control with regular blood tests (INR). Warfarin is now largely replaced by newer oral anticoagulants but is still used and must be understood.
• Typical results for someone on warfarin: PT 26 (ref 9-13 s), INR 2.5 (therapeutic range 2.0-3.0), APTT 42 (ref 22-34 s, also prolonged).
• Common clinical causes of warfarin control going out of range:
  • Changed vitamin K intake in food (vomiting, binge eating, sudden dietary change e.g. travellers).
  • Decreased vitamin K absorption (gastroenteritis, antibiotics, chronic malabsorption).
  • Change in warfarin clearance by the liver, especially via inhibition or induction of hepatic cytochrome P450 (e.g. cotrimoxazole inhibits P450), or any cause of liver cell injury (heart failure, hepatitis, alcohol).

Disseminated intravascular coagulation (DIC)

• Definition: widespread activation of platelets and coagulation throughout the blood, producing a coagulopathy from “consumption” of coagulation factors and platelets.
• Pathogenesis: tissue factor or other procoagulant cell products are released into blood vessels, or extensive endothelial damage occurs, leading to platelet activation and aggregation, activation of coagulation, and formation of microthrombi.
• Clinical features: variable, determined by the underlying condition; bleeding and/or microvascular thromboses/ischaemia. Moderate or severe DIC can produce bleeding and/or thromboses/ischaemia.
• Laboratory findings (consumptive coagulopathy): falling fibrinogen level, falling platelet count, increasing PT and APTT, and a high or rising level of fibrin degradation products (D-dimers).
• Causes:
  • Infections: gram-negative septicaemia, where endotoxin activates monocytes to express tissue factor (occurs in all gram-negative septicaemia, especially severe in meningococcal septicaemia due to very high endotoxin levels); platelets, fibrinogen and other coagulation factors are consumed.
  • Any severe/widespread tissue injury: prolonged shock with secondary hypoxic tissue injury, extensive tissue trauma releasing tissue products into blood, severe burns, severe viral infections causing endothelial injury (e.g. Ebola haemorrhagic fever).
  • Traumatic brain injury: brain myelin (a lipid from nerve sheaths) is released into blood, activating platelets and coagulation.
  • Obstetric complications: amniotic fluid embolism, placental abruption, intrauterine fetal death.
  • Severe allergic hypersensitivity reactions: severe anaphylaxis, incompatible blood transfusion reaction.
  • Malignancy: some acute leukaemias (e.g. acute promyelocytic leukaemia), mucin-secreting adenocarcinomas.
• Treatment of acute DIC:
  • Treat the underlying cause where possible and the DIC will stop (e.g. antibiotics for infection; treat shock then trauma).
  • If secondary bleeding occurs, replace the appropriate blood component: platelet transfusion for thrombocytopenia with bleeding; cryoprecipitate for low fibrinogen with bleeding; plasma infusion added for severe bleeding with a prolonged APTT.
  • Severe cases with multi-organ failure need Intensive Care Unit management.

Case: a patient 24 hours after abdominal surgery developed a rigor, temperature 39°C, and a falling blood pressure (145/95 to 100/55) over the last hour. Results: APTT 36 (ref 22-34 s), PT 15 (ref 9.0-13.0 s), INR 1.7 (ref 0.8-1.2), fibrinogen 0.9 (ref 1.8-4.0 g/L, low), D-dimers 7000 (ref <500 ug/L, markedly raised), platelet count 80 now vs 225 twenty-four hours ago (ref 150-400x10^9/L, falling). The slide left the differential diagnosis and treatment blank as a discussion prompt. This pattern (prolonged PT/APTT, low fibrinogen, falling platelets, very high D-dimers, in the context of post-surgical sepsis/rigors and hypotension) is consistent with DIC secondary to septicaemia.

Clinical case: meningococcal septicaemia

• A 21-year-old with meningococcal septicaemia had serial results over three timepoints (16:15, 18:22, 03:10): haemoglobin fell 137→116→67 g/L (ref 118-155), platelets fell 269→50→27x10^9/L (ref 150-400), neutrophils rose to 12.4x10^9/L (ref 1.9-7.5), APTT became >100 s (ref 24-35 s), and fibrinogen fell to <0.7 g/L (ref 1.8-4.0 g/L). Purpuric/haemorrhagic skin lesions developed (petechiae/purpura around the eye and large purpuric patches on the torso/limb). This illustrates severe DIC driven by meningococcal (gram-negative) septicaemia.

