Hemostatic derangement in Dengue infection...p < 0.001 : Dengue fever (DF) vs Dengue hemorrhagic fever (DHF) These are basic hematologic tests in our dengue patients and the controls.

Hemostatic derangement in Dengue infection

By Assoc. Prof. Darintr Sosothikul, MD

Pediatric Hematology-Oncology division, King Chulalongkorn Memorial Hospital,

Faculty of Medicine, Chulalongkorn University

Outline

• Overview of Severe Dengue • Hematologic and Hemostatic Changes in Severe

Dengue: Thrombocytopenia and platelet dysfunction Vasculopathy and endothelial dysfunction Changes in vWF parameters, ADAMTS13 and multimer Activation of coagulation and fibrinolysis • Management of significant bleeding in severe degue

1997 WHO dengue classification

Classical Dengue Fever (DF)

Fever,headache,retro-orbital pain,myalgia,arthralgias,+/- Haemorrhagic manifestrations

DHF Grade I Thrombocytopenia Haemoconcentration

DHF Grade II Spontaneous bleeding

DHF Grade III Pulse pressure 20 mmHg, Hypotension, cold clammy skin, restless

DHF Grade IV Profound shock, Undetectable BP and pulse

DSS

Fever of 2–7 days plus: ▫ Severe plasma leakage ▫ Significant bleeding ▫ Impaired consciousness ▫ Severe organ impairment

(AST or ALT > 1,000) ▫ Severe involvement of the

heart or others organs

Severe Dengue

Dengue: guidelines for diagnosis, treatment WHO 2009

Pathophysiology in severe dengue

Viremia

Risk factor for severe dengue;

Young age Female

High body mass index Virus strain

Genetic varients Secondary infection by

different serotypes Severe dengue

Transient and reversible imbalance of inflammatory mediators,cytokines and chemokines

Endothelial cell dysfunction

Derangement of coagulation system

Plasma leakage Shock Bleeding

Hematologic and Hemostatic Changes in Severe Dengue

Correlation between WBC and PMN in DHF

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10

Days of illness

WB

C c

ount

0

10

20

30

40

50

60

70

801 2 3 4 5 6 7 8 9 10

Phases of the diseases

PM

N c

ount

WBC countPMN count

Febrile Toxic Convalescent

Correlation between WBC count and ATL count in DHF

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 10

Days of illness

WB

C c

ou

nt

0

5

10

15

20

25

301 2 3 4 5 6 7 8 9

Phases of the diseases

Aty

pic

al l

ymp

ho

cyte

co

un

t

WBC countAtypical lymphocyte count

Febrile Toxic Convalescent

Correlation between WBC and PMN Correlation between WBC and ATL

Peripheral blood changes in DHF

PresenterPresentation NotesLeucopenia is thought to represent a direct effect of dengue virus on the bone marrow.Bone marrow biopsies of children with DHF revealed suppression of hematopoiesis early in the illness, with marrow recovery and hypercellularity in the late stage.In vitro studies have shown that dengue virus infects human bone marrow stromal cells and hematopoietic progenitor cells and inhibits progenitor cell growth

Correlation between Hct and platelet in DHF

37

38

39

40

41

42

43

44

1 2 3 4 5 6 7 8 9 10

Day of the illness

Hct (

%)

0

20000

40000

60000

80000

100000

120000

140000

1600001 2 3 4 5 6 7 8 9 10

Phases of the disease

Plat

elet

cou

nt

HctPlatelet

Febrile Toxic Toxic Convalescent

Correlation between Hematocrit &platelets in DHF

Thrombocytopenia I. Peripheral destruction or increased utilization • Dengue 2 virus can bind to human platelet, and

result in immune-mediated platelet destruction • Consumption of platelet during the process of

consumptive coagulopathy II. Decreased production ; dengue-virus-induced

BM suppression depressed platelet synthesis

Mitrakul C, etal Am J Trop Hyg 1977;26:975 La Russa VF. Baillieres Clin Haematol 1995; 8(1): 249-70 Krishnamurti C. Am J Trop Med Hyg 2002; 66(4): 435-41

Thrombocytopenia

• Decrease Platelet aggregation after stimulation with 5 µm M ADP in DHF patients during febrile or early convalescent period

• 9 in 10 children (90%) with DF and all of children with DHF have decrease platelet aggregation with ADP (DF 60%, DHF 100%), ristocetin (DF 40%, DHF 100%), collagen (DF 70%, DHF 100%) and arachidonic acid (DF 90%, DHF 66.7%)

• Plasma levels of platelet factor 4 and beta thromboglobulin were increased during the acute phase of DHF

Mitrakul C. Am J Trop Med Hyg 1977; 26: 975-84 Srichaikul T. Southeast Asian J Trop Med Public Health 1989; 20 (1): 19-25 Tanyong B, Sosothikul D. Abnormal Platelet Aggregation of Dengue fever in Thai Children 2011

Platelet dysfunction

Mechanism of platelet activation

Microparticles

Granule secretion

- Adhesive molecules - Chemokines/cytokines - Growth factors - Coagulation factors

