Journal of Virological Methods 61 (1996) 73-77

Journal of Virological Methods

Comparison of capillary blood versus venous blood samples in the assessment of immunity to measles

F. Novello”,“, B. Ridolf?, L. Fiore”, G. Buttinelli”, E. Meddab, A. Faveroc, D. Marchetti”, F. Gaglioppad

=Laboratory of Virology, Istituto Superiore di Sanitri, Viale Regina Elena, 299, 00161 Rome, Italy bLaboratory of Epidemiology and Biostatistics, Istituto Superiore di Sanitri, Rome, Italy

“Clinical Pathology Department, Bellaria Hospital, Bologna, Italy ‘Public Department of Pediatrics and Maternity, Bologna Sud, Bologna, Italy

Accepted 29 April 1996

Abstract

Seroepidemiological investigations are essential for assessing the efficacy of measles vaccination programmes. However, when large-scale sampling is needed, a major difficulty is the problem of taking venous blood, especially in children. An alternative method is the collection of capillary blood samples spotted on filter papers. The eluted extract from these ‘blood’ spots can be used instead of serum samples for measles laboratory diagnosis or investigations. Measles antibody dete’ction is readily carried out by ELISA on serum samples. The same technique can be used on eluates from capillary blood spots. Measles antibody titres determined on matched serum and blood spot samples from 27 children were compared. A strong correlation was found between the results obtained with the two methods of blood sampling.

Keywords: Measles; Antibody; Capillary blood spot; ELISA

1. Introduction

* Corresponding author: Tel.: + 39 6 49902664; fax: + 39 6 4453904.

Vaccination against measles has been recom- mended in Italy since 1979, and its widespread use began in 1988. Two live vaccines, the Schwartz and Edmonston-Zagreb strains, are currently available on the Italian market, alone or in com-

0166-0934/96/$15.00 0 1996 El sevier Science B.V. All rights reserved

74 F. Novel/o et al. /Journal of’ Virological Methods 61 (1996) 73-77

bination with mumps and rubella antigens. Sev- eral local mass vaccination campaigns have been carried out since 1980. These have demonstrated that a dramatic reduction in the spread of infec- tion can be achieved (Grandolfo et al., 1986). In the first phase of these campaigns, all children up to lo- 12 years of age without a history of measles were offered the vaccination. In the following years, all new-borns were vaccinated by the age of two.

Seroepidemiological investigations, together with active surveillance of measles cases, are es- sential to evaluate the efficacy of mass vaccination strategies, in particular to determine whether fu- sion between natural and induced immunity has occurred, and whether immunity is long-lasting.

Laboratory research has been carried out in order to improve the methods for detecting the humoral response in subjects with natural or vac- cine immunization. ELISA is the most widely used method because of its high degree of reliabil- ity (Voller and Bidwell, 1976).

In the organization of large seroepidemiological studies, difficulties are often encountered due to the resistance of both subjects and their parents to taking venous blood. This practice is also re- source-consuming in terms of personnel, treat- ment and transportation of samples. A valuable alternative is the collection of capillary blood by finger pricking. The samples are absorbed onto filter papers, punched out as disks, eluted and eventually processed by ELISA.

Several studies have been described on the reli- ability of antibody determination in eluates from absorbed capillary blood in comparison with serum samples (Brody et al., 1964; Karstad et al., 1957; Mathews, 1981; Nakano et al., 1983). A procedure for the application of ELISA to the determination of measles antibodies from capil- lary blood spot eluates is described. Statistical analysis of the results obtained on 27 matched serum samples and capillary blood eluates re- vealed a good correlation.

2. Materials and methods

A sample of serum and six drops of capillary

blood, obtained by finger-pricking and absorbed onto special filter paper cards (Serono, code 60011), were taken simultaneously from 27 chil- dren, with their parents’ consent. The subjects were aged from 2 to 12 years and had been vaccinated against measles with mono- or triva- lent vaccines at various ages. The filter paper cards, commonly used in blood sampling for the laboratory diagnosis of congenital hypothy- roidism, bear six imprinted circles within which blood drops are absorbed, and a marked space for sample identification. Care was taken to en- sure that each blood drop was absorbed com- pletely and that both sides of the filter paper were saturated. The cards containing the blood were air dried, placed inside separate plastic bags and stored at - 20°C until used. Serum samples were also stored at - 20°C.

One day before the assay, the filter papers were thawed at room temperature for at least 30 min and 5 mm o disks were extracted from each of the six imprinted circles with a paper punch (McGill Co., Illinois). Individual test samples for each subject were prepared by eluting two disks in 0.1 ml of 0.3 M Tris buffer at 4°C overnight. This procedure was developed on the basis of a previ- ous study by Nakano et al. (1983) and a prelimi- nary work carried out by us on different elution volumes of blood spots and serum samples from five subjects (unpublished data). The resulting elu- ates corresponded to a serum dilution of approxi- mately l/20.

