Von Willebrand Factor and Thrombocytopenia in Patients with

Dengue Haemorrhagic Fever

L. Mutiara

, Herman Hariman

, Stephen C. L. Koh

, Adang Bachtiar

Division of Tropical Medicine, Murni Teguh Memorial Hospital, Medan Indonesia

Department of Clinical Pathology, Faculty of Medicine, Universitas Sumatera Utara/Haj Adam Malik Hospital,

Medan Indonesia

Department of Public Health Program, Cipto Mangunkusuma Hospital, Universitas Indonesia, Jakarta, Indonesia

Keywords: DHF, endothelium status

Abstract: The clinical manifestation of DHF includes mild or marked febrile syndromes with abrupt onset of

headache, pain behind the eyes, muscle and bone pain, nausea, vomiting and rash. The pathophysiology of

DHF in human is complex involving endothelial cell activation and the impaired endothelial barrier leading

to plasma leakage triggering the activation of the haemostatic system. The objective of this study was to

determine Von Willebrand Factor levels and platelet effects on the vascular endothelium in patients with

dengue haemorrhagic fever. Fifty patients (males 34, females 16), were recruited, Grade 1 (n=41), Grade 2

(n=6), Grade 3 (n=2) and Grade 4 (n=1) DHF. Twenty patients at seventeen years and below (Grade 1) were

compared with 21 adults (Grade 1) showed no statistical differences and grouped together for analysis.

Blood sampling to determine haemoglobin, haematocrit, platelets and Von Willebrand Factor (VWF) at the

febrile, defervescence and convalescent phases was performed. The patients were aged between 4 and 54

years old with Grade 2 patients being significantly older (P=0.03) than Grade 1 DHF. Comparisons between

Grades 1 and 2 DHF showed no statistical differences in the parameters studied. Thrombocytopenia,

elevated VWF levels from normal was evident in all phases suggesting endothelial activation. In Grades 3

and 4 DHF, thrombocytopenia, elevated VWF was also seen. No mortality was observed in the study.

Endothelial activation was evident in dengue haemorrhage fever.

1 INTRODUCTION

Dengue fever is the most serious consequence of

mosquito-borne infection worldwide. There are

more than 2.5 billion persons at risk of infection and

occur mainly in the sub-tropical regions of Asia,

Africa and America (WHO 2008), the attacks have

shifted mainly to adults (WHO 2011). In Indonesia,

the overall incidence increased significantly from

0.05/100,000 in 1968 to 35-40/100,000 in 2013

(Karyanti et al 2014).

The actual numbers of dengue cases are

underreported or misclassified (WHO 2017).

Clinical manifestations of dengue include mild or

marked febrile syndromes of abrupt onset with

headache, pain behind the eyes muscle and bone

pain, nausea, vomiting and rash which is life

threatening. There is no specific treatment for

dengue fever but maintaining patients’ body fluid

volume is critical. Dengue as defined by WHO

(2009) as dengue with and without warning signs of

plasma leakage and defined into four grades (Grades

1 to 4) The endothelium plays an important

regulatory role in the circulation as a physical barrier

and involved in the control of thrombosis and

thrombolysis, vascular tone and growth of blood

vessels (Verhamme & Hoylaerts 2006). Endothelial

activation may be responsible for plasma leakage

and shock (De Castro et al 2007) and endothelial

injury is associated with elevated Von Willebrand

Factor (VWF) (Connolly 1991, Mohle et al 1997).

Thrombocytopenia is commonly observed in both

mild and severe dengue syndrome and associated

with clinical outcome (WHO 2009, Mourao et al

2007, Schexneiden & Reely 2005, Honda et al

2008). The level of platelet count correlates with

severity of DHF and high haematocrit with marked

thrombocytopenia support the diagnosis of dengue

shock syndrome (DSS) (WHO 2011) and has been

considered as an important factor responsible for

bleeding events in DHF (Diaz-Quijano et al 2010).

Platelet activation is significantly increased in

106

Mutiara, L., Hariman, H., Koh, S. and Bachtiar, A.

Von Willebrand Factor and Thrombocytopenia in Patients with Dengue Haemorrhagic Fever.