Self-test

1. Explain why clotting assays require plasma rather than serum, and describe how the sample is collected and activated for testing.
2. Describe what is added to plasma to perform the PT and which pathway this activates; state the reference interval and which factor deficiencies prolong it.
3. Describe what is added to plasma to perform the APTT and which pathway this activates; state the reference interval and which factor deficiencies prolong it.
4. Explain why the INR was introduced and how it is calculated.
5. State the reference interval for INR and the target range for a patient on warfarin.
6. Describe how the fibrinogen assay works and state its reference interval.
7. Using the sensitivity table, predict which screening test(s) would be abnormal in an isolated factor VII deficiency, and which in an isolated factor IX deficiency.
8. Explain why the APTT and PT are relatively insensitive to prothrombin and fibrinogen deficiencies.
9. Describe how a coagulation factor assay (e.g. for FVIII) is performed and how the result is expressed.
10. At approximately what factor concentration do the APTT and PT become prolonged?
11. List the four broad categories of causes of prolonged bleeding, with one example condition for each.
12. Distinguish the reference interval for platelet count from the level at which bleeding/bruising begins to appear.
13. Distinguish mucosal-pattern bleeding from joint/muscle-pattern bleeding in terms of the underlying defect they suggest.
14. List the tests in a standard bleeding-disorder work-up, and explain what important function none of them assesses.
15. Explain the genetic basis of haemophilia A and B, and why males are typically affected and female carriers usually are not.
16. Describe the three severity categories of haemophilia in terms of factor level and the trauma needed to trigger bleeding.
17. Explain why joints and muscles are especially prone to bleeding in haemophilia, referencing tissue factor levels and pathway reliance.
18. Describe the joint pathology that develops with recurrent haemarthrosis in poorly controlled haemophilia.
19. Describe how emicizumab works and how it differs in administration from standard factor replacement.
20. Describe the vitamin K cycle and explain how warfarin interferes with it.
21. List the coagulation factors and inhibitors that depend on vitamin K for their activation.
22. List three causes of vitamin K deficiency in adults, and explain the mechanism of the biliary-obstruction cause specifically.
23. Explain why neonates and premature infants are at particular risk of vitamin K deficiency bleeding, and describe how it is prevented.
24. State the benefits and risks of warfarin therapy, and list three clinical causes of a patient’s INR becoming unstable while on warfarin.
25. Define disseminated intravascular coagulation and describe its underlying pathogenesis.
26. List four categories of causes of DIC, with one example condition in each.
27. Describe the four characteristic laboratory findings of DIC and explain why each occurs.
28. For a post-surgical patient with sepsis, prolonged PT/APTT, low fibrinogen, high D-dimers and a falling platelet count, explain what disorder this pattern indicates and why.
29. Describe how acute DIC with bleeding is treated, matching each blood product to the deficiency it corrects.
30. A patient has an isolated prolonged APTT with a normal PT and normal fibrinogen. Using the sensitivity data in this lecture, explain what this pattern suggests and why the PT remains normal.