Measles antibody levels were determined by an ELISA kit ‘Enzygnost@ Anti-Masern-Virus-IgG’ by Behring, which makes it possible to express the antibody activity in terms of milli-International Units/ml (mIU/ml) on the basis of an internal reference serum (Anti-Measles Virus Reference Reagent P/N), which is processed together with the samples. The reference serum is calibrated on the First International Standard Preparation for Anti-Measles Serum of the WHO. For each serum sample, a single dilution point of l/231 is suffi- cient for calculating the final titre.

The ELISA was run in parallel on serum sam- ples and capillary blood eluates of the 27 subjects. All samples were processed according to the man- ufacturer’s procedure with a Behring ELISA Au-

F. Novello et al. /Journal of Virological Methods 61 (1996) 73-77 15

tomated Processor (or BEP II). Briefly, 0.02 ml of l/21 serum dilution or 0.02 ml of blood spot eluate were placed in each well of measles anti- gen sensitised microplates with 0.2 ml of 0.3 M Tris (Behring ‘Sample Buffer POD’). The result- ing dilution was thus l/231 for the serum sam- ples and approximately l/220 for the blood spot eluates. The reaction was allowed to proceed for 1 h at 37°C. After a thorough washing of the wells, peroxidase-conjugated anti-human IgG an- tibody was added and the plates were kept for 1 h at 37°C. The wells were washed again and TMB (3,3’,5,5’ Tetralmethylbenzidine) was added as the enzyme substrate. After 30 min at room temperature and protected from light, the reac- tion was stopped with 0.5 N H,SO,. The colori- metric reaction was measured at 450 nm. The ‘cut-off of this method is 0.2 Optical Density Units, that corresponded to 130 mIU/ml of anti- body activity in this assay.

The log titres of positive results were used for regression analysis to evaluate the relationship between the ELISA results obtained with the two sampling methods. The parameters of the inter- polating regression Iline were calculated together with their 95% ConAdence Interval (CI). The ra- tio of geometrical mean titre (GMT) values and its 95% CI were also calculated, as well as the GMT values for positive results. The probabili- ties of obtaining the observed estimates under the null hypothesis S = 1, c1= 0 and the ratio of the GMTs equal to 1 were also calculated.

3. Results

The ELISA results for the blood spot eluates and serum samples of the 27 subjects, expressed in mIU/ml, are shown in Table 1. The matched samples from T.J. and D.S. showed contrasting results, which is not unusual for values near the cut-off, and were excluded from the regression analysis.

Fig. 1 shows the scatter diagram of the posi- tive log,, ELISA titres obtained with the two methods. All values were close to the interpolat- ing line. The slope ‘b’ of the line was 0.99 (CI 0.88- 1. lo), P = 0.93 (Student’s t-test under the

hypothesis of B = 1); intercept ‘a’ was 0.07 (CI - 0.25-0.40), P= 0.67 (Student’s r-test under the hypothesis of a = 0). Table 2 reports the esti- mates of both the parameters of the interpolating regression line and the ratio of the GMT values with its 95% CI. The ratio of GMTs was 0.87 (CI 0.48-1.60), P= 0.65 (Student’s r-test for paired data). Considering only the positive re- sults, the GMTs were 3.06 (CI 2.10-4.03) for the serum samples and 3.00 (CI 2.06-3.95) for the blood spot samples.

Table 1 Measles antibody activity in 27 blood spot eluates and sera, by ELISA

Patient Antibody activity (mIU/ml)

Blood spot eluate Serum

P.C. A.A. P.L. CC. B.E. C.L. B.M. M.M. T.J. P.M. B.L. G.S. C.A. T.D. T.Y. L.L. Z.S. D.F. L.G. D.S. A.F. F.C. G.F. V.G. D.C.G. D.D. C.S.

2600 2800

600 1500 860 210

2100 7300

160 200

1400

*eg 4100

830 190 910

1300 210

5500

neg 1500 1200 1200

neg neg

170 930

2300 4600

580 1500 1200 280

2000 9700

neg 270

1500

neg 4400 410 240

1100 1400 360

7700 310

1400 1500 1600

neg neg

140 1400

Cut-off: 130 mIU/ml neg = below the cut-off

76 F. Nooello et al. /Journal of Virological Methods 61 (1996) 73-77

or..,.,..,,r,,“,‘,“,“~‘I 0 1 2 3 4 5

Log,, Blood Spot Titres

Fig. 1. Scatter diagram and regression line of serum and blood

spot determinations.

4. Discussion

Resistance to venipuncture by either subjects or their parents is a problem in seroepidemiological studies to assess the immunity of the population, e.g. after mass vaccination campaigns. Moreover, venous blood collection and treatment is cumber- some and requires specialised personnel and labo- ratories. The collection of capillary blood obtained by finger or heel pricking is better ac- cepted and sample handling is easier.

