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Clinica Chimica Acta 3

Letter to the Editor

IgM antibodies to dengue virus in dried blood on filter

paperB

Keywords: Dried blood; Dengue; UMELISA; IgM

Dear Editor,

Dengue fever (DF) is an acute infectious disease caused

by the dengue virus, which has 4 serotypes [1]. In terms of

morbidity, mortality, and economic costs, dengue is the most

important mosquito-borne disease in the world, with an

estimate of 50 to 100million cases/y [2]. The detection of

virus specific immunoglobulin M (IgM) antibody in serum

samples has been used successfully for the diagnosis of

dengue infection [3,4]. However, the requirement to take

blood samples is impractical, expensive, and limits the scale

of laboratory investigation, mainly in children [5].

The use of blood spots dried on filter paper for the

collection, shipment and storage of a large number of

specimens for serological investigation of infectious dis-

eases, offers many practical advantages over conventional

methods of serum collection and preservation [6,7]. The

dried samples are light, easy to store or mail, cannot be

broken or spilled, and take up little space. The major benefit

of this sample collection method is the facility to obtain

blood specimens from finger or heel punctures on infants,

children, or others from whom it is difficult to get venous

blood [8].

The ultramicroELISA (UMELISA) Dengue IgM tradi-

tionally uses serum samples. The purpose of the present

study was to evaluate whether dried blood specimens on

filter paper can be used to detect IgM dengue virus

antibodies using this kit, and whether this sample collection

method yields an acceptable specificity and sensitivity in

comparison with the standard method employing serum

samples.

Serum samples and whole blood spots on filter paper

were obtained from 189 patients clinically suspect to have

dengue. Venous blood was drawn and collected. Sera from

clotted blood were divided into aliquots and kept at �20 8C

0009-8981/$ - see front matter D 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.cca.2005.12.042

B The authors thank Lic Senia Bermudez for helping them concerning

revision of the manuscript.

until they were assayed. Blood was collected on filter paper

Schleicher and Schuell-2992 by lancet-induced finger

puncture. Filter-paper samples were air dried overnight at

room temperature, then, individually wrapped in plastic

wrap and stored in a refrigerator, in sealed plastic bags

containing desiccant, until testing.

UMELISA Dengue IgM assay was used as previously

described [9]. The specimens were applied into wells of

polystyrene ultramicrotiter strips coated with affinity-puri-

fied sheep anti-human IgM antibody. Three 1-h incubations,

each at 378C, were done, one to the samples and control

sera, another to the antigen (mixture of 4 serotypes of

dengue virus each 1 to 16 hemagglutinating units HU), and

the last one to the conjugate. After each incubation, 6

washing cycles were performed with 15mmol/l Tris–HCl,

0.05% Tween-20 buffer, pH 7.8 (washing buffer). Finally,

the substrate (4 methylumbelliferyl phosphate) was added

and incubated for 30min at room temperature. The eluate

and serum dilution from each patient were tested in

duplicate on the same plate. The results were expressed in

fluorescence units (FU) and calculated as the relation

M�B /P�B, where M, B (solution containing 5% sheep

serum in washing buffer) and P were, respectively, the

sample, blank and positive control serum fluorescence units.

The samples with a relation value z0.3 were considered

positive.

In order to optimize the assay using dried blood on filter

paper, preliminary experiments were carried out: different

diameter circles and different elution conditions were tested

to elute whole blood samples from the filter papers. For

testing purposes, 3 and 5mm circles were punched out of

the papers and eluted, respectively, with 0.04 and 0.07ml of

assay buffer. A fully impregnated disk of 7mm in diameter

contains ~5AL of serum [10]; therefore, the above combi-

nations were equivalent to a 1 :21 dilution. When we

compared the eluates obtained from 3 and 5mm circles

using 14 negative and 23 positive samples, results of

UMELISA Dengue IgM from both types of discs were

comparable (Table 1). This test demonstrated that it is

possible to use discs of 3 or 5mm in diameter with similar

effectiveness. With the aim of investigating the effect of

elution time on the UMELISA results, discs of 5mm of 16

filter-papers were left in agitation at room temperature for

30 and 60min, into 0.07ml of assay buffer. These samples

were selected to include a wide range of reactivities from

negative (3) to positive (13). The 2 elution conditions

67 (2006) 204–206

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2.5

Eluate from filter paper

Ser

um

ob

tain

ed b

y ve

nip

un

ctu

re

2

Fig. 1. Comparison of UMELISA test results obtained from serum dilutions

and filter-paper eluates from 189 patients with dengue suspects. The

Pearson correlation coefficient was 0.954.

