challenges in pediatric coagulation testing · • concentrations of the majority of coagulation...

©2015 MFMER | slide-1 ©2015 MFMER | slide-1

Challenges in Pediatric Coagulation Testing

Deepti M. Warad, MBBS Division of Pediatric Hematology Oncology

Division of Hematopathology, Special Coagulation Laboratory

©2015 MFMER | slide-2

Disclosures Relevant Financial Relationships

None

Off-Label/Investigational Uses

None


Objectives •  Recognize role of physiologic changes in hemostatic factors during

development

•  Identify age-related differences in coagulation parameters

•  Recognize the variation of normal ranges with laboratory techniques


Coagulation testing variables •  Age-related differences – developmental stage, sex, race

•  Pre-analytical factors – sample acquisition

•  Analytical factors – reagents, analyzers, reporting units


Developmental Hemostasis •  ‘‘Developmental Hemostasis” - late 1980s by Dr. Maureen Andrew

•  Single large cohort study of Canadian children

•  137 healthy pre-term, 118 healthy full-term, and 163 children

•  Concentrations of the majority of coagulation proteins, as measured by functional assays, varied significantly with age

•  Reference ranges preterm and term infants, children - PT/INR, APTT, bleeding time, fibrinogen, FII, FV, FVII, FVIII, vWF, FIX, FX, FXI, FXII, PK, HMW-K, FXIIIa, FXIIIs, plasminogen, TPA, PAI, antithrombin, a2-M, a2-AP, C1E-INH, a1-AT, HCII, protein C, protein S (total and free)

Andrew M et al. Blood 1987, Andrew M et al. Blood 1988, Andrew M et al. Blood 1987, Andrew M et al. Blood 1992


Age-related differences •  Procoagulant factors ---

•  Vit. K-dependent (II, VII, IX and X) and contact factors (XI, XII, PK), HMWK - <70% of the adult values at birth

- rapidly increase in the first few weeks of life and overlap substantially with the adult range by 6 months of age

- average values of most remain 20% lower until teenage years

•  Prothrombin - decreased by 20% throughout childhood

•  Fibrinogen, FV, FVIII, vWF, FXIII – >70% of the adult values at birth

•  vWF levels – remain elevated until about 3 months of age Andrew M et al. Blood 1987, Andrew M et al. Blood 1988, Andrew M et al. AM J Ped Hem Onc1990, Andrew M et al. Blood 1992


Age-related differences •  Coagulation inhibitors ---

•  AT and HCII - Vary with gestational and postnatal age -  50% of adult values at birth and increase to adult levels by 3 months

of age

•  Protein C and S are even lower at birth

•  Protein C - remains markedly low throughout childhood

•  α2-macroglobulin – elevated over adult values at birth, twice adult values at 6 months of age, and continue to be increased until the third decade of life

Andrew M et al. Blood 1987, Andrew M et al. Blood 1988, Andrew M et al. AM J Ped Hem Onc1990, Andrew M et al. Blood 1992


Age-related differences •  Fibrinolytic factors ---

•  Plasminogen (pro-fibrinolytic), and α2-antiplasmin (anti-fibrinolytic) - similar to adults throughout childhood (after 1 year of age)

•  Tissue plasminogen activator (TPA)- lower in children

•  Plasminogen activator inhibitor- elevated in children

Andrew M et al. Blood 1987, Andrew M et al. Blood 1988, Andrew M et al. AM J Ped Hem Onc1990, Andrew M et al. Blood 1992


Reagent differences •  Royal Children’s Hospital, Melbourne, Australia

•  Plasma samples from healthy – 400 children (1mo–16 yrs)

•  Neonates – of the 827 mothers approached, only 159 consented

•  Ethnic mix multiple racial backgrounds

•  Age groups – neonates - day 1 and day 3, children - 1 month to <1 year, 1-5 years, 6-10 years, 11-16 years and then adults.

•  At least 20 samples were tested for each analyte in each age range

Monagle P et al. Thromb Haemost 2006


Reagent differences Assay Monagle et al. Andrew et al.

APTT STA Compact analyser STA-PTT-A reagent – 4 reagents: PTT-A and CK-Prest (Diagnostica Stago), Actin FSL (Dade Behring) and Platelin L

ACL analyser Actin FS, Dade

TCT STA Compact analyser 1.5 – unit TCT

ACL analyser 2-unit TCT

PT / INR STA Compact analyser Neoplastine CI reagent

ACL analyser Thromborel S , Behring

Fibrinogen STA Compact analyser STA-Fibrinogen reagent

ACL analyser Reagent not specified

Factor Assays

STA Compact analyser One stage factor assays performed using STA deficient plasma samples

ACL one stage factor assays, using in house and commercial factor deficient plasma samples

Antithrombin Chromogenic Stachrom ATIII

Chromogenic Reagent not specified

Protein C Chromogenic-Stachrom Protein C Clotting-Staclot Protein C

Chromogenic Behring, Canada ELISA, Diagnostica Stago

Protein S Clotting (functional)-Staclot Protein S ELISA (total and free) Sheep antibodies Affinity Biologicals



