Week 6 - Haemostasis Flashcards
What are the indications for the evaluation of bleeding disorders?
- Asymptomatic patients undergoing a surgical procedure if personal or family history of bleeding is a concern.
- Patients with a history of increased bleeding, easy bruising, delayed wound healing.
- Patients with a previous bleeding disorder diagnosis.
• Detailed history is important. Family history, drugs etc.
• Lab tests: FBC, platelet count (platelets commonest cause of bleeding clinically), PTT, PT, fibrinogen/TT (thrombin time).
• Other factor assays, platelet function studies, bone marrow exam - only on special indications.
• Suspected VWD (combined platelet function and FVIII deficiency):
- VWF:Ag
- VWF:Rcofactor assay (plt function study)
- Factor VIII (levels)
Identify the clinical features of bleeding disorders.
• Superficial bleeding usually due to blood vessel/platelet disorder.
- Petechiae
- Purpura
- Echymosis
• Deep bleeding usually due to coagulation disorders (more common with congenital disorders).
- Haematoma
- Joint bleeds
• When severe - there is no differentiation i.e. a platelet disorder can give rise to a deep haematoma and severe coagulation disorders can give rise to superficial bleeding.
Outline the diagnosis of bleeding disorders.
- History
• Age, duration, spontaneous or induced, severity.
• H/o fever, drugs (aspirin, warfarin), other disease.
• Personal and family history of bleeding tendency. - Physical Examination - to differentiate between superficial bleed (platelet) or deep (coagulation).
• Site of bleeding - superficial or deep (plt/coag)
• Joints in haemophilia, oral cavity in platelet deficiency etc. - Laboratory Investigations
• FBC/platelet count, PT/INR, aPTT and TT, bleeding time.
• Urine and stool (occult) test for blood.
• Factor Assay, platelet function tests, bone marrow - rare - specialised investigations.
• Anticoagulants.
How are blood samples stored?
- Blood sample in sky blue cap: sodium citrate.
- Sample usually sent in sky blue cap which is sodium citrate → binds calcium and halts all coagulation processes. Therefore, preserves all the coagulation factors.
Describe the basic tests for bleeding disorders.
• Bleeding time
- Normal: < 10 minutes.
- Prolonged in blood vessel/platelet disorders.
- Not routine.
• Platelet count
- Important - platelets are the commonest cause of bleeding clinically.
• Prothrombin time (PT)
- Tests extrinsic and common pathway (factor VII + I, II, V, X).
- Prolonged in acquired disorders, liver disease, warfarin therapy, vitamin K deficiency.
- INR (international normalised ratio) - correction of different thromboplastin reagent (animal product). Normal 0.9-1.3.
• Partial thromboplastin time (PTT or aPTT)
- Tests intrinsic and common pathway (factor VIII, IX, XII + I, II, V, X).
- Prolonged in congenital bleeding disorders e.g. VWD, haemophilia.
- When PT and PTT both increased → suspect problem in the common pathway.
• Thrombin time (TT)
- Tests fibrinogen levels (common pathway).
- Useful in DIC.
- Not routine.
- Used when both PT and PTT are abnormal (mainly used for fibrinogen levels).
• FDP/D-dimer
- Fibrin degradation products.
- Increased in DIC.
- Specialised investigation.
Describe the test principle for PT and PTT.
- For both PT and PTT, take patient’s plasma which is preserved in sodium citrate (blocks all the calcium) - all coagulation factors are in their original non-activated state.
- PT - add tissue thromboplastin to start PT test → incubate and add calcium, immediately extrinsic pathway is activated. The time for clot formation is measured (the time from adding calcium to the formation of a clot).
- PTT - use cephalin and kaolin which activates the surface factors. Then add calcium and start the clock. Time taken to clot → PTT.
How do you differentiate a clotting factor deficiency from a clotting factor inhibitor?
• Prolonged PT/aPTT → mix patient’s plasma with normal plasma (1:1) and incubate.
• If PT/aPTT corrects → clotting factor deficiency (the normal plasma has provided the deficient factor).
• If PT/aPTT inhibited → inhibitor (inhibits the normal plasma too and therefore remains prolonged). Conduct further testing to determine type of inhibitor.
- Drug: heparin, direct thrombin inhibitor.
- Specific factor inhibitor e.g. FVIII or FV.
