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Indian Journal of Community Medicine

Collection of Blood on Filter Paper: Stability & Validation study for HIV Serology

Author(s): M.R. Thakar, M.V. Ghate, R.S.

Vol. 25, No. 4 (2000-10 - 2000-12)

Paranjape National AIDS Research Institute, Bhosari, Pune


Research question: Can blood collection on filter paper be used for serological diagnosis of HIV infection?

Objectives: 1) To compare HIV-antibody detection by EIA in serum and serum proteins eluted from dried blood spot on filter paper. 2) To check the stability of dried blood spots at ambient temperature.

Study design: Cross-sectional.

Results: The finger prick blood collection on filter paper was compared with the conventional venous blood collection for the detection of HIV antibodies in peripheral blood. 100% sensitivity and 100% and 98.8% specificity was observed in 2 phases containing 103 and 102 paired samples from STD and TB clinic attendees. The antibodies in dried blood spots were found to be stable at 37 C upto 7 weeks as no drop was observed in the optical density values of these eluted antibodies even after 7 weeks.

The dried blood spot could be tested for HIV-1 and HIV-2 antibodies by rapid tests and also by a line immunoassay.

Filter paper blood spot collection will be a useful tool in seroepidemiological surveys for HIV infection in remote areas and also in infants and small children from whom it is difficult to get blood sample by venipuncture.

Keywords: HIV serology, dried blood spots, filter paper elutes.


Over 3 million samples have been screened all over the country upto Aug. 1998, showing 2.36% seropositivity. The heterosexual transmission has been found to be the commonest route of HIV transmission accounting for 45.4% of total HIV infections1. High prevalence of HIV infection has been reported among persons with high risk behaviour such as commercial sex workers, STD patients, intravenous drug abusers etc and there has been significant increase in the prevalence of HIV infection2. The seroepidemiological surveys in HIV infection are thus necessary to know the extent of HIV epidemic in India, especially in general population. The data regarding HIV infection in urban areas is available through sentinel surveillance and through other studies. But no data is available on the extent of HIV infection in rural areas in India. Such studies will be helpful in forming a baseline data for intervention programmes and to plan for provision of medical facilities.

The serological diagnosis of HIV infection is made by detection of anti-HIV antibodies in the serum or plasma. Testing of serum requires collection of blood using a syringe and a needle, transportation of specimens to the testing laboratory and storage at 4° C. The disposal of used syringes and needles also poses problems. Hence implementation of seroepidemiological surveys becomes difficult and cumbersome. In addition, in case of infants and small children, it is difficult to draw blood by venipuncture.

Collection of blood on a filter paper from finger tip has been used as an alternative method of blood collection. The antibodies eluted from dried blood spot on filter paper have been used in serological assays for diagnosis of infectious diseases such as hepatitis B, measles, malaria and Kala Azar3-6. It has been also utilized for diagnosis of pathological conditions such as sickle cell disease, lactic acidemia, juvenile diabetes and atopic disorder7-10. The genetic material eluted from dried blood spot has been used successfully for amplification of HIV DNA11.

In this study, we report the feasibility of using dried blood spot on filter paper for serological diagnosis of HIV.

Material and Methods:

Specimen collection:

The filter paper (Whatman No. 1) was cut into 1cm x 5cm size strips and one end of each strip was used for sample identification. A 1cm x 1cm square was marked for sample collection at the other end. The marked area on the filter paper was allowed to soak blood from finger tip after puncturing it with lancet.

The feasibility of utilizing dried blood spot on the filter paper for the serological diagnosis of HIV infection was tested in two phases.

Phase 1: Validation of use of dried blood spots for serological diagnosis:

1. Determination of the minimum quantity of blood required for the assay: 2, 5, 10, 15, 20 and 25*ls of a HIV-1 seroreactive blood were applied on filter paper and allowed to dry. The serum proteins from dried blood spots were eluted in 0.5 ml of sterile Phosphate Buffer Saline (PBS) (0.01 M, with 0.1% sodium azide) by agitating overnight at room temperature and tested for anti-HIV antibody by commercial EIA kit (UBI HIV-1/HIV-2 EIA, United Biomedical Company, USA) at 1/5, 1/10 and 1/20 dilutions.