Answers

Reveal answers

1. Plasma retains fibrinogen because an anticoagulant prevents clotting; in serum the fibrinogen would already be converted to fibrin and removed with the clot. Blood is collected into sodium citrate (blue-top) tubes with a precise 1:9 citrate-to-blood ratio, and clotting is then triggered in the assay by adding back a precise amount of Ca++.
2. PT: Ca++, phospholipid and tissue factor are added, activating the extrinsic pathway (which converges with the common pathway). Reference interval ~9-13 sec. Prolonged by deficiency of FVII, FX, FV (less so prothrombin/fibrinogen).
3. APTT: Ca++, phospholipid and silica are added, activating the intrinsic pathway (which converges with the common pathway). Reference interval ~22-34 sec. Prolonged by deficiency of FVIII, IX, XII, XI, X, V (less so prothrombin/fibrinogen).
4. The INR was introduced to correct for differences between PT reagent kits, particularly differences in tissue factor/thromboplastin source (rabbit, human, recombinant). It is calculated as the ratio of the patient’s PT to a normal plasma PT, corrected via the reagent’s Sensitivity Index to match the result that would be obtained using the International Reference Thromboplastin (from human brain).
5. Reference interval for INR: 0.8-1.2. Target range on warfarin: 2.0-3.0.
6. Thrombin is added to plasma and the rate of fibrin formation is measured, then converted to a mass concentration. Reference interval 1.8-4.0 g/L.
7. Isolated FVII deficiency: PT prolonged, APTT normal (VII is only in the extrinsic pathway tested by PT). Isolated FIX deficiency: APTT prolonged, PT normal (IX is only in the intrinsic pathway tested by APTT).
8. Because both tests can still generate a clot end-point with reduced levels of prothrombin or fibrinogen; clinically significant reductions in these need to be quite marked before PT/APTT become abnormal, so they are not very sensitive indicators of prothrombin or fibrinogen deficiency (the dedicated fibrinogen assay is used instead).
9. The patient’s plasma is mixed with a synthetic plasma lacking only the factor being tested, the clotting time is measured, and the result is read off a standard curve of clotting time vs % factor present, giving the factor level as a percentage of normal (normal plasma = 100%, reference interval ~50-150%).
10. Roughly <40-50% factor concentration.
11. Defective vessel wall disorders (e.g. scurvy, vasculitis), platelet disorders (e.g. thrombocytopenia, defective platelet function), von Willebrand disease, and coagulation disorders (inherited or acquired).
12. Reference interval for platelet count is 150-400x10^9/L, but bleeding/bruising only becomes progressively apparent once the count falls below 100x10
13. Mucosal bleeding (nose, heavy periods, gut) suggests platelet dysfunction; joint/muscle bleeding suggests a coagulation defect such as haemophilia; skin bleeding could indicate either.
14. Blood screen (platelet count and morphology) and coagulation screen (APTT, PT, fibrinogen); if needed, second-stage specialist tests (vWF assays, platelet function studies, factor assays). None of the routine screen tests assess platelet function.
15. Haemophilia A (FVIII deficiency) and B (FIX deficiency) are X-linked recessive; both genes are on the X chromosome. Males have only one X chromosome so a single affected copy causes disease, while female carriers have a second normal X and are usually unaffected or only mildly affected.
16. Severe (≤1% factor level): spontaneous bleeding and bleeding after minor trauma. Moderate (2-5%): bleeding after minor/moderate trauma. Mild (5-50%): bleeding after moderate/major trauma.
17. Joints and muscle have low levels of tissue factor, so they rely on the intrinsic pathway (which requires FVIII/FIX) for haemostasis; when this pathway is deficient, these sites bleed preferentially.
18. Recurrent joint bleeding causes chronic inflammation, cartilage damage and wear, with the synovium becoming stained brown from haemosiderin-laden macrophages; without adequate treatment this progresses to end-stage joint disease often needing reconstructive surgery (e.g. synovectomy) by middle age.
19. Emicizumab is a monoclonal antibody that bridges activated FIX and FX on the phospholipid membrane in the same way as activated FVIIIa, restoring FXa generation. Unlike intravenous factor replacement, it is given as a subcutaneous injection every 1-4 weeks.
20. Carboxylase uses vitamin K (reduced) to convert glutamic acid residues on target factors into γ-carboxyglutamic acid (their active, calcium-binding form), producing vitamin K epoxide as a by-product; epoxide reductase (VKOR) recycles the epoxide back to reduced vitamin K. Warfarin inhibits VKOR, blocking this recycling, which reduces carboxylase activity and so reduces production of functional factors.
21. Factors II, VII, IX and X, plus the coagulation inhibitors Protein C and Protein S.
22. Low dietary intake (uncommon alone), low intake combined with antibiotics, and malabsorption disorders. In biliary obstruction (e.g. gallstones), reduced bile salt delivery to the gut impairs emulsification of fats and fat-soluble vitamins, reducing vitamin K absorption.
23. Only limited vitamin K crosses the placenta, and oral intake is negligible in the first 24 hours after birth, so vitamin K-dependent factor levels fall to their lowest around 48 hours, risking Vitamin K Deficiency Bleeding (especially gastrointestinal and brain). It is prevented by giving vitamin K at birth (0.5 mg IM, or 3 oral doses); premature infants cannot absorb enough orally so must receive the IM dose.
24. Benefits: effective oral treatment for venous clots and prevention of clots on heart valves and in atrial fibrillation, used for decades. Risks: bleeding if over-anticoagulated, clotting if under-anticoagulated, due to a narrow therapeutic window needing regular INR monitoring. Causes of instability: changed dietary vitamin K intake, decreased vitamin K absorption (e.g. antibiotics, gastroenteritis), and changes in hepatic clearance of warfarin (e.g. P450 inhibition/induction, liver injury).
25. DIC is widespread activation of platelets and coagulation throughout the blood, causing a coagulopathy from consumption of coagulation factors and platelets. It arises from tissue factor or other procoagulant material entering the circulation, or extensive endothelial damage, leading to platelet activation, coagulation activation, and microthrombi formation.
26. Infections (e.g. gram-negative/meningococcal septicaemia), severe/widespread tissue injury (e.g. burns, trauma, prolonged shock), obstetric complications (e.g. placental abruption), and malignancy (e.g. acute promyelocytic leukaemia).
27. Falling fibrinogen (consumed in widespread clotting), falling platelet count (consumed by activation/aggregation), increasing PT and APTT (factors consumed, less remaining to clot), and rising D-dimers (breakdown products of the excess fibrin being formed and degraded).
28. This pattern indicates DIC: sepsis triggers widespread tissue factor exposure and endothelial damage, causing consumption of platelets and coagulation factors (low fibrinogen, falling platelets), impaired clotting on screening tests (prolonged PT/APTT), and excess fibrin degradation (high D-dimers).
29. Treat the underlying cause (e.g. antibiotics for infection, treat shock/trauma) so the DIC resolves; for bleeding, give platelet transfusion for thrombocytopenia, cryoprecipitate for low fibrinogen, and plasma infusion for severe bleeding with prolonged APTT; manage severe multi-organ failure in ICU.
30. An isolated prolonged APTT with normal PT and fibrinogen suggests deficiency of an intrinsic-pathway-only factor (VIII, IX, XI, or XII), since these are not part of the extrinsic pathway tested by PT; the PT remains normal because the extrinsic and common pathway factors it tests (VII, X, V, prothrombin, fibrinogen) are unaffected.