- Non-specific inhibitor: lupus anticoagulants.
What is a thromboelastogram (TEG)?
- Quick test before and during major surgery e.g. open heart surgery.
- Measures clot formation, strength and its lysis.
- Global assessment of haemostatic function.
- Line diagram of clot formation - shape indicates type of disorder e.g. fibrinolysis (excess fibrinogen - clot breaks down faster).
Identify the lab findings of common haemostasis disorders:
- ITP
- VWD
- Haemophilia
- DIC
- Aspirin
- Warfarin or heparin
Platelet count, BT, PT, PTT:
• ITP - decreased, increased, normal, normal.
• VWD - normal, increased, normal, increased.
• Haemophilia - normal, normal, normal, increased.
• DIC - decreased, increased, increased, increased.
• Aspirin - normal, increased, normal, normal.
• Warfarin or heparin - normal, normal, increased, increased.
What is the commonest cause of bleeding?
Thrombocytopenia (decreased platelets). • Immune - autoantibodies to platelets. • Drugs. • Infections - especially viral. • Marrow disease.
Describe the production of platelets.
- Megakaryocytes under the influence of thrombopoietin hormone → mature and divide endomitotically and the cytoplasm fragments separate out as platelets.
- The platelets are loaded with coagulation factors that help in haemostasis.
- Lifespan is 10 days - either used up in haemostasis or destroyed in spleen.
- Bleeding usually superficial and small blood vessels (capillary damage). Clinically produce petechiae, purpura, ecchymosis.
Outline platelets.
• Normal wear and tear of endothelium (microscopic damages) healed by platelet adhesion and aggregation, no need for coagulation.
• Platelets contain many factors (of haemostasis) - ADP, calcium, VWF, PF4 (platelet factor 4), fibrinogen.
- Disorders of platelet granules: platelets are present but non-functional or surface glycoproteins.
- Platelet disorders usually lead to small capillary bleeds.
• Platelet counts (x10^9/L)
- Normal: 150-450
- Excess bleeding: <50
- Spontaneous bleeding: <20
• Thromobocytopenia: - Autoimmune, drugs, infections - Bone marrow suppression - MBA - Increased consumption - DIC, TTP & HUS. • Platelet function disorders (less common): - VWD - Bernard Soulier syndrome - Glanzman thrombasthenia.
What is Immune Thrombocytopenic Purpura (ITP)? What are the 2 types of ITP?
- Autoimmune disorder - IgG antibody against platelets.
- Platelets destroyed in spleen → thrombocytopenia.
- Petechiae, purpura, ecchymosis, easy bruising.
2 types - acute and chronic:
• Acute ITP - common in children, post infection, self limited.
• Chronic ITP - females, 20-40 years, chronic and severe.
Outline the laboratory diagnosis for ITP.
- FBC - thrombocytopenia, giant immature platelets on blood film.
- PT, PTT - normal (coagulation normal - only platelets decrease).
- Bone marrow - increased immature megakaryocytes (in response to loss of platelets).
Describe the aetiology and pathogenesis of viral haemorrhagic fevers?
Aetiology: • Dengue • Chikungunya • Measles • Malaria • Yellow fever • Ebola
Pathogenesis:
• Endothelial damage by virus and anti-viral antibodies - vasculitis → platelet activation → thrombocytopenia → bleeding.
Outline the laboratory diagnosis of viral haemorrhagic fevers.
- Thrombocytopenia.
- PT and PTT usually normal.
• Rarely DIC e.g. Dengue shock syndrome. Activation of coagulation and platelet aggregation.
- Very severe viral infections can also give rise to activation of coagulation.
- Platelets also stimulate coagulation and in that case → would produce DIC with abnormal PT and PTT as well as thrombocytopenia.
Outline haemophilia A and B.
• X-linked recessive. Factor 8 (A) or 9 (B).
• Rare congenital bleeding tendency due to coagulation factor 8 or 9 (both X-linked recessive).
• Haemophilia A - factor 8 deficiency.
• Haemophilia B - factor 9 deficiency.
• Females carriers, males affected.
- Females have 2 X chromosomes - usually have factor levels around 55% and don’t produce any bleeding tendency.
- Males have a single X chromosome - 1% factors → severe bleeding.
Identify the clinical features of haemophilia.
