Coagulation tests in children. Katrien Devreese, MD, PhD Coagulation Laboratory Ghent University Hospitial

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1 Coagulation tests in children Katrien Devreese, MD, PhD Coagulation Laboratory Ghent University Hospitial 25 April

2 Introduction The young are not just miniature adults Physiology of pediatric hemostasis differs from adults Course of hemostatic disorders may differ Correct interpretation of coagulation test results 2 2

3 Introduction Hemostasis Coagulation Anticoagulation 3 3

4 Introduction Primary hemostasis Platelets interact with damaged endothelium Primary hemostatic plug to arrest bleeding Primary platelet plug: fragile and easily dislodges Secundary hemostasis Formation of insoluble fibrin strands Secundary hemostatic plug: a stable plug Involves the coagulation cascade proteins that interact with each other and the platelet plug Fibrinolysis Once healing occured, the clot has to be lysed to prevent occlusion of vessel 4 4

5 Introduction Primary hemostasis Platelet count Platelet morphology Platelet function Secundary hemostasis Clotting assays: aptt, PT, thrombin time Fibrinogen Coagulation factors (FXII, FXI, FIX, FVIII; FII, FV, FVII, FX; FXIII; VWF) Coagulation inhibitors (AT, PC, PS) Fibrinolysis Plasminogen, t-pa, α2-antiplasmin, PAI 5 5

6 Laboratory tests for hemostasis Testing hemostasis in children Bleeding Screening tests: complete blood count prothrombin time (PT) activated partial thromboplastin time (aptt) (Platelet function analysis (PFA)) Further laboratory evaluation: additional tests Thrombosis Inherited thrombophilia markers (antithrombin, protein C, protein S, FVLeiden, FIIG20210A) Issues related to coagulation testing in children 6 6

7 Laboratory tests for hemostasis Sample acquisition for coagulation tests Blood collection Anticoagulated blood with citrate Pre-analytical variables Sufficient blood Not from a heparinised line Not in heparinised syringe No air bubbles No fluid contamination Heparin strongly disturbs coagulation tests! 7 7

8 Laboratory tests for hemostasis Citrated tubes - buffered sodium citrate 0,109 mol/l: 3,2% - proportion 1 part citrate/9 parts blood - allows recalcification for testing False prolonged aptt Normal High hematocrit / underfilling of tube Hct ~ 45% 1 part citrate Plasma ~ 55% Citrate:plasma ~ 1:5 9 parts blood Citrate:plasma ~ 1:3 -dilution effect on plasma -excess of citrate that binds to calcium added in reaction mix 8 8

9 Hemostasis in children Developmental hemostasis Fib (Jaffray et al, Pediatr Clin N Am 2013, 60, ) 9 9

10 Developmental hemostasis Neonatal versus Adult Hemostasis Component Neonatal Functions Global Effect on Hemostasis Coagulation factors Primary hemostasis Fibrinolysis FII, FVII, FIX, FX, XI, XII Fibrinogen, FV, FXIII FVIII VWF Normal Platelet count Platelet Function Plasminogen, t-pa, α 2 - antiplasmin PAI Decreased thrombin generation Enhanced primary hemostasis Hypofibrinolytic state Natural coagulation inhibitors AT, PC, PS Reduced capacity to inhibit activated coagulation proteins (Lippi et al, Sem Thromb Hemost 2007, 33, Guzzetta et al, Ped Anesthesia 2010, 21, 3-9) 10 10

11 Hemostasis in children Developmental hemostasis Maureen Andrew ( ) Mc Master University Hamilton, Canada Reference values for coagulation tests: Healthy full-term infants (day1-6 months) Healthy children (1y-16y) Healthy prematures (30-36 weeks) Healthy fetuses (19-27 weeks) Blood 1992, 80, Blood 1987, 70, Ped J Hematol/oncol 1990,12, Journal of Pediatric Hematology/Oncology, 2001, 23, Tribute To Dr. Maureen Andrew Monagle et al, Blood Reviews 2010, 24,

12 Routine coagulation assays intrinsic neg charged surface XII XII a aptt contact activator PT extrinsic thromboplastin XI PL, CaCl 2 IX XI a IX a VIII a PL Ca++ common TF VII a PL Ca++ X X a V a PL Ca++ activator II thrombin Citrated anticoagulated plasma fibrin 12 12

13 Routine coagulation assays 13 13

14 Routine coagulation assays aptt aptt= is the time it takes to form a clot in activated plasma measured in seconds 6 mth. FXI FXII PK HMWK (M. Andrew et al, Blood 1992, ) 14 14

