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Clinical Chemistry 49: 275-280, 2003; 10.1373/49.2.275
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(Clinical Chemistry. 2003;49:275-280.)
© 2003 American Association for Clinical Chemistry, Inc.

Detection of Anti-Human T-Lymphotropic Virus Type I Antibody in Whole Blood by a Novel Counting Immunoassay

Kazunari Yamaguchi1a, Yuji Yonemura1, Hiroaki Okabe1, Yoichi Takahama2, Shinya Nagai2, Haruki Yamaguchi2 and Kojiro Hirai2

1 Blood Transfusion Service, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan.

2 Product Development Division, Sysmex Corporation, 4-4-4 Takatsukadai Nishi-ku, Kobe 651-2271, Japan.

aAuthor for correspondence. Fax 81-096-373-5813; e-mail kyama{at}gpo.kumamoto-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Assays to screen for and confirm the presence of the antibody for human T-lymphotropic virus type I (HTLV-I) are currently performed with serum or plasma. We developed and evaluated a new counting immunoassay (CIA) for the detection of HTLV-I antibody in whole blood, using recombinant and synthetic peptide antigens.

Methods: We assessed the CIA for detection of HTLV-I antibody in whole blood and plasma. The CIA is an immunity-measuring method that combines latex agglutination with particle-counting technology. The numbers of agglutinated latex particles, single latex particles, and blood cells in a sample are measured based on differences in particle size between latex particles and blood cells.

Results: The CIA and ELISA methods were in agreement for all 24 plasma samples tested, including those from 6 patients with HTLV-I-associated diseases, 6 HTLV-I carriers, and 12 HTLV-I antibody-negative individuals. The concordance between the ELISA (plasma) and the CIA (whole blood) for samples from 24 patients was 100%. The concordance between a particle agglutination method (plasma) and the CIA (plasma or whole blood) for 1065 patients was 99.5%. The concordance between results obtained for 1065 pairs of plasma and whole blood samples with the CIA method was 100%. HTLV-I antibody in whole blood was stable for 3 days after blood collection. With this CIA method, results were available within 15 min.

Conclusions: The CIA method can be used in screening for HTLV-I. The use of whole blood rather than serum or plasma reduces the sample volume and number of blood collections required, as well as assay time.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Human T-lymphotropic virus type I (HTLV-I),1 a member of the retroviridae family, infects primarily CD4-positive cells (1)(2)(3). HTLV-I provirus DNA and anti-HTLV-I antibody coexist in infected individuals, and the presence of infection is documented by demonstration of the antibody.

Gelatin particle agglutination (PA) assays (4); ELISAs (5) with disrupted whole viruses, synthetic peptides, or recombinant proteins; indirect immunofluorescence; radioimmunoprecipitation assays; and Western blot analysis are widely used for anti-HTLV-I antibody detection. These tests are carried out in numerous facilities worldwide to diagnose HTLV-I-associated diseases, such as adult T-cell leukemia (ATL), HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) (6), and HTLV-I uveitis (7), and to prevent infection by blood transfusion and mother-infant transmission (8). Although the gold standard methods for HTLV-I antibody screening are PA and ELISA, until now, HTLV-I antibody detection has been performed in serum or plasma.

We have developed a new counting immunoassay (CIA) method for the detection of antibody to HTLV-I in whole blood samples.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
patients
Samples of ATL (4 cases) plasma, HAM/TSP (2 cases) patient plasma, HTLV-I carrier (6 cases) plasma, and anti-HTLV-I antibody-negative plasma (12 cases), in which the existence of the anti-HTLV-I antibody had already been confirmed by ELISA (New Eitest ATL-EIA; Eisai), were used for the experiments. In addition, 1065 samples collected for routine testing in hospitals and from volunteer blood donors were screened by the CIA method for whole blood and the PA method (Serodia HTLV-I) for plasma. We also assessed antibody stability in 24 whole blood samples stored at 4 °C for 7 days without preservative with the CIA method.

methods
We measured the HTLV-I antibody concentrations in whole blood and plasma with the CIA method on the PAMIA-50 (Sysmex Co.) (9)(10). We used three HTLV-I antigens (p21 recombinant antigen, p19 peptide antigen, and gp46 peptide antigen). After these HTLV-I antigens were coated to the particles, particles were washed with a buffer solution containing bovine serum albumin. The antigenic activities of these recombinant proteins and peptides were evaluated by the CIA method in panels of anti-HTLV-I/II-positive sera.