• Mild >5%, severe <1% (>30% factor - no bleeding) - only when coagulation factors <30%, patient may produce mild bleeding. Severe bleeding when coagulation factors <1%.
• Increased wound bleeding and deep haematoma.
- Commonest presentation is excess bleeding following wounds.
- Coagulation important for deep haemorrhages therefore develop deep haematoma and not petechiae or purpura.
• Joint bleeds* (skin/mucosal bleeding - in severe).
- Muscular bleeding, internal haemorrhages all common in coagulation disorders.
- Deeper bleeds in muscles or joints. Young kids develop haematoma in joints - get destroyed as they grow up.
Describe the diagnosis and treatment of haemophilia.
Diagnosis:
• PTT - prolonged (suggests it is affecting intrinsic pathway).
• FVIII assay - deficient.
Treatment:
• DDAVP/FVIII concentrate - DDAVP stimulates endothelium to release the stored factors or FVIII or FIX concentrate can be used.
Outline vitamin K deficiency.
- Acquired.
- Vitamin K - present in green veg and intestinal bacteria.
- Necessary for carboxylation of factors 2, 7, 9, 10 in liver.
- Causes - diet, drugs and liver disease.
- Warfarin blocks vitamin K action* - some drugs such as warfarin specifically block vitamin K action - prevent thrombophilia.
- Lab - increased PT, aPTT normal/increased (late).
- Although it affects all factors, factor 7 has the shortest half-life. Consequently, warfarin therapy first affects factor 7 and later affects other factors. Therefore, use PT value to detect early warfarin excess. aPTT will usually be normal until late stage.
Outline Von Willebrand disease (VWD) and the function of VWF.
• Congenital bleeding disorder. VWF useful for platelet adhesion to the damaged epithelium.
• VWF by endothelial cells - platelet and coagulation - double function.
- VWF produced by both platelets and endothelial cells.
- Helps in adhesion of platelets (not aggregation - aggregation is via fibrinogen).
• Platelet adhesion to collagen and carry/protect FVIII.
- Usually VWF is in the subendothelial region and in plasma. In the plasma, carries FVIII and prevents its early degeneration. Therefore, deficiency of VWF also leads to deficiency of FVIII.
• VWD is a combination of both a lack of platelet adhesion and FVIII deficiency → causes superficial and deep bleeds (both platelet and coagulation abnormal).
• VWD is an autosomal dominant disorder - many subtypes. Mild to severe bleeding clinically.
• Both males and females are affected (not X-linked).
- Congenital bleeding tendency in female → most likely VWD. Only in males → likely haemophilia.
Describe the diagnosis and treatment of VWD.
Diagnosis:
• Normal platelet count but increased bleeding time (platelets cannot function without VWF).
• Normal PT but aPTT is increased (due to decreased FVIII levels - VWF carries and protects FVIII).
Treatment:
• Fresh frozen plasma/cryoprecipitate - contain VWF.
Outline the aetiology of disseminated intravascular coagulation (DIC).
• Systemic activation of coagulation → consumption of all factors and platelets → severe bleeding.
Aetiology - infection, trauma, cancer (common causes).
• Infection - widespread endothelial damage.
• Trauma - tissue factor release into circulation.
• Common causes - release of tissue factor into circulation or widespread endothelial damage leading to activation.
Explain the pathogenesis of DIC.
- Activation of coagulation → microthrombi - infarction.
- Consumption of haemostatic factors - bleeding.
- Activation of fibrinolysis - excess fibrin breakdown products.
• Some stimulus leading to activation of coagulation forming microthrombi and breakdown of the thrombi by fibrinolysis ultimately using up all the coagulation factors and platelets → results in severe bleeding (thrombi → bleeding → loss of factors).
Describe the morphology/clinical features of DIC.
- Extensive thrombosis.
- Severe bleeding.
- Shock.
- Renal/organ failure.
Outline the laboratory diagnosis of DIC.
- Decreased coagulation factors and platelets - all tests abnormal* (everything gets used up - DIC is life-threatening - extensive severe bleeding both superficial and deep).
- Increased fibrin degradation products (FDP and D-dimer) - extensive breakdown of thrombi, both FDP and D-dimer (also breakdown product of fibrin) raised.
- Release of tissue factor → widespread microvascular thrombosis → leading to ischaemic tissue damage and consumption of clotting factors and platelets → ultimately leading to extensive bleeding and also fibrinolysis.
