Revista new lab artigo 6 inglês

Article

Evaluation of Method for Detection of Anti-HIV Antibodies on Filter Paper

Tatiane Funari Chrusciak¹, Amanda Regina Nichi de Sá¹, Letícia Mika Fusano¹, Alvaro Largura², Alisson Marassi³

1 - Students in the Clinical and Toxicological Analysis Specialization program at the Faculdade Assis Gurgacz

2 - Biochemical and Pharmacological Director of the Alvaro Laboratory – Cascavel/PR

3 - Biochemical Pharmacist at the Alvaro Laboratory in Cascavel/PR, professor and advisor for this final course project

Resumo

Avaliação de método para detecção de anticorpos Anti-HIV em papel filtro

O presente trabalho foi realizado a fim de confirmar a reprodutibilidade do teste para HIV utilizando-se de amostras coletadas de recém-nascidos em papel filtro. Para o teste, utilizou-se de sangue total sem anticoagulantes, sendo duas das amostras conhecidamente reagentes (A1 e A4) e duas conhecidamente não reagentes (A2 e A3) para o HIV. A avaliação foi realizada no setor Neonatal do Laboratório Alvaro – Cascavel/PR, através do método de Elisa. No teste intraensaio, A1 e A2 foram dosadas 20 vezes consecutivas em uma única rotina. No teste interensaio, A3 e A4 foram dosadas cinco vezes consecutivas, em quatro rotinas diferentes e também consecutivas. Os resultados foram avaliados através de sistemas não numéricos (reagentes e não reagentes). Na análise intraensaio, ambas as amostras tiveram resultados coerentes, sendo A1 reagente e A2 não reagente, tendo reprodutibilidade satisfatória com o coeficiente de variação das amostras e do controle igual a zero. No teste interensaio, as amostras A 3 (não reagente) e A4 (reagente) apresentaram todas as leituras corretas, tendo também reprodutibilidade confirmada. Os resultados demonstram que as amostras se mantiveram estáveis, garantindo a detecção de anticorpos anti-HIV em amostras coletadas em papel filtro.

Palavras-chave: HIV, papel filtro, estabilidade, Elisa

Abstract

Evaluation of method for detection of anti-HIV antibodies on filter paper

The present work was performed in order to confirm the reproducibility of an HIV test using samples collected from newborns on filter paper. Samples of total blood were used for the test without anticoagulants, two of them being reactive (A1 and A4) and two nonreactive samples (A2 and A3) for HIV. The evaluation was performed in the Neonatal section of the Laboratory Alvaro - Cascavel/PR, using the ELISA method. In the intra-assay test, A1 and A2 were measured 20 times in a single routine. In the inter-assay test, A3 and A4 were measured 5 times, in 4 different and also consecutive routines. The results were evaluated through non numerical systems (reactive and nonreactive). In the intra-assay analysis, both samples had consistent results, being reactive A1 and nonreactive A2, having satisfactory reproducibility with the variation coefficient of the samples and control equal to zero. In the inter-assay test, the samples A3 (nonreactive) and A4 (reactive) presented all the correct readings and also confirmed reproducibility. The results showed that the samples remained stable, guaranteeing the detection of anti-HIV antibodies in samples collected on filter paper.

Keywords: HIV, filter paper, stability, Elisa

Introduction

Qualification of an analytic method, its implementation and adaptation involves an evaluation process that assesses its efficiency in laboratory routine (1). Validation is essential to determine whether the methods developed are completely adequate for the objectives they are designed for, in order to obtain reliable results that may be satisfactorily interpreted.

In this way it is possible to determine the limitations and reliability of the measurements made in the analyses (2).

For the results to be reliable it is necessary for the analytical variations to be kept within the limits pre-established by criteria based on the purpose of the analysis, analytic systems and statistical studies (1, 3).Validation of a method assures the specificity, accuracy and precision of an analytic test and estimates the stability of the analyte during storage and handling of the sample (4).

Statistics

The criteria to be adopted for validation of a particular method depend on its nature and purpose. Acquired immunodeficiency syndrome (AIDS) takes, on average, eight years to appear (5). According to Unaids/WHO (16), the number of people estimated to be infected by the human immunodeficiency virus worldwide is 33.2 million, with one-third of them living in Brazil where 470 thousand cases of the disease had already been identified between 1980, when the first case of AIDS was recorded, and June 2007.

Until the mid-1990s, the rate of occurrence was growing, reaching close to 20 cases per 100 thousand people in 1998. Approximately 80% of total cases were concentrated in the southeastern and southern regions. However, in spite of having high occurrence rates and being the regions most affected since the beginning of the epidemic, the southeast and south are the regions that have continued a process of stabilization, although slow, since 1998 (5).

Detection

The ideal for an HIV test would be to combine a sensitivity of 100% with a specificity of 100%. Given the impossibility of reaching this, screening favors sensitivity over specificity due to the consequences of a false-negative test for the patient (6).

The techniques usually employed for diagnosis of HIV infection are based on detection of anti-HIV antibodies and are used for initial screening.