Table 2

Parameters of the regression line and comparison of the two

determinations

Significance test and CI for b and a

b = 0.99 a = 0.07

SE”(b) = 0.05 SE(a) = 0.17

CIb(95%)=0.88-1.10 CI(95%) = -0.250.40

P = 0.93 P = 0.67

Ratio of the GMT” values (spot/serum)

Ratio = 0.87

CI(95%) = 0.48- 1.60

t-test for paired data t = -0.46 P = 0.65

“Standard error.

bConfidence interval.

“Geometrical mean titre.

The use of capillary blood absorbed onto filter papers has been used extensively for neonatal screening studies to detect the presence of proteins, hormones, etc., in the blood (Harada et al., 1994; Carta Sorcini et al., 1982). The applica- tion of this method to antibody investigations was described by Nakano et al. (1983), who compared the detection of measles antibodies on both capil- lary blood spot eluates and serum samples by haemoagglutination inhibition. In the present study, measles antibody levels were determined by the ELISA test, which is the most widely used technique to detect serum antibodies. In a prelim- inary study on five subjects we could establish that two 5 mm o disks eluted in 0.1 ml of buffer corresponded to a serum dilution of about l/20.

Twenty-seven sets of specimens (filter paper blood and serum) were analysed by ELISA, and the results were compared and evaluated by statis- tical analysis. As shown in Tables 1 and 2 and Fig. 1, the correlation found between the data from peripheral blood samples and matched sera is high. Even though the number of subjects inves- tigated was small, the accuracy of the sampling guaranteed the reliability of the results. This type of blood collection, coupled with ELISA process- ing, appears to be very useful for large-scale seroinvestigations because it greatly simplifies sample collection, storage and transport. It could be particularly helpful in areas where the need for seroepidemiological investigation is not matched by the availability of laboratory equipment and trained staff, and when venipuncture is not well accepted. The WHO in its Expanded Programme on Immunization has recommended this method of collection and processing for the detection of anti-poliovirus antibodies, when necessary, in de- veloping countries (WHO, 1990).

The proper collection and storage of the capil- lary blood samples absorbed on the filter papers is essential to carry out correct determinations. Blood from the finger puncture must completely soak through both sides of the paper and until each imprinted circle is completely filled. This ensures that the quantity of blood eluted from the disks is consistent from sample to sample. Once dried, filter papers must be kept at - 20°C until used. The extracts from blood spots also must be

F. Novello et al. / Journal of Virological Methods 61 (1996) 73-77 71

Acknowledgements

We thank M.E. G:randolfo for critical reading of the manuscript; Sabrina Tocchio and Deborah Wool for editorial assistance.

References

Brody, J.A., McAlister, R.! Haseley, R. and Lee, P. (1964) Use

stored at - 2O”C, if they are to be used for subse- quent determinations.

We are investigating presently approximately 4000 blood spot samples collected from vacci- nated subjects living in areas where vaccination campaigns have been carried out. The analysis of the results will shed light on the use of this method of blood sam:pling to monitor the efficacy of measles vaccinations.

of dried whole blood collected on filter paper disks in adenovirus complement fixation and measles haemaggluti- nation-inhibition tests. J. Immunol. 92, 854-857.

Carta Sorcini, M., Moschini, L., Fiore, L., Tomarchio, S., Di Iorio, M.G., Gilardi, E., Romagnoli, C., Cur& V. and Carta, S. (1982) Familial thyroxine-binding globulin defi- ciency detected in a pilot screening program for congenital hypothyroidism. J. Endocrinol. Invest. 5, 21-25.

Grandolfo, M.E., Santoro, R., Polo, M., Scardellato, U., Alberti, A.M. and Pasquini, P. (1986) A pilot measles vaccination campaign in Italy. Public Health 100, 208-213.

Harada, F., Ireland, J.H., Hsia, Y.E. and Chui, D.H. (1994) Anti-zeta antibody screening for alpha-thalassemia using dried filter paper blood. Biochem. Med. Metab. Biol. 51, 80-84.

Karstad, L., Spalatin, J. and Hanson, R.P. (1957) Application of the paper disk technique to collection of whole blood and serum samples in studies on eastern equine en- cephalomyelitis. J. Infect. Dis. 101, 259-299.

Mathews, H.M. (1981) Parasitic disease: testing with filter-pa- per blood spots. Lab. Manage. 9, 55-62.

Nakano, J.H., Miller, D.L., Foster, S.O. and Brink, E.W. (1983) Microtiter determination of measles haemagglutina- tion-inhibition antibody with filter papers. J. Clin. Micro- biol. 17, 860-863.

Voller, A. and Bidwell, D.E. (1976) Enzyme-immunoassays for antibodies in measles, cytomegalovirus infections and after rubella vaccination. Br. J. Exp. Path. 57, 243-247.

WHO Manual for the Virological Investigations of Poliomyeli- tis (1990). Expanded Program Immunization and Division of Communicable Diseases, WHO.