- All this leads to decreased platelets, increased PT, PTT, TT and increased FDP and D-dimer.
Outline thrombotic microangiopathy: TTP and HUS.
• Widespread thrombosis in microcirculation leading to organ infarctions and microangiopathic haemolytic anaemia (fragmented RBC - breakdown of RBCs due to fibrin threads).
• Unlike DIC, coagulation factors remain normal or mild decrease (there is only activation of platelets, very little activation of coagulation).
• 2 major clinical syndromes: related and usually together in adults.
- Thrombotic Thrombocytopenic Purpura (TTP) - more common in adults.
- Haemolytic Uraemic Syndrome (HUS) - more common in children.
Outline Thrombotic Thrombocytopenic Purpura (TTP).
• Widespread thrombosis with thrombocytopenia, red cell fragmentation, fever, transient neurologic deficits (stroke) and kidney failure.
- Predominantly neurological deficits in TTP (widespread thrombosis with neurological deficits - thrombi in brain causing stroke) and predominantly kidney failure in HUS.
- Deficiency of ADAMTS13, a protease that normally breaks down VWF multimers to stop platelet activation (breakdown of VWF after the formation of thrombi to prevent further activation).
- Deficiency leads to increased VWF, platelet activation and loss → to extensive systemic thrombosis (opposite of VWD - excess VWF).
• 2 types (both due to deficiency of ADAMTS13):
- Acquired - antibody to ADAMTS13 → deficiency (more common).
- Congenital - mutation → deficiency of ADAMTS13.
• Treatment - plasma exchange with FFP or cryoprecipitate to remove antibody and replace ADAMTS13 (previously 90% fatal).
- Plasma exchange to remove excess VWF.
Outline Haemolytic Uraemic Syndrome (HUS).
• Similar to TTP but kidney failure is the major disorder. More common in children.
• Childhood and adult type (TTP)
- Toxic endothelial damage → thrombosis (toxic endothelial damage leading to thrombosis).
- Predominant acute renal failure.
Childhood HUS:
• Normal ADAMTS13 levels.
• E. coli or Shigella infection + Shiga toxin.
• Childhood HUS - enzyme levels normal unlike TTP. More due to infections and the Shiga toxin causing extensive endothelial damage activating only platelets (not much coagulation).
Adult HUS (TTP with predominant renal failure):
• Infection, inherited or drugs.
• Inappropriate activation of complement, endothelial damage.
• Acute renal failure (HUS like).
Differentiate between DIC, TTP and HUS (systemic activation of haemostasis).
- DIC - more common, activation of both platelets and coagulation.
- TTP - activation of only platelets with the neurological deficits.
- HUS - activation of only platelets with renal failure.
Identify inhibitors of coagulation.
- Hypercoagulability - opposite of bleeding disorders.
- Haemostasis → fibrin formation.
- Normal fibrin production stimulates anticoagulant mechanism to prevent further clotting.
- System of anticoagulants prevent further activation. Disorders of this known as hypercoagulability.
Natural anticoagulants:
• Protein C + S - inhibit activated FVIII and Va (inhibit both intrinsic and common pathway).
• Heparin - blocks activated FX.
- Drugs are also used to prevent excess coagulation. Most important coumarins are warfarin - anti-vitamin K drug which is necessary for production of factors II, VII, IX and X. Also use anti-platelet drugs such as aspirin.
- Normally coagulation and anticoagulation are well balanced.
Outline the aetiology of hypercoagulability.
- Excess coagulation tendency (x. bleeding tendency) - state of increased coagulation and decreased anti-coagulation - more common in lifestyle disorders such as hypertension, diabetes (due to damage to the endothelium by glucose), valvular heart disorders, MI, atherosclerosis - many conditions clinically produce hyper coagulability.
- Hereditary (rare):
- Factor V Leiden (resistant to protein C - anticoagulant) - mutated FV, uncontrolled activation.
- Protein C or S deficiency.
• Acquired (Virchow’s triad - BV, blood, blood flow):
- More common clinically - factors which stimulate coagulation - Virchow’s triad abnormalities - any damage to vessel or tissue injury stimulates coagulation.
- Trauma, inflammation, cancer.
- Heart disease, atherosclerosis etc.
