Paediatrics Paul Monagle Department of Clinical Haematology Royal - PowerPoint PPT Presentation

Normal and Abnormal Haematology in Paediatrics Paul Monagle Department of Clinical Haematology Royal Children’s Hospital, Melbourne Stevenson’s Chair, Head of Department of Paediatrics University of Melbourne Group Leader, Haematology Research Murdoch Children’s Research Institute

Disclosures • Stago • Bayer • Paediatrician • Swans supporter • Ironman

Why does it matter ? • Appropriate diagnosis of children with bleeding and clotting disorders • Appropriate use of anticoagulant drugs in children • Understanding biology of coagulation disorders • Implications for diseases in adults

Why does it matter ? • Appropriate diagnosis of children with bleeding and clotting disorders • Appropriate use of anticoagulant drugs in children • Understanding biology of coagulation disorders • Implications for diseases in adults

Appropriate diagnosis

Appropriate diagnosis Age 1 month – 1 – 5 6 – 10 APTT 11-16 Adults results (sec) 1 year years years years PTT-A 39.3* 37.7* 37.3* 39.5* 33.2 (35.1-46.3) (33.6-43.8) (31.8-43.7) (33.9-46.1) (28.6-38.2) CK Prest 34.4* 32.3* 32.9* 34.1* 29.1 (31.1-36.6) (29.8-35.0) (30.8-34.8) (29.4-40.4) (25.7-31.5) Actin FSL 37.4* 36.7* 35.4* 38.1* 30.8 (33.4-41.4) (31.8-42.8) (30.1-40.4) (32.2-42.2) (27.1-34.3) Platelin L 36.5* 37.3* 35* 39.4* 31.3 (33.6-40.4) (32.5-43.8) (31.0-39.3) (32.6-49.2) (27.2-35.4)

Appropriate diagnosis AGE <1 YEAR 1-5 years 6-10 years 11-16 years ADULT APTT 32.2 31.6 33.1 33.8 33.6 Cephascreen (29.1 - 35.5) (28.6 - 35.8) (29.8 - 35.3) (28.0-37.9) (26.3 - 40.3) (seconds)

Appropriate diagnosis • Family history • Personal history • Pre surgical workup • Family anxiety • Non accidental injury

Appropriate diagnosis

Why does it matter ? • Appropriate diagnosis of children with bleeding and clotting disorders • Appropriate use of anticoagulant drugs in children • Understanding biology of coagulation disorders • Implications for diseases in adults

Heparin therapy in Adults • Current recommended therapeutic range for the treatment of venous TEs in adults is an Activated Partial Thromboplastin Time (APTT) that reflects a heparin level by Anti-Factor Xa level of 0.35 to 0.7 units/ml. • This is based on correlation of Anti-Xa levels to protamine titration levels of 0.2 to 0.4 u/ml of UFH.

Heparin Therapy in Children: APTT 300 250 200 APTT (sec) 150 <1 100 1-5 6-10 11-16 50 Adults 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Measured Anti FXa (IU/ml)

Heparin Therapy in Children: APTT range Adults 55 - 118 11-16 54 - 142 6-10 75 - 154 78 - 200 1-5 82 - 177 <1 0 50 100 150 200 250 APTT (sec)

Heparin in children • Newall et al – PK study of UFH in children – Cardiac catheter scenario – 64 children enrolled – 75 - 100 u/kg bolus of UFH – No infusion – Bloods taken at baseline, 15, 30, 45 120 minutes

Correlation of Heparin monitoring tests in children • PSO 4 : Anti Xa: r 2 = 0.47 • PSO 4 : APTT: r 2 = 0.56 • Anti Xa: APTT: r 2 = 0.72 • But >60% APTT values > 999 second so not included in comparison

Therapeutic ranges : How should they be determined? APTT RANGE MEAN APTT (SECONDS) 66 ± 10* † 1.5 to 2.5 times 54-90 Baseline Anti-Xa assay 97-287 217 ± 114* (0.35 to 0.7IU/mL) 236 ± 186 † Protamine 49-287 Titration (0.2 to 0.4IU/mL)

Anti Xa: IIa ratio following single bolus of UFH Age Group Anti-Xa:Anti-IIa Anti-Xa:Anti-IIa Ratio (15mins) Ratio (120mins) <1yr 1.9 1 1-5yrs 1.5 1.1 6-10yrs 1.6 1.2 11-16yrs 1.3 1.2

Which anti Xa method is appropriate? Coamatic (-AT, +DS) Coatest (+AT, -DS) Modified (-AT, -DS) Ignjatovic et al. Thromb Res . 2007;120: 347-51.

anti Xa in children (< 1 year) on UFH - in vivo Anti-Xa activity (IU/ml) Age-group 0 - <0.35 0.35 - 0.7 >0.7 Anti-Xa (-AT, +DS) N = 89 N=73 N=5 <1 year N=11 Anti-Xa (+AT, -DS) N = 87 N=57 N=26 N=4 Ignjatovic et al. J Thromb Haemost 2006;4:2280-3

PK of UFH in children MEAN ± SD PARAMETER AUC/dose/kg 2.1 ± 0.97 2.9 ± 1.1 cMax Elimination Rate Constant 0.017 ± 0.006 (k) 45.6 ± 14 t½ (mins)

Binding of Heparin to plasma proteins * * Up to three-fold difference across the age-groups.