DOI: 10.5220/0009861401060110

In Proceedings of the 2nd International Conference on Tropical Medicine and Infectious Disease (ICTROMI 2019), pages 106-110

ISBN: 978-989-758-469-5

dengue-patients especially with thrombocytopenia

which exhibited signs of apoptosis pathway

activation (Hottz et al 2013)

The objective of the study was to determine Von

Willlebrand Factor levels and platelet effects on the

vascular endothelium in patients with dengue

haemorrhagic fever.

2 MATERIALS AND METHODS

The study received ethical approval from the Health

Research Ethical Committee (No.438/TGL/KEPK

FK USU-RSUP-HAM/2018), Faculty of Medicine,

University of North Sumatera, Jakarta, Indonesia.

The study was conducted at the Murni Teguh

Memorial Hospital, Medan Indonesia. Subjects.

Fifty patients (males 34, females 16) admitted to the

hospital with fever were recruited and diagnosed

according to WHO protocol (2009) to have Grade 1

(n=41), Grade 2 (n=6), Grade 3 (n=2) and Grade 4

(n=1) DHF. The Inclusion criteria: patients who met

WHO criteria for dengue fever and willing to take

part in the study and had one or more dengue

serology positive for either IgM/IgG or NS1,

Exclusion criteria: patients with other infections and

systemic diseases and not willing to take part in the

study. Normal Controls. Fifteen normal subjects

(males n=14, female n=1) who are normotensive and

not taken any medication, no history of health issues

was recruited to serve as normal controls for the

DHF study.

Blood Sampling and Laboratory Investigation.

EDTA blood was used to determine Haemoglobin

(Hb), Haematocrit (Hct) and platelets in the Siemens

high volume haematological analyser (Advia

2120/1); Citrated plasma for Elisa analysis of Von

Willebrand Factor (VWF) (USCN Life Sciences,

Wuhan, China). Statistical Analysis. The Statistical

Package for Social Sciences (SPSS 22 IBM Corp)

was used to perform statistical analysis. The

independent t-test for differences between groups at

different phases of febrile, defervescence and

convalescence and Analysis of Variance (ANOVA)

was performed. A P-value of <0.05 was considered

statistically significant.

3 RESULTS

Comparison of parameters (mean ± SD) studied in

dengue haemorrhagic fever at febrile phase (Grade

1) between cohorts at age seventeen and below and

above 17 years.

There were twenty cohorts (males n=13, females

n= 7) at seventeen years and below and twenty-one

cohorts (males n=14, females n=7) above 17 years

old. Except for the significance in age (P=<0.001)

there were no statistical significance in haemoglobin

(Hb), haematocrit (Hct), platelets and VWF studied

(Table 1). They were therefore combined (Grade 1)

for further statistical analysis.

Table 1. Comparison of parameters (mean and SD) in dengue haemorrhage fever at febrile stage (Grade 1) between cohorts

at age seventeen years and below and above.

≤ 17 years

>17 years P

N (male/female) 20 (13/7) 21 (14/7)

Age years 10.9 (3.9) 29.8 (9.0)

<0.001

Range years 4 – 17 18 - 54

Haemoglobin g//L 13.1 (1.3) 14.1 (2.1) 0.06

Haematocrit % 39.0 (4.4) 42.2 (6.2) 0.07

Platelets x10

/L 111.7 (82.5) 73.1 (50.7) 0.05

VWF ng/mL 108.4 (29.0) 111.0 (30.7) 0.78

Normal values for VWF. Of the fifteen normal

controls studied, the level of VWF were non-

detectable in fourteen controls and one at 83.1

ng/mL (sensitivity of assay <0.94 ng/mL) mean 11.9

± 31.4 ng/mL. The VWF levels of DHF at various

phases are shown in Figure 1.

Von Willebrand Factor and Thrombocytopenia in Patients with Dengue Haemorrhagic Fever

107

Figure 1.VWF in Grades 1 and 2 DHF at febrile, defervescence and conva;escence phases compared to normal controls

(mean +2SD)

Dengue haemorrhagic fever: Comparison of Hb,

Hct, platelets and VWF (mean ± SD) between

Grades 1 and 2 at febrile, defervescence and

convalescence phases and comparison to febrile

phase. Thrombocytopenia (platelets <100 x10

/L)

was evident in both Grades 1 and 2 DHF at febrile,

defervescence and convalescence phases with

normal haematocrit and haemoglobin levels seen.