- Stasis of blood (decreased blood flow e.g. long flights - stimulation for activation of coagulation.
- Drugs/antibodies (AAS - antiphospholipid antibody syndrome).
What are the clinical features of hypercoagulability?
• All due to thrombosis - excess coagulation.
• Vein (DVT).
- DVT - clot in deep vein - if dislodged → can go through the heart (right side) and into the pulmonary circulation blocking the pulmonary veins to produce pulmonary embolism.
• Artery (MI, stroke).
- Particularly in patients with heart failure and AF.
• In placenta → abortions.
- In pregnancy - placenta undergoes thrombosis → leading to early first trimester abortions.
Outline antiphospholipid syndrome.
- Antibodies to platelet phospholipids - reasonably common clinically in which antibodies develop to the platelet phospholipids in patients with autoimmune disorders. Can also develop in children post-infection.
- Following infections in children or in patients with autoimmune disorders or drugs. Antiphospholipid Ab syndrome (AAS) e.g. anticardiolipin antibody and lupus anticoagulant (production of these autoantibodies activate coagulation in the body by stimulating the coagulant).
- 30% of SLE patients develop AAS.
- These autoantibodies activate coagulation in body but bind to phospholipid part of reagent in the laboratory to prolong PTT.
- In the lab, they bind to the phospholipid part of the PTT reagent → prolong PTT.
- Very characteristic of ASS - clinically they have excess coagulation but also prolong PTT in the lab - looks like a deficiency but clinically it will be hypercoagulability.
- Clinical: thrombosis - ischaemia, gangrene, DVT and early abortion. Rarely → TTP.
- Lab diagnosis: normal PT but prolonged PTT and remains prolonged even after adding 50% normal plasma, unlike factor deficiency.
Describe the blood products used for blood transfusion.
- Life saving products of blood - blood is the most commonly transplanted tissue but the transfusion carries risk from mild allergy to fatal haemolysis.
- RBC antigens and plasma antibodies cause life-threatening reactions.
- Foreign proteins to infections.
- Mild allergy to fatal haemolysis.
Blood products:
• Red cell concentrate (RCC) - lifespan of 35 days, contains packed RBCs. Used for anaemia.
- Fresh frozen plasma (FFP) - can be stored for up to 12 months, has all the coagulation factors and the plasma proteins. Used for bleeding - coagulation.
- Cryoprecipitate - contains concentrated factors 1, 8, 9 and VWF. Used for specific deficiencies.
- Platelets - only product that has to be stored at approx. 22˚C, can only be stored for 5 days. As the temp is higher → infections common. Used in thrombocytopenia.
- Whole blood - rare used these days. Contains no WBC, platelets or coagulation factors. Used for acute blood loss and major surgeries.
Outline immunohaematology.
• Science of immune reactions relating blood transfusion.
• Transfusion adverse reactions: immune, infection, allergy etc. (transfusion carries the risk of many adverse reactions. Therefore, transfusion should always be weighed against the risk).
• Fatal transfusion reactions still occur (rare).
• WBC, platelets - HLA Ag, difficult to match. More reactions (WBCs, plts most difficult to match as they carry HLA antigens → more reactions, rejection possible).
• RBC - no HLA Ag, only major blood groups, easy to match.
• 30+ blood group systems, >330 antigens.
• Only ABO and Rh are clinically significant blood groups (others MN, Kell, Duffy, P etc.) - important because they have naturally occurring strong IgM antibodies.
• Naturally occurring strong IgM antibodies in ABO system.
• Common blood group O +ve, rare AB -ve.
• Landsteiner law - when a person has a blood group antigen, corresponding antibody is absent from plasma.
- When you transfer blood from one patient to another, patients who lack the antigen on exposure to donor antigens → they develop Abs.
Describe the ABO blood group system.
- ABO system - 3 genes - A, B and O.
- A and B genes produce enzymes which modify the red cell membrane antigen known as H substance.
- O is non-functional. O group - the H substance is not modified.
- When H substance is converted to A antigen due to A gene → blood group known as A group.
- ABO system - only have naturally occurring IgM antibodies.
- In ABO system, there is a H antigen on RBC membrane → converted to A or B → A or B group.
- If it is not converted → known as O group.
Describe the Rh blood group system.
- Rh system also has 3 antigens known as C, D and E. If the genes are non-functional/absent - denoted as c, d and e. Many different Rh groups.