Anticoagulant Drugs

True quantitative differences

True quantitative differences

True quantitative differences

True quantitative differences

Implication of antigenic differences

Qualitative differences

Qualitative differences β -AT Activity SD p-value (% Adult Value) Neonates 78 0.63 45.8 <1 year old 114 0.54 38.4 1  5 yrs 101 0.95 35.4 6  10 yrs 116 0.53 41.4 11  16 yrs 105 0.86 41.1 Adults 100 N/A 36.3

Qualitative Age-Related Differences Protein Author Year Findings • Existence of ‘foetal fibrinogen’ Fibrinogen Kunzer et al, 1961-3, Witt et al. 1969 • ‘Foetal fibrinogen’ has greater negative charge • Different molecular structure as compared to adults

Qualitative Age-Related Differences (Animal models) Protein Author Year Findings • Unique foetal isoform of AT Antithrombin Niessen et al. 1996 was detected in sheep • Foetal fibrinogen in lamb Fibrinogen Andrew et al. 1988 has high sialic acid content • Different glycan structure • Foetal ovine protein C is Protein C Manco-Johnson et 1989 ~4 kDa larger than the al. adult form (70 kDa) • Detection is only during 6 days before birth and 4 days post birth

Fibrinogen Assay Fibrinogen 5 4 3 2 1 Day 1 Day 3 1 month-1 year1-5 years 6-10 years 11-16 years Adults Age groups Broken lines represent previously published range: mean and 95% population Table 1. Fibrinogen reference values for different age-groups compared to results determined by Monagle et al . *Denotes values that are significantly different from adult values (p<0.05). Fibrinogen levels were determined by the STA-R Evolution analyzer.

RP-HPLC Purification of Fibrinogen Adult 11 to 16 years old 6 to 10 years old 1 to 5 years old <1 years old neonates 10.5 10.0 9.5 9.0 8.5 8.0 7.5 Adult 11 to 16 6 to 10 1 to 5 <1 years neonates years old years old years old old Age Group Figure 1. Retention times of fibrinogen for different age-groups. Results are represented as Mean  SD (n=6). Comparisons: Adult vs. Children and Neonates. All values are p<0.01.

SDS-PAGE Analysis of the whole peak of fibrinogen Standard Fibrinogen MW marker (kDa) 11 - 16 years old 6 - 10 years old 1 - 5 years old < 1 years old Neonates Adult 250 148 A  100% 123.5% 105.9% 94.1% 64.7% 64.7% 64 B   50 36 22

Adult Infant Pore size Fibrin Median Minimum Maximum Median Minimum Maximum (pm 2 ) (nm) Adults 352.3 26.1 997.5 Adults 0.052 0.001 15.24 Infants 343.3** 61.2 997.1 Infants 0.057** 0.0041 19.97 **P<0.001 **P<0.001

Adult Infant Baseline + UFH Baseline + UFH Fibrin Fibrin Median Minimum Maximum Median Minimum Maximum (nm) (nm) Contro 352.3 26.1 997.5 Control 343.3 61.2 997.1 l UFH 334.9** 109.1 998.4 UFH 339.0** 66.9 996.4 Pore size Pore size Median Minimum Maximum Median Minimum Maximum (pm 2 ) (pm 2 ) Control 0.057 0.0041 19.97 Control 0.052 0.001 152.4 UFH 0.055* 0.0041 24.25 UFH 0.054** 0.0041 218.74 *P<0.05, **P<0.001

Adult Infant Baseline + tPA Baseline + tPA Fibrin Fibrin Median Minimum Maximum Median Minimum Maximum (nm) (nm) Control 352.3 26.1 997.5 Control 343.3 61.2 997.1 tPA 355.8* 70.0 999.6 tPA 343.0 65.6 998.8 Pore size Pore size Median Minimum Maximum Median Minimum Maximum (pm 2 ) (pm 2 ) Control 0.052 0.001 152.4 Control 0.057 0.0041 19.97 tPA 0.049 0.0041 202.60 tPA 0.050** 0.0041 19.34 *P=0.02 **P<0.001

Summary of clot structure differences Adults Infants  BASELINE Fibrin fiber  Pore size   + UFH Fibrin fiber   Pore size  + tPA Fibrin fiber No change  Pore size No change

Why does it matter ? • Appropriate diagnosis of children with bleeding and clotting disorders • Appropriate use of anticoagulant drugs in children • Understanding biology of coagulation disorders • Implications for diseases in adults

Incidence of Thrombosis increases with age < 1 / 100,000

Who controls the controllers? In a novel study of children who received livers transplanted from adult donors, Lisman and colleagues describe how plasma levels of coagulation proteins remain at pediatric levels posttransplantation, suggesting that control of the plasma levels is not primarily driven by the liver itself. This study raises numerous important questions about the biology and regulation of the coagulation system, a key control system in our bodies, and should be the stimulus for much further research.

Why does it matter ? • Appropriate diagnosis of children with bleeding and clotting disorders • Appropriate use of anticoagulant drugs in children • Understanding biology of coagulation disorders • Implications for diseases in adults

Adult Day 1 neonate