principle of cia analysis
The CIA method is an immunity-measuring method that combines a latex-coagulating method with particle-counting technology. An antigen-antibody reaction occurs when latex particles, which bind to antigens, are mixed with a fluid containing the antibody. The action of the antibody leads to the formation of coagulated latex particles (Fig. 1 ). A sheath-flow mechanism then sends these latex particles through the flow cell in a single line. Laser light illuminates the particles, and a photodiode detector element detects the light that is scattered (Fig. 2 ).



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  		Figure 1. Formation of coagulated latex particles as a result of antibody action.

When the latex particles coated with HTLV-I antigens are mixed with a fluid containing the antibody, an antigen-antibody reaction occurs.



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  		Figure 2. Sheath flow mechanism for counting latex particles that react.

The CIA method combines latex coagulation with particle-counting technology.

The intensity of the scattered light becomes greater with an increase in the size of the coagulated latex particles. The degree of coagulation is determined from the values counted for each burst of scattered light. The latex particles are 0.75 µm in diameter, and the concentration used to analyze is 105 particles/test (Fig. 3 ). The degree of coagulation was calculated as follows:



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  		Figure 3. Example of particle counting based on the size of the particles.

Monomers and polymers, which are shown on the analyzer screen, and platelets (PLT), red blood cells (RBC), and other particles, which are not shown on the screen, are counted based on the principle that the intensity of the scattered light becomes greater as particle size increases.


where P (polymer) is the number of coagulated latex particles counted; M (monomer) is the number of uncoagulated latex particles counted; and T (total) = M + P (P/T of sample) - (P/T of negative control).

The degree of coagulation increases relative to the amount of antibodies. A cutoff index (COI) is calculated using the following formula:


where a COI >=1 is considered positive and a COI <1 is considered negative.

analysis process
The following process of analysis is carried out automatically by the PAMIA-50 (Fig. 4 ). Briefly, after 80 µL of buffer solution is aspirated, 10 µL of sample is aspirated from the sample container and dispensed into the reaction cuvette. At this stage, the sample is stabilized, and no antigen-antibody reaction occurs. Approximately 80 s later, 10 µL of the latex reagent is dispensed into the cuvette. At this point, the antigen-antibody reaction starts, and the coagulation of latex particles begins.



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  		Figure 4. Schematic of analysis automatically carried out on the PAMIA-50.

p/t (t1) analysis
To perform the T1 analysis, 19.2 µL of the reaction sample in the cuvette is aspirated ~30 s after the reaction starts. The reaction sample is dispensed for particle counting, together with 960 µL of the analysis diluent (CLEANSHEATH). T1 analysis is the degree of coagulation (P/T) at 30 s after starting the reaction. T1 analysis is performed to check whether the sample concentration is in the excess range (prozone). Therefore, the analysis results are not used.

p/t (t2) analysis
T2 analysis is the degree of coagulation (P/T) at 15 min after the reaction is initiated. The reaction sample is aspirated in the same manner as that used for the T1 analysis and is then dispensed for particle counting together with the analysis diluent. The data from the T2 analysis provide the analysis results.

For both T1 and T2 analyses, the reaction sample is diluted by a factor of ~51 and is sent through a flow cell and an optical detector unit. A laser beam, at a wavelength of 780 nm, is focused on the reaction sample flowing through the optical detector unit flow cell. The intensity of scattered light corresponding to the size of the latex particles is detected. Each burst of scattered light is counted as a pulse signal.