2. Stability of the dried blood spots at different temperatures: Filter paper dried blood spots of a HIV-1 seroreactive blood sample were stored at 4° C and 37° C for different time periods upto 7 weeks. One dried blood spot stored at both the temperatures were used each week (from week 1 to 7) to elute serum proteins. The elutes were stored at -20° C, and tested in a single assay by an EIA kit (Detect HIV, Biochem Immuno systems, Canada) at a later date.

3. Testing on blood samples collected from STD and TB clinic attendees: Paired venous blood samples and finger prick blood samples on filter paper were collected from one hundred and three STD and TB clinic patients. Ten microliters (*l) of blood was collected by a finger prick using a micropipette and applied on the filter paper strip. After drying, the blood was eluted in PBS. The sera from venous blood were separated and stored at -20° C. The eluted samples and sera from these patients were tested for the presence of anti-HIV antibodies using same EIA kit. (Innotests HIV-1/HIV-2 Ab. sp, Innogenetics, Belgium). The kappa's agreement test was applied to compare the results of conventional serum EIA and EIA done on elutes of dried filter paper spots.

Phase 2: Feasibility of collection of sample without use of micropipette:

In the second phase, finger prick blood samples were collected directly on a filter paper strip so as to saturate a designated area (1cm x 1cm) with the blood. This approximately corresponds to 10*l blood. This avoided use of micropipette and tips. One hundred and two samples from STD and TB clinic attendees collected in a period of 2 months were tested in this phase. The elutes and the serum samples were tested by the same EIA (Detect HIV, Biochem Immuno systems, Canada). The kappa's agreement test was applied to compare the results of conventional serum EIA and EIA done on elutes of dried filter paper spots.

Use of dried blood spot for detecting HIV antibodies in rapid tests and Western Blot:

Seventeen EIA reactive samples and 8 EIA non-reactive samples from dried blood spots were tested by Immunocomb II HIV 1 and 2 bispot test kit (Orgenics, Israel). Another rapid test, a latex agglutination test (Capillus HIV-1/HIV-2, Cambridge Diagnostics, Ireland) was also performed on 10 EIA reactive and 5 EIA non-reactive elutes. A confirmatory test: line immunoassay (Innolia HIV-1/HIV-2 Ab. Innogenetics, Belgium) was performed on 10 EIA reactive and 5 non-reactive dried blood samples.

Results and Discussion:

The antibodies eluted from the blood dried on filter paper could be detected in various serological assays. If we consider that all antibodies from 10*l of dried blood are eluted in 500*l of buffer, the elute corresponds to 1/100 dilution of serum (considering serum from 50% of blood volume). This elute when used in EIA at three dilutions; 1/5, 1/10, 1/20 (effective dilutions 1/500, 1/1000 and 1/2000), 1/5 dilution (i.e. effective dilution 1/500) gave results comparable with the respective serum EIA (standard dilution: 1/20). Thus it was decided to use 1/5 dilution of the elute. But in first phase of standardization, one sample that was reactive in serum EIA was found non-reactive at 1/5 dilution of eluted sample; however, it was found reactive at 1/2 dilution. Thus, it was decided that all the filter paper elutes should be tested at 1/2 (effective dilution; 1/200) dilution.

Table I: Comparison of HIV-EIA results of serum and dried blood spots.

Phase 1: Use of micropipette for addition of blood on filter paper Phase 2: Addition of blood on filter paper without using micropipette
Serum results (1:20) + - Total Serum results (1:20) + - Total
Dried blood spot results* Dried blood spot results*
Positivity 48 - 48 Positive 18 - 18
Negative - 55 55 Negative 1 83 84
Total 48 55 103 Total 19 83 102
Sensitivity 100% Sensitivity 98.8%
Specificity 100% Specificity 100%
Kappa value 1 Kappa value 0.96

*Effective dilution is 1:200.