The conventional strategy for diagnosis of HIV-1 infection in Brazil includes the combination of two different enzyme immunoassays (EIA) and a corroborative test, the Western Blot or immunofluorescence, also known as the “gold standard”. This diagnostic system is highly specific and sensitive for detection of anti-HIV-1 antibodies, requiring highly trained laboratory technicians and advanced laboratory infrastructure.

The Elisa (Enzyme-Linked Immunosorbent Assay) method is the screening test for HIV most utilized since its automation is simple, its cost is low and its sensitivity and specificity are high (7).

The operational characteristics of a methodology are expressed in terms of analytic parameters that should be determined during its validation. These parameters are designed to verify the linearity, precision, accuracy, specificity, detection and qualification limits and the robustness of the analytic method proposed (8).

Depending on the purpose of the method, some of the parameters may not need to be evaluated. It should be emphasized that the method may be considered valid even if some of the parameters do not fall within the limits established in the literature, but they should be clearly understood and, consequently, adequate for the objectives of the study being conducted (2, 9). According to the Ministry of Health and the National Sanitary Surveillance Agency (6), the characteristics of a test that may be considered for a screening program are its precision, accuracy and reproducibility, which is a test's ability to always reproduce the same results when the technical and biological conditions are the same when the test is repeated. The fixed and inherent operational characteristics of a diagnostic test which are represented by its sensitivity and specificity are also considered, where these refer to the agreement rate in comparison to a standard (percentage of reactive and nonreactive samples in relation to the reactive and nonreactive samples according to the standard test or condition).

The predictive values refer to the proportion of correct tests out of the total reactive and not reactive tests (percentage of correct reactive and not reactive tests in relation to the total number of tests).

The objective of this work was to evaluate the stability of a method for detecting anti-HIV antibodies in blood samples on filter paper for newborn patients using the Elisa method, widely used as an initial test for detection of antibodies against HIV in blood.

1

4

Methodology

The evaluation took place in the Neonatal section of the Alvaro Laboratory - Cascavel/PR, using whole blood samples without anticoagulant collected on filter paper (dried blood sample) pertaining to the routine of the section. Two reactive samples (A1 and A4) and two nonreactive samples (A2 and A3) for HIV were selected.

The quality of the collection procedure and the integrity of the sample were checked, or rather, samples showing any indication of contamination or dilution (alcohol, moisturizers, oils or creams that remain on the skin of the newborn during collection, or even accidents during transport of the sample to the laboratory), forced drying, dirty filter paper, as well as filter paper on which the blood is not found on both sides, were rejected.

The methodology used for performing the tests was manual, following the Elisa method (enzyme immunoassay), with reagents from Q-Preven HIV 1+2 – DBS commercial kits, from Symbiosis Diagnóstica Ltda., specific for detection of antibodies against HIV 1+2 in samples collected on filter paper with capacity for 96 tests. All the tests were performed using kits from the same lot (1022000100) with 08/05/08 as the date of manufacture and 08/29/09 as the expiration date.

In the intra-assay procedure both the A1 (reactive) and A2 (nonreactive) samples were measured 20 times consecutively in a single routine. The objective of this procedure was to verify the reproducibility of the results of the tests in a single routine, conducted on the same day, using the same controls. These measurements were performed on 04/28/09.

In the inter-assay procedure, the A3 (nonreactive) and A4 (reactive) samples were measured five times consecutively, in four different routines and also consecutively. The objective of this procedure was to verify the reproducibility of the results of tests in different routines, conducted on different days and, consequently, using different controls. These measurements were performed in routines on 04/28, 04/30, 05/05 and 05/07 of 2009.

Since this is a qualitative method, the results were evaluated with reproducibility of nonnumeric systems (reactive and nonreactive). The results were tabulated and analyzed.

Table 1. Results of the intra-assay for reactive and nonreactive HIV samples

No Date Sample 1 (A1) Sample 2 (A2)

1 28/4/2009 Reactive Nonreactive




















1

2

Results

The intra-assay measurements made with the A1 e A2 samples on 04/28/09 are represented on Table 1. Of the 20 tests performed during the same routine, both samples had coherent results, with the A1 sample being reactive and the A2 sample being nonreactive in all the tests, as expected. The analysis of the samples and controls was considered satisfactory with regard to reproducibility since the variation coefficient was equal to zero.

To evaluate the reproducibility of the inter-assay test, the A3 (nonreactive) and A4 (reactive) samples showed all correct readings in the five measurements in four different and consecutive routines, as shown in Table 2.

Table 2. Results of the inter-assay analyses for reactive and nonreactive HIV samples

No Date Sample 3 (A3) Sample 4 (A4)





















Discussion

Confidence has been growing for measurement of antibodies against HIV using filter paper. The analyte has shown stability, in addition to the ease that filter paper collection offers, with no need for refrigeration of the material, and the ease of transport of the sample to the support laboratory, which can even be sent by mail without causing any alteration of the results.

Similar results were obtained in research conducted by the Universidade de Caxias do Sul (UCS), where a note from the Ministry of Health (10) showed that transport of conventional blood samples, in test tubes, may alter the characteristics of the blood if there is not adequate refrigeration and that there are even chances of accidents occurring with the material. The collection of the material is also considered more acceptable by the parents of the newborn, in the majority of cases, when compared to venous collection (10).