In vitro study of dengue infection showed that platelet –derived IL-1B was chiefly released in microparticles and

correlated with sign of increased vascular permeability

Hottz ED,et al. Blood 2013; 122: 3405-14

Platelet derived microparticles in Dengue infection

P = 0.007

Sosothikul D, et al Poster presentation at European Hematology Association 2014 , Milan ,Italy

(Dengue : N=20)

(N=40)

Interleukin-1 beta in Dengue infection

P < 0.001


Platelet Activation in Dengue Infection

• The levels of PDMP, and IL-1β were significantly increased in patients with dengue infection compared to the unaffected controls

• Platelet activation can be one of the

mechanism that leads to platelet dysfunction and increased vascular permeability in patients with dengue infection


Vasculopathy • Plasma leakage, due to an increase in

capillary permeability, is a cardinal feature of DHF but is absent in dengue fever.

• Appear to be due to endothelial cell dysfunction rather than injury, as electron microscopy demonstrated a widening of the endothelial tight junctions.

• Dengue virus-infected monocytic cells produce TNF-alpha and activate endothelial cells in vitro.

Anderson R. et al. J Virol 1997;71(6): 4226-32 Bunyaratvej A, et al. southeast Asian J Trop Med Public Health 1997;28 Suppl 3:32-7

Activation of endothelial cells in dengue infection

controlConvalescentToxicFebrile

Stages of the illness

8.00

6.00

4.00

2.00

sTM

(ng/

ml)

27

ControlDHFDF

GROUP DIAG(DF,DHF)P=0.04

controlConvalescentToxicFebrile

Stages of the illness

400

300

200

100vW

F: A

g (%

)

ControlDHFDF

GROUP DIAG(DF,DHF)

P=0.01

Soluble thrombomodulin Von Willebrand Factor antigen

Sosothikul D, et al. Thromb Haemost 2007; 97: 627-634

Changes in ADAMTS 13 and VWF parameters in dengue infection

Febrile Toxic Convalescent control

Phase of the illness

0

50

100

150A

DA

MT

S 1

3 (%

)

DFDHFcontrolP=0.039

P=0.002

P=0.003 ADAMTS 13


Normal

Abnormal Multimers

Abnormal Multimers

Normal pool plasma

Normal

Abnormal Multimers

Larger than normal

Normal pool plasma

VWF Multimers in DHF

Febrile

Toxic

Convales.

Febrile

Toxic

Convales.

Sosothikul D., et al. Thromb Haemost 2007;97: 627-634

High MW Low MW

TM

Dengue/Cytokines

Activated endothelial cells

t-PA vWF Platelets seqestration

TF/FVIIa FXa+FV

FIXa+FVIII

Prothrombin Thrombin

FXIa

AT

TAT

Fibrinogen Fibrin D-Dimer

Plasmin Plasminogen + t-PA

- PAI-1

TAFIa

TM

- Fibrinolysis

Coagulopathy in DHF


TM: thrombomodulin vWF: von Wellibrand factor t-PA: tissue plasminogen PAI: plasminogen activator inhibitor

TF: tissue factor TAT: thrombin anti-thrombin complex AT: anti-thrombin III TAFI: thrombin activatable fibrinolysis inhibitor

PresenterPresentation NotesFrom our previous study in 2007, we have shown that the endothelial cells are activated by Dengue virus and/or cytokines. They can lose protective properties by expressing TF, TM and vWF. The release of plasma VWF might be the mechanism for platelet sequestration causing thrombocytopenia. Furthermore, TF/FVIIa complex activate the clotting system, leading to thrombin generation. The fibrinolysis is initially activated by t-PA. TAFI might be a secondary inhibitor of fibrinolysis in dengue patients.

Thromboelastometry

CT = clotting time, CFT = clot formation time , MCF = maximum clot firmness

Coagulation tests in dengue patients during febrile phase

Tests Controls Dengue fever Dengue hemorrhagic fever (n = 30) (n = 22) (n = 19) Hematocrit (%) mean + SD 41.1 + 3.3 37 + 4.4** 44.1 + 3.4 Range (34 - 46.4) (28.2 - 45.8) (37.3 - 50.9) Platelets (x109) mean + SD 289.4 + 832 99.6 + 48.3* 40.8 + 31.4 Range (152 - 485) (0 - 196.2) (0 - 103.6)

Prothrombin time (sec) mean + SD 12.5 + 1.3 14 + 1.8* 16.3 + 4.6 Range (10.6 - 14.7) (10.4 - 17.6) (7.1 - 25.5) APTT (sec) mean + SD 35.9 + 3.1 42 + 6.5* 48.4 + 9.6 Range (29.7 - 40.3) (29 - 55) (29.2 - 67.6) Fibrinogen (mg/dL) mean + SD 429.4 + 110.3 307 + 70.9 306.3 + 94 Range (220 - 678) (165.2 - 448.8) (118.3 - 494.3)

*p

Results from thromboelastometry

EXTEM

INTEM


Sosothikul D, et al. Oral Poster presentation at ISTH meeting 2013

PresenterPresentation NotesRotem parameters in our dengue patients showed that DHF patients had a significant longer time in CT ,CFT in EXTEM and INTEM than DF patients. In addition,, DHF patients had significant lesser clot firmness (Lower MCF) and lower amplitude (A10,a20) than DF patients.