Differences in assays Test Day 1 Day 3* 1 mo-1 yr 1-5 yr 6-10 yr 11-16 yr

APTT (4 reagents)

↑ ↑ ↑ ↑ ↑ ↑

↑ ↑ ↔ ↔ ↔ ↔

PT ↑ ↑ ↔ ↑ ↑ ↑

↔ ↔ ↔ ↔ ↔ ↔

INR ↑ ↑ ↔ ↑ ↑ ↑ ↓ ↓ ↓ ↔ ↔ ↔

Fibrinogen ↔ ↔ ↓ ↓ ↔ ↔

↔ ↔ ↔ ↔ ↔ ↔

Monagle et al. Andrew et al. Monagle P et al. Thromb Haemost 2006



APTT (4 reagents)

↑ ↑ ↑ ↑ ↑ ↑

↑ ↑ ↔ ↔ ↔ ↔

PT ↑ ↑ ↔ ↑ ↑ ↑

↔ ↔ ↔ ↔ ↔ ↔

INR ↑ ↑ ↔ ↑ ↑ ↑ ↓ ↓ ↓ ↔ ↔ ↔


↔ ↔ ↔ ↔ ↔ ↔



Differences in procoagulants •  Similarities to Andrew et al. --

•  Vitamin K dependent factors (factors II, VII, IX, X) ~50% adult values in the newborn, and remained 10–20% reduced throughout childhood.

•  For most assays - absolute values proportionally increased in all age-groups •  factor VIII - decrease in newborns, nadir between 1 month- 1 year of age, and

gradually increased to adult levels







•  Differences compared to Andrew et al. – •  Factor XII - lower values; however trending up







•  Differences compared to Andrew et al. – •  Factor XII - lower values; however trending up

•  New data – •  D-dimers – markedly elevated in newborns, remains elevated in childhood •  TFPI and Endogenous Thrombin Potential – significantly lower in children



Differences in coagulation inhibitors Test Day 1 Day 3* 1 mo -1 yr 1-5 yr 6-10 yr 11-16 yr

AT ↓ ↓ ↑ ↑ ↑ ↑

↓ ↓ ↔ ↔ ↔ ↔

PC chromo ↓ ↓ ↓ ↓ ↓ ↓

↓ ↓ ↓ ↓ ↓ ↓

PC clot ↓ ↓ ↓ ↓ ↓ ↓

na na na na na na

PS clot ↓ ↓ ↑ ↑ ↑ ↑

↓ ↓ ↔ ↔ ↔ ↔




AT ↓ ↓ ↑ ↑ ↑ ↑

↓ ↓ ↔ ↔ ↔ ↔

PC chromo ↓ ↓ ↓ ↓ ↓ ↓

↓ ↓ ↓ ↓ ↓ ↓

PC clot ↓ ↓ ↓ ↓ ↓ ↓

na na na na na na

PS clot ↓ ↓ ↑ ↑ ↑ ↑

↓ ↓ ↔ ↔ ↔ ↔



Conclusions •  Reagent and analyzer cause differences

•  Confirm trends of developmental hemostasis and physiologic principle

•  Age-related reference ranges need to be determined for each reagent/analyzer combination

•  More studies are needed in <1 year-old children

•  ? Differences due to racial composition#

Monagle P et al. Thromb Haemost 2006, #Attard et al. J Thromb Haemost 2013


Author; Year Assays Study age groups (n) Perlman; 1975

PT, TT, APTT, fibrinogen, FDP, platelet count, haematocrit, FV, FVIII, plasminogen, hemoglobin

Healthy infants (35), Small-for-dates infants(26), Post-mature infants (30)

Beverley, D.W. et al.; 1984

APTT, FII-VII-X, fibrinogen, a2-antiplasmin, platelet count, MPV, megathrombocyte index, plasminogen

Cord blood Newborns (48 h) (n=80)

Boos, J. et al.; 1989 PIVKA II, FVII, FII, FII:Ag Day 1, 2, 3 neonates (57)

Reverdiau-Moalic P. et al.; 1996

PT/INR, APTT TCT, FI, FII, FVII, FVII, FIX, FX, FV, FVIII, FXI, FXII, PK, HMWK, AT, HCII, TFPI, PC (Ag, Act), PS (free and total), C4b-BP

Fetuses wks gestation 19–23 (20), 24–29(22), 30–38 (22), Newborns (60), Adults (40)

Ries, M. et al.; 1997 TAT, F1+2, PAP, D-dimer 1–6 years (20), 7–12 years (20), 13–18 years (20), Adults (20)

Cargo, M.D. et al.; 2002 PFA100, Hb, platelet count Neonates (17), Children (57), Adults (31)

Salonvaara, M.et al.; 2003 FII, FV, FVII, FX, APTT, PT/INR, platelet count Premature infants 24–27 wks (21), 28–20 wks (25), 31–33 wks (34), 34–36 wks (45)

