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

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

Appropriate diagnosis

AGE <1 YEAR 1-5 years 6-10 years 11-16 years ADULT

APTT Cephascreen (seconds) 32.2 (29.1 - 35.5) 31.6 (28.6 - 35.8) 33.1 (29.8 - 35.3) 33.8 (28.0-37.9) 33.6 (26.3 - 40.3)

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

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

<1 1-5 6-10 11-16 Adults 50 100 150 200 250 APTT (sec)

Heparin Therapy in Children: APTT range

55 - 118 54 - 142 75 - 154 78 - 200 82 - 177

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

• PSO4: Anti Xa: r2 = 0.47
• PSO4: APTT: r2 = 0.56
• Anti Xa: APTT: r2 = 0.72
• But >60% APTT values > 999 second so not

included in comparison

Therapeutic ranges : How should they be determined?

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

Anti Xa: IIa ratio following single bolus of UFH

Age Group Anti-Xa:Anti-IIa Ratio (15mins) Anti-Xa:Anti-IIa 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?

Ignjatovic et al. Thromb Res. 2007;120: 347-51. Coamatic (-AT, +DS) Coatest (+AT, -DS) Modified (-AT, -DS)

anti Xa in children (< 1 year) on UFH - in vivo

Age-group Anti-Xa activity (IU/ml) 0 - <0.35 0.35 - 0.7 >0.7 Anti-Xa (-AT, +DS) N = 89 <1 year Anti-Xa (+AT, -DS) N = 87 N=73 N=5 N=11 N=57 N=26 N=4

Ignjatovic et al. J Thromb Haemost 2006;4:2280-3

PK of UFH in children

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

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

Qualitative Age-Related Differences

Protein Author Year Findings

Fibrinogen Kunzer et al, Witt et al. 1961-3, 1969

• Existence of ‘foetal fibrinogen’
• ‘Foetal fibrinogen’ has greater

negative charge

• Different molecular structure as

compared to adults

Qualitative Age-Related Differences (Animal models)

Protein Author Year Findings

Antithrombin Niessen et al. 1996

• Unique foetal isoform of AT

was detected in sheep

Fibrinogen Andrew et al. 1988

• Foetal fibrinogen in lamb

has high sialic acid content

• Different glycan structure

Protein C Manco-Johnson et al. 1989

• Foetal ovine protein C is

~4 kDa larger than the adult form (70 kDa)

• Detection is only during 6

days before birth and 4 days post birth

Fibrinogen Assay

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.

1 2 3 4 5 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

Fibrinogen

RP-HPLC Purification of Fibrinogen

7.5 8.0 8.5 9.0 9.5 10.0 10.5

Adult 11 to 16 years old 6 to 10 years old 1 to 5 years old <1 years

• ld

neonates Age Group Adult 11 to 16 years old 6 to 10 years old 1 to 5 years old <1 years old neonates

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

MW marker (kDa) Neonates Adult 11 - 16 years old < 1 years old 1 - 5 years old 6 - 10 years old Standard Fibrinogen

64 50 250 148 36 22 A B 

100% 123.5% 105.9% 94.1% 64.7% 64.7%

Adult Infant

Fibrin (nm) Median Minimum Maximum Adults 352.3 26.1 997.5 Infants 343.3** 61.2 997.1 Pore size (pm2) Median Minimum Maximum Adults 0.052 0.001 15.24 Infants 0.057** 0.0041 19.97

**P<0.001 **P<0.001

Adult Infant

Baseline + UFH Baseline + UFH Fibrin (nm) Median Minimum Maximum Control 343.3 61.2 997.1 UFH 334.9** 109.1 998.4 Fibrin (nm) Median Minimum Maximum Contro l 352.3 26.1 997.5 UFH 339.0** 66.9 996.4 Pore size (pm2) Median Minimum Maximum Control 0.052 0.001 152.4 UFH 0.054** 0.0041 218.74 Pore size (pm2) Median Minimum Maximum Control 0.057 0.0041 19.97 UFH 0.055* 0.0041 24.25

*P<0.05, **P<0.001

Adult Infant

Baseline + tPA Baseline + tPA Fibrin (nm) Median Minimum Maximum Control 343.3 61.2 997.1 tPA 343.0 65.6 998.8 Fibrin (nm) Median Minimum Maximum Control 352.3 26.1 997.5 tPA 355.8* 70.0 999.6 Pore size (pm2) Median Minimum Maximum Control 0.057 0.0041 19.97 tPA 0.050** 0.0041 19.34 Pore size (pm2) Median Minimum Maximum Control 0.052 0.001 152.4 tPA 0.049 0.0041 202.60

*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

• ur 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

Day 1 neonate Adult

Increase in abundance with age Clusterin Haptoglobin Alpha-1-antitrypsin Hemopexin precursor Kininogen 1 Variant Chain A, Human Factor H Decrease in abundance with age Alpha-2-macroglobulin Complement C3 Complement Factor B Fibrinogen Vitamin D-binding protein Fibronectin 1 variant Heparin Cofactor II Alpha-1-antichymotrypsin Bikunin

Molecular Functions

1 protease binding 1 peptidase activity 3 serine-type endopeptidase activity 3 hemoglobin binding 2 protein binding 1 calcium ion binding 1 vitamin transporter activity 1 vitamin D binding 1 actin binding 11 protein binding 11 eukaryotic cell surface binding 12 receptor binding 2 endopeptidase inhibitor activity 2 wide-spectrum protease inhibitor 2 tumor necrosis factor binding 3 serine-type endopeptidase inhibitor 2 interleukin-8 binding 2 interleukin-1 binding 2 enzyme binding