FIBTEM


Results from thromboelastometry

Sosothikul D, et al. Oral Poster presentation at ISTH meeting 2013

Thromboelastometry in Dengue

• ROTEM® showed significant changes in hemostasis in both groups of dengue patients, and it was correlated well with standard coagulation studies and severity of the disease • ROTEM® can early detect abnormal fibrinogen function in DHF patients • It may become a useful bedside tool for an early detection and a quick guide to choose appropriate blood products in treatment of DHF patients with bleeding

Sosothikul D, et al.Oral Poster presentation at ISTH meeting 2013

Management of significant bleeding in DHF

Risk factors for Hemorrhage in DHF

• A study of risk factors for hemorrhage in 114 patients with DHF/DSS showed no correlation between bleeding and platelet count

• The strongest risk factor for hemorrhage were prolonged duration of shock and a low level of hematocrit at the time of shock

Lum LC, et al. Journal of Pediatrics 2002: 140; 625-31

Management of significant bleeding

• Significant internal bleeding should be suspected in patients with signs of intravascular hypovolemic without elevation of hematocrit

PRC 5 ml/kg and clinical response/post transfusion hematocrit should be monitored • Do not wait for the hematocrit to drop too low

before deciding on blood transfusion

Thomas L, et al Transfusion 2009; 49:1400 Dengue: guidelines for diagnosis,treatment WHO, Geneva 2009 Comprehensive guidelines for prevention/control DHF WHO Regional Office for Southeast Asia 2011

FFP and/or platelet –no different in bleeding Increase pulmonary edema, longer hospitalization

Platelet • Benefit in DIC with

platelet < 50,000 or undergoing procedure

• Platelet 0.1 unit/kg/dose • Not indicate for prevent

spontaneous bleeding

FFP • PT or aPTT ratio >1.5 +

DIC or severe bleeding • FFP 10-15 ml/kg

Update on pediatric infectious diseases 2016

Transfusion in dengue with hemorrhage

NG tube/ foley catheter: • Great care should be taken when inserting a NG tube

or bladder catheters • A lubricated orogastric tube may minimize the trauma

during insertion

Central line • should be done with USG guidance or by an

experienced person

WHO; clinical management of dengue 20121ex

Transfusion in dengue with hemorrhage

• rFVIIa may have role in case of massive bleeding unresponsive to conventional blood component therapy.

• rFVIIa enhances thrombin generation and also enhances the activity and function of both patients and transfused platelets.

Chuansumrit A, et al Blood Coagul Fibrinolysis 2005; 16(8):545-55

Role of rFVIIa in control of dengue bleeding

Dengue and prophylactic transfusions

• 106 DSS children with thrombocytopenia and coagulopathy, there was no difference in hemorrhage between patients who received preventive transfusions compared with those who did not

• Patients who received transfusion had higher frequency of development of pulmonary edema and increased length of hospitalization

• Preventive transfusions did not produce sustained improvements in the coagulation status in DSS

Lum LC, et al. Journal of Pediatrics 2003: 143; 682-4

Conclusions • Bleeding in DHF patient is caused by vasculopathy,

thrombocytopenia, platelet dysfunction, abnormal VWF multimers and coagulopathy (DIC)

• ROTEM® may become a useful bedside tool for an early detection and a quick guide to choose appropriate blood products in treatment of DHF patients with bleeding

• Prophylactic platelet transfusions have not been shown to be effective at preventing or controlling hemorrhage

Conclusions

• Platelet transfusion should be reserved for dengue patients with major bleeding

• Early recognition of severe dengue, with prompt

correction of hemodynamic status, remains the mainstay for good clinical outcome

THANK YOU FOR YOUR ATTENTION

Hemostatic derangement in Dengue infection�Outline 1997 WHO dengue classificationSlide Number 4 Pathophysiology in severe dengueSlide Number 7Slide Number 8Slide Number 9ThrombocytopeniaSlide Number 11Slide Number 12Platelet derived microparticles�in Dengue infectionInterleukin-1 beta in �Dengue infection�Platelet Activation in �Dengue Infection VasculopathyActivation of endothelial cells in �dengue infection Changes in ADAMTS 13 and VWF parameters in dengue infectionSlide Number 20 Coagulopathy in DHFSlide Number 22 Coagulation tests in dengue patients �during febrile phaseSlide Number 26Slide Number 27Thromboelastometry in Dengue �Management of significant bleeding in DHF Risk factors for Hemorrhage in DHFManagement of significant bleedingSlide Number 32Slide Number 33Slide Number 34Dengue and prophylactic transfusionsConclusionsConclusionsSlide Number 38

Hemostatic derangement in Dengue infection...p < 0.001 : Dengue fever (DF) vs Dengue hemorrhagic fever (DHF) These are basic hematologic tests in our dengue patients and the controls.

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