Letter to the Editor 205

yielded similar results, and by Student t test, they were

statistically compared.

Overall, there was agreement in the outcomes obtained

with the 2 sample collection methods. There were 47

specimens positive for both and 136 negative for both

specimens. There were discrepancies found in the assignation

of diagnoses in individual cases. Dengue infection was not

detected in 4 out of 51 (7.84%) dried blood samples, whereas

only 2 out of 138 (1.45%) patients with no dengue in serum

were found to have IgM antibodies in whole blood spots. In

order to guarantee good quality in the results is necessary to be

careful when collecting blood samples on the filter paper [11].

In the case of the 4 false negative specimens on filter paper,

blood samples were not well taken, the blood spots were not

uniform and they did not penetrate adequately the paper.

The 2 false positive results could have been caused due

to the breaking of the red blood cells during the elution

process and thus provoking the presence of hemoglobin in

the eluates. Many manufacturers recommend that operators

avoid hemolyzed serum samples [6,12].

Table 1

Results from 37 dried blood spots punched out of the filter paper in 3 and

5mm diameter circles

Samples Diameter 3mm Diameter 5mm

1 0.02 0.03

2 0.08 0.09

3 0.08 0.11

4 0.09 0.11

5 0.10 0.12

6 0.10 0.13

7 0.11 0.14

8 0.12 0.15

9 0.18 0.19

10 0.20 0.21

11 0.22 0.21

12 0.24 0.21

13 0.25 0.24

14 0.27 0.26

15 0.38 0.41

16 0.42 0.41

17 0.46 0.48

18 0.47 0.51

19 0.56 0.55

20 0.58 0.61

21 0.61 0.61

22 0.67 0.71

23 0.69 0.71

24 0.73 0.79

25 0.73 0.80

26 0.77 0.80

27 0.84 0.81

28 0.88 0.90

29 0.90 0.92

30 0.90 0.94

31 0.99 0.95

32 1.04 1.00

33 1.25 1.20

34 1.45 1.39

35 1.63 1.63

36 1.78 1.75

37 1.95 1.95

The sensitivity and specificity of the new procedure were

92.1% and 98.6%, respectively. The relation between

M�B /P�B from eluates and serum dilutions was esti-

mated with Spearman correlation coefficient. The correla-

tion coefficient was 0.954 as shown in Fig. 1, which

suggests that filter-paper results reflect those in serum from

venous blood [8] over a wide range of concentrations of

IgM antibody to dengue virus. The comparison of the results

obtained by using both collection methods has demonstrated

close agreement with a kappa concordance index of 0.918

[13]. These outcomes demonstrate that the UMELISA

Dengue IgM also provides reliable results for early detection

of dengue infection when dried blood spots collected on

filter-paper are used.

The effect of delay in testing specimens on IgM

antibodies was evaluated. 38 out of 189 samples (28

positive and 10 negative) were stored at 48C and at room

temperature (26 to 358C), and then tested. A slight

decrease of FU was detected when whole blood specimens

were tested after 30 days of being kept at room

temperature, and after 2 months of storage at 48C, but

this did not cause any positive samples to became negative.

No significant differences were revealed when we com-

pared the results obtained from whole blood specimens

stored as above mentioned with the sera taken from the

same patients and stored at �208C. The data of this study

support the suitability of collecting blood specimens on

filter paper for dengue diagnosis using the UMELISA

Dengue IgM kit.

References

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Letter to the Editor206

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Regla de la C. Herrera

Infectious Diseases Laboratory,

Center for Immunoassay, Cuba

E-mail address: iqtorch3@cie.sld.cu.

Corresponding author. Centro de Inmunoensayo,

Calle 134 y Ave 25, Playa. Apto 6945,

Ciudad de la Habana, Cuba.

Marıa V. Cabrera

SUMA Laboratory from Santiago the Cuba City,

Center for Immunoassay, Cuba

Susana Garcıa

SUMA Laboratory from Havana City,

Center for Immunoassay, Cuba

Mayra Gilart

Provincial Hygiene and Epidemiology

Center from Santiago the Cuba City, Cuba

26 September 2005