The study was done in 2 phases - in the first phase, the blood was collected using micropipette. In the second phase the use of micropipette was avoided to further simplify the specimen collection. 100% specificity was obtained during both the phases when the HIV-EIA results of dried blood spot elutes were compared with the results of the serum samples (Table I). The sensitivity as observed in HIV-EIA was found to be 100% and 98.8% in both the phases respectively. The comparison between the results of conventional serum EIA and EIA on elutes of dried filter paper spots showed excellent agreement for both the phases by the kappa's agreement test (kappa's values 1 for phase I and 0.96 for Phase II). But when the results of the rapid test done on the serum sample were considered, the sensitivity was also found to be 100% as the EIA reactive serum sample with non-reactive results on filter paper elute, was found to be negative in Immunocomb bispot assay. The antibodies eluted from dried blood spots on filter paper could be detected in rapid tests also. The Immunocomb bispot assay gave the identical results and intensity of the spots when both the elutes and the corresponding serum samples were tested. The amount of the elute used in the assay was the same as serum. But the Capillus rapid latex agglutination assay failed to detect antibodies in case of one out of the 10 EIA reactive filter paper elutes. This may be due to different sensitivities of the tests. It is known that the EIA and rapid test kits available commercially differ considerably in their sensitivity and specificity12. Thus, the choice of rapid kits to test filter paper elutes should be done cautiously.

The western blot or lien immunoassay (LIA) is a confirmatory test which needs to be performed on samples giving ambiguous results. Our results show that antibodies eluted from dried blood spot can to be tested by LIA. The Innolia LIA done on filter paper elutes revealed the same band patterns as obtained in the Innolia LIA of the corresponding serum samples.

The results of 2 phases of the validation experiments showed enough evidence that the dried blood spots can be used for detecting HIV antibodies. It was important to see the feasibility of this technique in the seroepidemiological surveys at remote places. Thus stability of HIV antibodies in the dried blood spot was tested. It was found that the reactivity of the antibodies from dried blood spots was not affected during the storage of dried blood spots at 37° C for 7 weeks; as no drop in optical density (O.D.) values was observed in EIA testing of these elutes.

These preliminary experiments demonstrate and confirm the usefulness and feasibility of the filter paper blood collection method for testing of HIV antibodies. Farzadegametal et al tested smaller no. of samples for validation of the methodology13. They did not apply the rapid tests for antibody detection in the eluted samples. We report here the additional advantage of using dried blood spot by rapid tests for testing HIV antibodies. The storage of the dried blood spots at 37° C upto 7 weeks without affecting test reactivity showed suitability of this method in field conditions where ambient temperature is often high. This will enable and facilitate collection and transportation of samples for surveys in remote areas without requirement of cold chain. We have used ordinary filter paper and simple elution buffer such as PBS which would be cost-effective and available in any laboratory. Avoiding the use of vacutainers, needles (cost Rs. 10/-) will reduce the cost of the test further where lancet costing Rs. 2/- is sufficient to collect blood on filter paper. This will also minimize the problems of disposal of vacutainers and needles. For blood collection by conventional venupuncture, maintenance of cold chain is essential for transportation and storage in the laboratory. Use of centrifuge is essential for serum separation. Taking these factors into consideration, the cost of dried blood spot on filter paper is 40% less than the conventional blood collection method.

The other major advantage of this technique is the suitability of the finger prick blood sample for testing HIV antibodies in the populations in whom there is resistance to blood collection by venipuncture.

The methodology has a promise of its effective use in seroepidemiological surveys on large number of samples in remote areas and among the population from whom it may be difficult to obtain blood by venipuncture such as infants and small children.


Authors sincerely thank Dr. Anand Divekar and Dr. S.P. Tripathy and staff members of STD and TB out patient Deptt. for their prompt help in sample collection. Authors acknowledge Ms. Shubhangi Kamat and Ms. Aarti Goverdhan for their technical assistance.


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