15 Routine coagulation assays Contactactivator and PL CaCl 2 aptt Incubation 37 C Clotting time PK HMWK XII Kall IX XIa Fibrinogen Fibrin IXa Tissue damage HMWK XI XIa XII XIIa VIII X VIIIa Ca ++ PL Tenase complex Xa aptt= is the time it takes to form a clot In activated plasma measured in seconds 6 mth. FXI FXII PK HMWK (M. Andrew et al, Blood 1992, ) 15 15

16 Routine coagulation assays intrinsic neg charged surface PT XII XII a extrinsic XI IX XI a FII FVII FIX FX IX a VIII a PL Ca++ TF VII a PL Ca++ X II X a V a PL Ca++ thrombin 6 mth. (M. Andrew et al, Blood 1992, ) fibrin 16 16

17 Routine coagulation assays Reference Values for Coagulation Tests in the Healthy Full-term Infant During the First 6 Months of Life day 1 day 5 day 30 day 90 day 180 adult PT (s) 13.0± ± ± ± ± ±0.78 aptt(s) 42.9 ± ± ± ± ± ± 3.44 significantly different from adults (M. Andrew et al, Blood 1987, 70, ) Day 5 FII FVII FIX FX (M. Andrew et al, Blood 1992, ) 17 17

18 Routine coagulation assays Developmental hemostasis Age-dependent reference ranges Definition of healthy children Diagnosis of bleeding and thrombotic disorders Monitoring anticoagulant therapy Labs use published reference ranges Blood samples: ethical stand points, costs Reported reference ranges on small groups Not reagent or instrument adapted 18 18

19 Coagulation assays Age-dependent reference ranges Neonates 1 month-1year 1-5 y 6-10 y 11-16y <1 year Premature neonates Low birth weight vs healthy children (Ignjatovic et al, J Thromb Haemost 2012, 10, ) 19 19

20 20 20

21 Routine coagulation assays Example of aptt Adult reference range: 28,9-38,1 sec 5-30 dag reference range: sec abnormal aptt Repeat testing Mixing test FVIII, FIX, FXI, FXII, VWF, lupus anticoagulant Unnecessary costs, cancellation of surgery, additional clinical consults, overtreatment Misdiagnosis of bleeding or clotting disorder 21 21

22 Prolonged aptt Prolonged aptt and bleeding Increased bleeding tendency No bleeding Thrombosis Von Willebrand Artefact F XII def. LAC Factor XI def. anticoagulants Def. prekallikrein LAC FX, FV, FII, Fg Hemophilia 22 22

23 Mild decreased FXI: normal aptt 23 23

24 Prolonged aptt Sensitivity for factordeficiencies Procoagulant Fg Prothrombine (FII) F V FVII F VIII F IX F X F XI F XII Reference value mg/dl % % % % % % % % Level for normal aptt* 60 mg/dl 15% 40% - 35% 20% 25% 30% 20% * Depends on the reagent used 24 24

25 Von Willebrand disease Screening tests Confirmation tests Tests for further classification aptt VWF:Ag RIPA (2B) Closure time (PFA) VWF:act (VWF:RCo; CBA) Multimer analysis Thrombocytes FVIII:C VWF-FVIII binding assay (2N) Mutation (type 2) 25 VWF:pp 25

26 Von Willebrand disease Screening tests Confirmation tests Tests for further classification aptt VWF:Ag RIPA (2B) Closure time (PFA) VWF:act (VWF:RCo; CBA) Multimer analysis Acute phase proteins Repeat testing! Thrombocytes FVIII:C VWF-FVIII binding assay (2N) Mutation (type 2) 26 VWF:pp 26

27 Prolonged aptt Prolonged aptt and bleeding Increased bleeding tendency No bleeding Thrombosis Von Willebrand Hemophilia Artefact Factor XII def. LAC Factor XI def. anticoagulants Def. prekallikrein LAC FX, FV, FII, Fg Heparin contamination Under filling tube 27 27

28 Prolonged aptt as artefact CRP Acute phase protein Affinity for phospholipids to recognize pathogens and apoptotic cells Prolonged aptt in intensive care patients (van Rossum et al, Br J Haematol, 2012, 157, ) Analytical interference with CRP: binds to phospholipids Falsely prolonged aptt (Devreese, et al Clin Chem Lab Med, 2015, 53(5): 141-5) 28 28

29 Prolonged aptt as artefact 1,30 Normalized ratio aptt 1,20 1,10 1,00 0, sec (refw <38.1 sec) 16.9 mg/dl 0, CRP (mg/dl) PTT-A Pathromtin SL Cephascreen SynthASil C.K. Prest (Schouwers, Delanghe, Devreese. Thromb Res 2010,125:102-4) (Devreese, et al Clin Chem Lab Med, 2015, 53(5): 141-5) 29 29