The pulse signals are divided into coagulated (P) and uncoagulated (M) latex particles, according to the size of the pulse. The sum of M and P is T. The degree of coagulation (P/T) is calculated. The COI (P/T of cutoff control) is set as the value ~10 SD from the P/T distribution of negative samples.

pamia-50
PAMIA-50 is an automated immunochemical analyzer for use in clinical laboratories in examinations such as those for infectious illnesses and tumor markers. This instrument processes 120 tests/h using the CIA method to measure proteins in the sample. The volume of sample required for analysis is 10 µL. Analysis time is 15 min.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
comparison of cia and pa/elisa
The positivity of all 24 samples, including that of the ATL cases, was determined with both the CIA and ELISA methods (Table 1 ). The results of the CIA and ELISA methods were in agreement for all 24 plasma samples. The concordance between the ELISA for plasma and the CIA for whole blood in 24 patients was also 100%. The concordance between the PA method, using plasma, and the CIA, using plasma or whole blood, for samples from 1065 patients was 99.5%. The concordance between plasma and whole blood for the CIA method in 1065 samples was 100%.


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  		Table 1. Comparison between ELISA using plasma and CIA using whole blood.

Among 1065 samples, 1050 were CIA- and PA-negative for HTLV-I antibody, 10 were CIA- and PA-positive, 3 were CIA-negative/PA-positive, and 2 were CIA-positive/PA-negative.

For an anti-HTLV-I/II mixed-titer panel (PRP 205), the CIA and PA methods agreed completely not only when plasma was used but also when whole blood was used for the CIA method.

Using five discordant samples, we performed Western blot analysis for HTLV-I (HTLV-I Problot; Fujirebio) and found no positive bands in any of the samples (Table 2 ). In addition, we analyzed two CIA-positive/PA-negative samples, although the COI of the CIA was low, using recombinant p21, peptide p19, and peptide gp46 as antigens. The CIA-positive/PA-negative samples reacted only with the p19 antigen.


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  		Table 2. Western blot analysis of discrepancies between two assays.

daily stability test
Although results from 24 whole blood samples remained constant (as positive or negative) on days 1, 2, and 3 after blood collection, one sample (sample 22) became positive on day 7 after collection, with the COI increasing from 0.7 to 1.4.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
HTLV-I is a RNA retrovirus that primarily affects CD4+ T cells. The HTLV-I provirus genome is 9032 bp long and contains the gag, pol, and env genes, which encode the viral matrix; capsid and nucleocapsid proteins; enzymes such as reverse transcriptase, integrase, and protease; and envelope protein consisting of a surface glycoprotein and a transmembrane protein. HTLV-I-infected individuals produce antibodies against protein products of the viral env, gag, and tax genes. Serum titers of anti-HTLV-I antibodies are usually higher in HAM/TSP patients than in asymptomatic carriers and ATL patients.

HTLV-I infection is causally associated with a variety of human diseases, including leukemia/lymphoma, myelopathy, and uveitis (1). HTLV-I-associated diseases such as ATL, HAM/TSP, and HTLV-I uveitis are contracted from a carrier, and carriers can become a source of infection in others through contact with infected lymphocytes. Three means of infection, i.e., mother-infant transmission through the mother’s milk, infection of a woman by a man, and blood transfusion are the main pathways of infection. It is estimated that 1 to 2 million individuals are infected with HTLV-I in Japan, where the virus is most prevalent in the world. ATL develops in a small proportion of HTLV-I-infected individuals (1 in 1000–2000 seropositive individuals per year) after a long latent period.

In this report, using whole blood, HTLV-I recombinant antigen, synthetic peptide antigens, the CIA method, and a PAMIA-50 analyzer (10), we established a novel anti-HTLV-I antibody detection system. The PAMIA system, based on CIA technology, involves a measuring principle that combines particle counting with antibody-based latex agglutination. The main advantages of this technology are (a) the possibility of easy automation of the assays and (b) a comparatively short analysis time. In addition, no special reaction facility or solid phase is needed, thus allowing the use of an automatic dilution protocol.

When whole blood samples are used, the degree of coagulation is measured and the number of blood cells in a sample is counted simultaneously, making use of the difference between the size of latex and a blood cell (Fig. 3Up ). The ratio of a blood cell component and a plasma component is calculated by the result obtained from the above process, and the COI (concentration) is compensated. Therefore, hematocrit has no effect on the assay, and the correlation between plasma and whole blood is good (Fig. 5 ). The analysis results are displayed on the screen, stored in the main unit, and output to a printer and/or host computer.



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  		Figure 5. Correlation between plasma (or serum) and whole blood results obtained by CIA.

The least-squares method was used for regression analysis: y = 1.105x + 0.015 COI; R2 = 0.968. Ab, antibody.