Flanders, M. et al. 2004 PT, APTT, FVIII, FIX, FXI, antithrombin, RCF, vWF, PC, PS 7–9 years, 10–11 years, 12–13 years, 14–15 years, 16–17 years, Adults (124 per age grp)

Chan, K.L. et al.; 2007 Thromboelastography <1 years (24), 1–5 years (24), 6–10 years (26), 11–16 years(26), Adults (25)

Sosothikul, D. et al.;2007 PT, APTT, fibrinogen, TAT, PC:Ac, TF, FVIIa, sTM, vWF (Ag & RCo), D-dimer, tPA, PAI-1, TAFI

1–5 years (19), 6–10 years (26), 11–18 years (25), Adults (26)

Mitsiakos, G. et al.; 2008 INR, PT, APTT, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, antithrombin, PC, PS, APCr, tPA, PAI-1, VWF

Small for growth newborns (90), Appropriate for growth Newborns (98)

Newall, F. et al.; 2008 PF4 and Vitronectin <1 years, 1–5 years, 6–10 years, 11–16 years, Adults (15 per age grp)

Sosothikul, D. et al.;2012

ADAMTS 13, TFPI, homocysteine, VWF:CB, PC activity and antigen, total PS, free PS and AT III

<1 year (18), 1-5 years (30), 6-10 years (38), 11-16 years (41), adults (30)

Appel IM et al.; 2012 PT, APTT, TT, fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, XIII, AT, PC, PS, VWF:Ag, VWF:RCo, D-dimer, Plasminogen, α2-Antiplasmin

1–6 months (29), 7–12 months (25), 1–5 years (57), 6–10 years (57), 11–18 years (50), > 19 years (52)

Attard C et al.; 2013 Factor II, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, plasminogen, PC and total and free PS

neonates (day 1 and day 3) (10), 28 days to 1 year (10), 1–5 years (20), 6–10 years (19), 11–16 years (20), and adults (20)


Physiologic relevance of assays •  AT supplemented anti-Xa assays may overcome physiologic differences

•  Methods: •  4 anti-Xa kits – Stachrom; Diagnostica Stago; Actichrome Heparin American

Diagnostica; IL Test Heparin IL; Coatest Heparin Chromogenix •  1 anti-IIa - IL Test Heparin (IIa) •  Purified AT added to immune depleted AT plasma –

final concentrations: 0, 0.5, 1.0, 1.5, 2.0, 2.5 u/ml. •  Heparin added to each of the AT concentrations -

final concentrations: 0, 0.2, 0.4 u/ml.



Physiologic relevance of assays •  Results:

•  Heparin added: Heparin concentrations were dependent on plasma concentrations of AT (p < 0.001)

•  No heparin added: Anticoagulant activity detected at plasma AT 1.0 u/ml Significant correlation between plasma AT concentrations and the amount of anticoagulant activity (p < 0.001)



Correlation between assays

•  APTT – •  Age dependent and overly sensitive to heparin •  unreliable measure for heparin anticoagulation in children

Ignjatovic V et al. J Thromb Haemost 2006, Newall F et al. Thromb Res 2008, Ignjatovic V T et al. Thromb Res 2008

Author Year Samples Type of AT assay Agreement between APTT and anti-Xa

Andrew et al. 1994 In-vivo Added AT r2=0.51

Chan A et al. 2007 In-vivo No added AT

< 2 years age(r2 = 0.08) > 2 years age (r2 = 0.27)

Kuhle S et al. 2007 In-vivo Added AT No added AT

r2=0.22 r2=0.21

Schechter T et al. 2012 In-vivo No added AT τ = 0.47, P < 0.0001

Hanslik A et al. 2015 In-vivo No added AT k = 0.04


Challenges •  Ethical difficulties in obtaining blood from healthy children

•  Controlling pre-analytical variables – small volumes, high hematocrit

•  Cost involved in laboratory assays

•  Time-consuming labor intensive process

•  New analyzers and reagents


Strategies •  ISTH SSC: Perinatal and Paediatric Haemostasis Subcommittee 2012

recommendations for diagnostic laboratories •  Generate age, analyzer and reagent appropriate reference ranges

•  Use tests/reagents as physiologically relevant as possible (i.e. no additional AT in anti-Xa assay)

•  Use standard age groups (i.e. neonates, 1 month to 1 year, 1-5 years, 6-10 years, 11-16 years)

•  If unable to generate reference ranges, laboratories should only use published ranges using the identical analyzer and reagent system

•  If published unavailable, then laboratory should consider referral of samples

Ignjatovic V et al. J Thromb Haemost 2012


Summary •  Children are not mini adults

•  Hemostatic system evolves with developmental stage

•  Children have physiologically lower levels of both procoagulation and anticoagulant proteins in a balance

•  “Abnormal” lab value may be physiologic

•  Assay reagent and analyzer variation should be considered during interpretation of lab results

•  All diagnostic laboratories should generate own pediatric reference ranges


Questions & Discussion

challenges in pediatric coagulation testing · • concentrations of the majority of coagulation...

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