Elevated VWF levels are seen but they were not

significantly different between Grades 1 and 2.

There were also no significant differences between

the two groups of cohorts in the parameters studied.

Similarly, no differences when compared to febrile

phase (Table 2). Analysis of Variance (ANOVA)

One-way ANOVA analysis for Hb, Hct, platelets

and VWF. in either Grades 1 or 2 between different

DHF phases showed no significant differences (not

shown).

Table 2. Dengue haemorrhage fever: Comparison of parameters studied (mean and SD) between Grades 1 & 2 at febrile,

defervescence and convalescence phases and compared to febrile phase.

Grade 1

Grade1 – P vs

Febrile phase

Grade 2 Grade 2- P vs

Febrile phase

(Gr1 vs Gr 2)

Febrile

(male/female)

41 (28/13) 6 (4/2)

0.03

Age years 20.6 (11.8) 30.8 (8.7) 0.46

Haemoglobin

g//L

13.6 (1.8) 14.3 (2.1) 0.45

Haematocrit % 40.6 (5.6) 42.8 (6.1) 0.4

Platelets x10

/L 94.8 (70.9) 70.5 (60.4)

VWF ng/mL 109.7 (29.6) 120.0 (29.1) 0.4

Defervescence

Haemoglobin

g//L

13.6 (1.7) 0.93 14.2 (2.0) 0.91 0.49

Haematocrit % 41.2 (6.6) 0.64 41.4 (8.3) 0.75 0.96

Platelets x10

/L 78.4 (51.3) 0.23 66.5 (413) 0.90 0.54

VWF ng/mL 114.0 (24.9) 0.48 112.3 (26.1) 0.37 0.89

Convalescence

Haemoglobin

g//L

13.2 (1.7) 0.35 13.6 (1.8) 0.65 0.63

Haematocrit % 40.1 (5.3) 0.68 40.4 (5.6) 0.50 0.91

Platelets x10

/L 101.8 (58.8) 0.63 97.0 (55.3) 0.45 0.85

VWF ng/mL 113.7 (24.8) 0.51 114.3 (13.0) 0.68 0.9

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4 DISCUSSION

Dengue fever is the most serious consequence of

mosquito-borne infection worldwide. Endothelial

damage may also be caused by the virus itself.

Thrombocytopenia is responsible for bleeding events

in DHF (Diaz-Quijano et al 2010, Orsi et al 2013)

but many factors can contribute to the onset of

thrombocytopenia from a reactive immune response

against platelets and decreased platelet production

(Lin et al 2001, De-Castro et al 2007, Saito et al

2004), platelet activation and apoptosis (Hottz et al

2013). Bleeding manifestations and plasma leakage

are complications seen in dengue and bleeding

manifestation in adults may occur in the absence of

plasma leakage (Wichmann et al 2004). In our study,

thrombocytopenia was observed in all phases of

DHF even though in convalescence phase the mean

platelet numbers were higher than febrile and

defervescence phases they did not reach statistical

significance. Normal haemoglobin level and no

haemoconcentration was observed indicating no

bleeding episodes occurred despite the

thrombocytopenia. Endothelial activation as evident

by elevated VWF level suggest the possibility of

plasma leakage in Grades 1 and 2 DHF. Other

evidence like activation of the coagulation system

and thrombocytopenia with haemoconcentration will

support the cause of dengue shock syndrome (WHO

2011) and bleeding episodes (Diaz-Quijano et al

2010) which was not seen in our cohorts. Identifying

the mechanisms affecting DHF would improve

diagnosis and management therapy. No mortality

was recorded in our study.

5 CONCLUSION

Thrombocytopenia and elevated VWF are seen in

dengue haemorrhagic fever despite normal

haemoglobin and haematocrit levels suggesting

endothelial activation.in dengue haemorrhagic fever.

ACKNOWLEDGEMENTS

The authors wish to express their sincere gratitude to

the staff of the research laboratories at the Medical

Faculty, University of Sumatera Utara and Murni

Teguh Memorial Hospital for their expert technical

assistance.

CONFLICT OF INTEREST

The authors declared that they have no Conflict of

Interest.

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