Protein binding Cell surface binding Receptor binding

Biological Processes

1 lipid metabolic process 1 innate immune response 1 apoptosis 1 blood coagulation 1 acute-phase response 3 proteolysis 1 positive regulation of type IIa receptor 1 positive regulation of phagocytosis 1 vitamin transport 11 signal transdcuction 11 response to calcium ion 11 protein polymerization 11 platelet activation 1 G-protein coupled receptor process 2 complement activation, classical 1 complement activation, alternative 2 protein homooligomerization

Platelet activation Protein polymerization Response to Calcium ion Signal transduction

Protein Locations

1 nucleolus 3 extracellular region 1 No GO ID 1 centrosome 3 soluble fraction 11 fibrinogen complex 11 external side of plasma membrane 14 platelet alpha granule lumen 5 nucleus 6 extracellular space

Platelet alpha granule lumen External side of plasma membrane Fibrinogen complex

Platelet Proteome

A. Adult Sample (Cy3) B. Paediatric sample (Cy5)

• C. Overlay

~837 protein spots per gel

Platelet Proteome 10 protein spots (1.2%) differentially expressed; majority up regulated in children

Protein ID

Average Ratio

(children : adults)

P value Thrombospondin-1

• 1.51

0.012 Serotransferrin +1.78 0.037 Fibrinogen alpha chain +1.57 0.028 Serum Albumin +2.45 0.010 Glyceraldehyde-3-phosphate dehydrogenase +1.68 0.013 Transgelin-2 +2.64 0.050 Calponin-2 LIM SH3 domain protein 1 +1.69 +1.69 0.030 0.030 HCG2039797 (Possible J 56 gene segment) +1.68 0.019

Platelet Secretome

A. Paediatric sample (Cy3) B. Adult sample (Cy5)

• C. Overlay

~718 protein spots per gel

Platelet Secretome

• 11 protein spots (1.5%) differentially expressed

Protein ID Average Ratio

(children:adults)

P value Thrombospondin-1

• 1.73

0.041 Gelsolin

• 1.73

0.041 Factor XIII A chain

• 2.17

0.015 Factor XIII B chain

• 2.17

0.015 Gelsolin

• 2.87

0.0115 Serotransferrin +11.10 0.0076 Fibrinogen gamma chain

• 4.07

0.0018 Actin, cytoplasmic 1 Beta-actin; +2.71 0.031 Plasminogen activator inhibitor 1 +1.85 0.047 Tropomyosin 3 (TPM3) +2.00 0.042 Tropomyosin-4 +2.00 0.042 Basement-membrane protein 40

• 1.82

0.0007 Secreted protein acidic and rich in cysteine

• 1.82

0.0007

Canonical Pathways

Common Proteins

Proteome

(children to adults)

Secretome

(children to adults)

Thrombospondin-1   Serrotransferrin  

Implications

• There are significant differences in the expression of platelet

proteins and proteins secreted in response to platelet activation in healthy children compared to healthy adults.

• Identified proteins play important roles in processes such as

platelet aggregation and plaque stabilization.

• In this context they could be important in:
• Normal Growth and Development
• Protective mechanism against disease
• Potential biomarkers for susceptibility to disease
• Targets for pharmacological therapies

Thrombospondin-1 (TSP-1)

• Decreased expression in the platelet proteome and secretome of children

Role Disease setting Expression

(Adult studies)

Clinical Significance in children Promotes platelet aggregation CVD Atherosclerosis Thromboembolic events

↑

↓ TSP-1 levels may contribute lower incidence of thrombosis Cancer aetiology

Tumour cell adhesion, migration, invasion and angiogenesis

Cancer

↑

↓ TSP-1 levels may contribute to the decreased incidence

• f particular cancers

in children

Serotransferrin

• Increased expression in the platelet proteome and secretome of children

Role Disease Setting Expression (Adult studies) Clinical Significance in children Suppresses Platelet Activation Acute Coronary Syndromes (ACS) Thrombus formation

↓

↑ Serotransferrin levels may contribute to thromboprotection in children Antioxidative effects Alzheimer’s disease

↓

↑ Serotransferrin levels are protective against Alzheimer’s disease

Circulating MPAs are increased in healthy children compared to adults (Yip et al, 2013)

MPAs P-Selectin

Monocyte-Platelet Aggregates (MPAs) Adults Children Platelets activated Platelets not activated Binding of platelets to monocytes is via P-Selectin on platelets P-selectin independent Plaque instability & increased thrombotic risk ???

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

Thanks to the Team

• Vera Ignjatovic
• Chantal Attard
• Vicky Karlaftis
• Fiona Newall
• Sophie Jones
• Christina Yip
• Michelle Hepponstall
• Charmaine Cini
• Clinical Haematology team RCH
• Anaesthetic /surgical teams RCH/RWH
• Maastricht University, Maastricht, The

Netherlands

• A/Prof Bas de Laat, Dr Raed al Dieri
• University of Murcia, Murcia, Spain
• Professor Javier Corral
• Henderson Research Centre, Hamilton,

Canada

• Professor Anthony Chan and Mr Leslie

Berry (Patent)

• University of Santiago de Compostela,

Spain

• Dr Angel Garcia Alonso
• The University of Western Australia
• A/Prof Matthew Linden