In addition, the adoption of an appropriate strategy can improve the availability and acceptance of HIV counseling and testing, allowing a greater number of individuals to be reached and motivating them to adopt risk reduction behaviors (6) as shown in Graph 1. It must be remembered that, just as for the other tests conducted by neonatal services, this measurement is considered only a screening procedure and in no way should be used as a corroborative test, given that, in the case of a positive sample, the patient should be referred for gold standard tests, only then confirming the diagnosis.

It is important to emphasize that the diagnostic protocol currently used by the Ministry of Health is valid for

patients older than two years of age since antibodies detected for children below this age may still be those of the mother (vertical transmission) and not necessarily produced by the child itself, which would give a false positive result.

Thus, confirmation of infection in these cases is provided by conducting a RNA viral plasma quantification test, following a Ministry of Health flowchart for diagnosis of HIV infection in children, in accordance with Decree No. 488/98/SVS/MS (11), or also through fourth generation tests.

[Graph 1]

HIV measurements in 2008 x 2009

Jan Feb Mar April May June

Months

Graph 1. Comparison of the measurement of samples of anti-HIV antibodies received by the Neonatal section of the Alvaro Laboratory during the first semester of 2008 and the same period of 2009

According to Rouet (17), definitive diagnosis of HIV infection is only possible using an advanced technique like PCR to confirm the presence of the virus in the blood. Our studies of Elisa treatment using filter paper support the results obtained by LPHA (HIV/AIDS Research Laboratory – Universidade Caxias do Sul) researchers which also confirmed the results by comparing the initial data, obtained using the Elisa method, comparing and confirming that the results are the same as those obtained using Western Blot, one of the corroborating tests for HIV (10).

As shown previously, the HIV measurement appeared stable with 100% reproducibility in the same routine (intra-assay), as well as in different routines (inter-assay). Even if for validation of the test the reproducibility could present, at best, only a discordant reading (with 95% agreement), the results showed that all readings were accurate, or rather, there were no disagreements between one and other reading.

According to the Ministry of Health (10), for the research conducted at UCS testing the use of filter paper in HIV diagnosis, validation confirmed that this has the same collection efficiency as venous puncture and pointed out other safety factors. The evaluation of filter paper sensitivity for HIV testing showed 100% successful application, and that for specificity had 99.5% favorable results.

In all, the studies showed that post-collection, the filter paper was stable for up to 35 days without special storage conditions, or rather, it adapts well to room temperature and can be stored for up to 53 weeks at temperatures of 4ºC, - 20º e -70ºC. (12)

The filter paper has positive points when compared to conventional collection (in vials), such as the ease of collection and storage, need for little structure or human resources, and the ability to provide quick answers at low cost (13, 14).

De Souza (18), in a study similar to ours where the Elisa method is used with the same Q-Preven HIV 1+2 – DBS kit, confirms that this methodology performs well requiring a small amount of blood, in addition to facilitating transport and storage through collection on filter paper, and concludes that this test is ideal for samples that are sent from locations distant from the testing location, especially when resources are limited.

New technologies have been regularly developed and made available by clinical laboratories and it is fundamentally important to understand the performance of each new methodology, like the use of dried samples collected on filter paper for HIV diagnosis. Factors like the collection, sample storage and choice of diagnostic method are instrumental, directly affecting the reliability of the laboratory results.

The fourth generation immunoassay was developed from the combination of the third generation test, which detects anti-HIV-1 antibodies, including subtype O, anti-HIV-2 antibodies, and, simultaneously, also detects the HIV-1 p24 antigen. With this method, the immunological window is diminished and the number of false positive results is reduced, like the detection of maternal antibodies in the newborn’s sample and in populations with low prevalence, such as blood donors (15).

The samples were shown to be stable during the study. Reproducibility was 100% in both evaluation methods. In other words, the results had no variation coefficient between the consecutive measurements and also between the measurements from different routines, meaning that the samples maintained their stability with total reproducibility during this period, guaranteeing reliable results.

One of the difficulties found in conducting studies such as this for neonatal screening is in the traceability of the patient, since when the filter paper sample arrives at the support laboratory, it generally uses the mother’s name

preceded by “RN of”. When the corroborative sample (serum) is sent, the name used for identification is already that of the newborn, making it, therefore, impossible to compare the results (filter paper x serum).

It can be concluded, therefore, that this work highlights the need for a comparative study of diagnostic methodologies in neonatal screening, in which there would be complete patient traceability so that the methods used could be compared. It is also relevant to conduct a study with samples collected on filter paper using fourth generation testing in order to prove that selection of the collection (filter paper) method will not interfere with results.

This would build confidence in filter paper samples, which are more economical, stable and provide advantages that samples in vials do not have, such as ease of transport, mainly for specific tests which are generally only conducted in support laboratories.

Correspondence to: Tatiane Funari Chrusciak

[email protected]

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Revista new lab artigo 6 inglês