30 Prolonged aptt Prolonged aptt and bleeding Increased bleeding tendency No bleeding Thrombosis Von Willebrand Hemophilia Artefact Factor XII def. LAC Factor XI def. anticoagulants Def. prekallikrein LAC FX, FV, FII, Fg 30 30

31 intrinsic Prolonged aptt as artefact neg charged surface XII XII a extrinsic XI IX XI a APA IX a VIII a PL Ca++ common TF VII a PL Ca++ X X a V a PL Ca++ II thrombin fibrin Antiphospholipid antibodies: lupus anticoagulant 31 31

32 Prolonged aptt as artefact Lupus anticoagulant Criterion for antiphospholipid syndrome persistently positive > 12 weeks Risk for thrombosis Transient LAC Incidentally found prolonged aptt Infection No association with thrombosis Repeat testing 32 32

33 aptt as screening test A normal aptt is not always an indication of normal hemostasis Mild factor deficiencies with normal aptt can be associated with significant bleeding history (eg FXI >30%, type I VWD) Be aware of the sensitivity of the aptt reagent If bleeding history is suggestive: perform factor dosage even with normal aptt 33 33

34 intrinsic neg charged surface XII Routine coagulation assays XI XII a IX aptt XI a IX a VIII a PL Ca++ common extrinsic TF VII a PL Ca++ X PT X a V a PL Ca++ II trombin fibrin 34 34

35 Normal aptt, normal PT -FXIII activity -Immunoassay for subunit A and subunit B 35 35

36 Routine coagulation assays intrinsic neg charged surface PT XII XII a extrinsic XI IX XI a IX a VIII a PL Ca++ vitamin K dependent factors TF VII a PL Ca++ X II X a V a PL Ca++ trombin fibrin 36 36

37 Prolonged PT Vitamin K deficiency Vitamin K deficiency 37 37

38 Prolonged PT Precursors of factors II, VII, IX, X, protein C and protein S => PIVKA Complete forms of factors II, VII, IX, X, protein C en protein S Ca 2+ Bind to phospholipids 38 38

39 Prolonged PT Vitamin K deficiency bleeding (VKDB) Bleeding: factors <30% Mild vitamin K deficiency: PT prolonged (FVII) Severe vitamin K deficiency: aptt and PT prolonged Diagnosis: PT, aptt, FII, FVII, FIX, FX dosage < PIVKA, vitamin K dosage FV: differentiates liver dysfunction/vit K deficiency 39 39

40 Routine coagulation assays (Sarnaik et al, Clin Ped, 2010, 49, ) 40 40

41 Platelet function Neonates: Hyporeactive to thrombin, ADP, tromboxane first 2-4 weeks 41 41

42 Laboratory tests for platelet function Platelet aggregation LTA on PRP -250 µl PRP/agonist= 10 ml blood -platelet aggregometry should be repeated once to ensure reproducibility of results. ADP 2.5µM ADP 5µM Collagen 2.5µg/ml Collagen 5 µg/ml Ristocetine 1.5 mg/ml Ristocetine 0.5 mg/ml Arachidonic acid 1.5 mm Epinephrine 10µM Tromboxane-analogue 42 42

43 Laboratory tests for platelet function Screening platelet function 2x 800µl citrated blood Hct>35% and > plts/µl Closure time in seconds is a measure for function of thrombocytes Adult: Collagen/epinephrine sec Collagen/ADP sec Shorter in neonates 43 43

44 Laboratory tests for platelet function The PFA 100 : a potential rapid screening tool for the assessment of platelet dysfunction (P. Harrison et al. Clin Lab. Haem :25-232) a small number of false negative results (6%) obtained with various milder platelet defects (e.g. Hermansky Pudlak syndrome, storage pool and release defects, type I VWD and macrothrombocytopenia) EPI EPI PFA-100 collagen/adp (CADP) (a) and collagen/epinephrine (CEPI) (b) closure times in patients classified as either normal (N) or diagnosed with von Willebrand's disease (VWD), Bernard Soulier sydrome (BS) and Glanzmann's thrombasthenia (GT); Hermansky Pudlak syndrome (HPS), Storage Pool Disease (SPD), Release Defects (RD), Wiskott Aldrich syndrome (WAS) and Grey Platelet syndrome (GPS). The normal range values are shown (NR) with 2 SD normal range limits (dotted lines).

45 Interpretation of coagulation tests Interpretation with caution Diagnosis of bleeding disorder or thrombophilia based on Clinical symptoms Family history Reproducible laboratory results 45 45

46 Coagulation tests in children: Conclusions Immature hemostatic system 3-6 months Differences between adults and children: physiological Age appropriate reference ranges Sample integrity, repeat testing Routine coagulation tests and additional testing An abnormal laboratory test result is not sufficient to define a disease A normal test result does not exclude a disease 46 46

47 THANK YOU FOR YOUR ATTENTION 47 47

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