- If D antigen is present - known as Rh positive. When D antigen is absent - known as d or Rh negative.
- Only the D antigen is strong and clinically significant.
What is the Bombay blood group?
Rare condition of absence of H substance in the ABO system. These patients have anti-H antibodies. Clinically, they will be seen as O group but they will have anti-H antibodies.
Differentiate between IgG and IgM immunoglobulins.
IgG: • Small, monomer. • Optimum reaction at 37˚C. • Able to cross the placenta. • Coat cells but not agglutinate. • Main type produced following pregnancy or transfusion (alloantibodies).
IgM: • Large pentamer. • Optimum reaction at 0-20˚C. • Do not cross the placenta. • Agglutinate red cells. • Often 'naturally occurring' antibodies - ABO groups.
Outline pre-transfusion testing - cross match (major and minor).
- Major cross match - donor RBC with patient plasma.
- Minor cross match - donor plasma with patient RBC.
- To prevent haemolytic transfusion reactions.
- Before transfusion, the patient’s blood is matched with the donor’s blood known as cross matching.
- Major cross match is when we take donor’s RBCs and mix with patient plasma. If the patient’s plasma has some Abs against the donor’s RBCs → results in agglutination (not compatible).
- Minor - opposite - donor’s plasma with patient’s RBC. Does not cause severe reactions - minor cross match.
Differentiate between antibody screening and cross matching.
• Cross matching of donor RBC and patient plasma is designed to prevent major immune reaction - major cross match.
• Cross match compatible units are not the safest blood*
• There are many other antigens on donor RBC. So additional antibody screening is done to detect alloantibodies in patient plasma with known reagent RBC (past transfusion or pregnancies).
- Many times, patients may have been exposed to previous transfusions or pregnancies and this would’ve resulted in the patient developing Abs against many minor blood groups.
What are the types of transfusion reactions?
- Occur because blood has so many components.
- Allergic reaction - plasma proteins (plasma proteins causes the minor/mild allergic reactions) - facial flushing, hives/rash. Severe - anxiety, wheezing, dyspnoea, hypotension.
- Febrile reaction - antibody to leucocytes (antibodies to donor leucocytes results in febrile reaction) - fever, chills, anxiety, headache, tachycardia, tachypnoea.
- Haemolytic reaction - immune/mismatch (most dreaded/fatal - haemolytic reactions due to mismatch) - fever, chills, tachycardia, tachypnoea, hypotension, haemoglobinuria, chest pain, apprehension, lower back pain.
Transfusion transmitted infections.
• TRALI: acute lung injury (ARDS) - rarely transfusion related acute lung injury.
• GVHD: graft vs. host disease - rare - donor lymphocytes attacking patient’s tissues.
Outline transfusion transmitted infections.
- Common in the past - most haemophilia cases were HIV, hepatitis +ve.
- With the introduction of pre-transfusion testing, donor screening and leucocyte depletion - safe (transfusion are safe today).
- Bacterial contamination of blood product more common with platelets (22˚C - because kept at higher temp). But can occur with other products rarely.
- Prions (CLD/mad cow disease). Cannot be destroyed with current methods.
Describe donor screening for transfusion transmitted infections.
• HIV-1 and HIV-2 - screening for HIV antigens or antibodies.
- Chances of getting HIV following blood transfusion - 1 in 5 million.
• Hepatitis B - screening for hepatitis B surface antigen (HBsAg).
• Hepatitis C - HCV RNA testing and HCV antibody.
• Syphilis - treponemal antibody.
• Malaria test - on epidemiological evidence only.
• CMV negative - on request for immunosuppressed patients.
Outline the procedure for transfusion adverse reactions.
- Every transfusion has risk - benefit must outweigh risks.
- Donor blood testing - syphilis, HIV-1 & 2, HBV, HCV and HTLV 1 & 2.
- CMV negative and leucocyte depleted: neonatal and immunosuppressed.
Procedure for transfusion adverse reactions:
• Stop transfusion immediately.
• Check patient ID and blood product ID and cross match details.
• Report to the Blood Bank immediately with patient blood sample and remaining blood product - for further investigations.
• “Lookback” program is to identify and notify recipients who have received blood products from a previously negative, screened donor who has now become positive for an infectious agent.