We evaluated this new CIA system using clinical specimens and panels of HTLV-I/II-positive sera. Four antibody screening assays were included in the evaluation: New Eitest ATL-EIA (Eisai), Serodia HTLV-I (Fujirebio), Western blot (Fujirebio), and CIA. The Eitest represents a new generation of HTLV-I screening tests with an electrochemiluminescence immunoassay; Serodia also represents a new generation of particle agglutination methodology.

In this study, agreement between the results obtained by these four HTLV-I antibody assays was quite good. Results from all 24 patient samples were in agreement with regard to the correlation between the ELISA and CIA methods. The results were also good with regard to the correlation between the PA and CIA methods.

In two samples that reacted with p19 antigen, as shown by CIA, it was difficult to judge whether there had been a nonspecific reaction or whether the results reflected an early stage of HTLV-I infection. To resolve this issue, a new assay system using PCR or the monitoring of individual cases will be required.

A determination by only one method is dangerous in the case of HTLV-I antibody detection. Reactive samples were confirmed by additional assays.

We determined seropositivity by an HTLV-I antibody screening method as follows: If the results of the screening were negative, it was concluded that the sample was negative. If the sample was positive, we confirmed the positive results by a different assay method based on a different principle, using the same sample. In addition, we used a third method (e.g., Western blot) when a negative or an equivocal result was obtained by the second method. Detection by only one method should be regarded only as a screening process. The CIA method should not be used as an exception to this approach.

The results obtained for all 24 examples agreed within 3 days after blood collection, as observed by the daily stability test. The CIA method could be used to validate detection within 3 days after blood collection.

Although PA and ELISA methods are widely used in screening for HTLV-I carriers, the samples required by these assays are either serum or plasma, and ~2–3 h are required until the results are definitive. The CIA method automatically provides results in ~15 min for whole blood. Similar methods of antibody detection to screen for other retroviruses (e.g., HIV) and hepatitis viruses (e.g., hepatitis B and C viruses) may be expected in the future.

The availability of virologic assays that use whole blood samples makes combining these assays with routine blood examination possible; this is advantageous because it reduces the required volume and number of blood collections. An additional advantage of the present assay system would be the reduction in time required for detection, which is achieved by the omission of centrifugation. It is expected that the results will be similar for whole blood or serum samples. Use of this automated system could substantially change the clinical laboratory.


   Footnotes
 
1 Nonstandard abbreviations: HTLV-I, human T-lymphotropic virus type I; PA, particle agglutination; ATL, adult T-cell leukemia; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; CIA, counting immunoassay; and COI, cutoff index.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Yamaguchi K. Human T-lymphotropic virus type in Japan. Lancet 1994;343:213-216.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  2. Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto T, Kinoshita K, et al. Adult T-cell leukemia; antigen in an ATL cell line and detection of antibody to antigen in human sera. Proc Natl Acad Sci U S A 1981;78:6476-6480.[Abstract/Free Full Text]
  3. Yamaguchi K, Seiki M, Yoshida M, Nishimura H, Kawano F, Takatsuki K. The detection of human T-cell leukemia virus proviral DNA and its application for classification and diagnosis of T-cell malignancy. Blood 1984;63:1235-1240.[Abstract/Free Full Text]
  4. Ikeda M, Fujino R, Matsui T, Yoshida T, Komoda H, Imai J. A new agglutination test for serum antibodies to adult T-cell leukemia virus. Gann 1984;75:845-848.[ISI][Medline] [Order article via Infotrieve]
  5. Taguchi H, Sawada T, Fujishita M, Morimoto T, Niiya K, Miyoshi I. Enzyme-linked immunosorbent assay of antibodies to adult T-cell leukemia-associated antigens. Gann 1983;74:185-187.[ISI][Medline] [Order article via Infotrieve]
  6. Osame M, Usuku K, Izumo S, Ijichi N, Amitani H, Igata A, et al. HTLV-I associated myelopathy, a new clinical entity. Lancet 1986;i:1031-1032.
  7. Mochizuki M, Tajima K, Watanabe T, Yamaguchi K. Human T lymphotropic virus type-I uveitis. Br J Ophthalmol 1994;78:149-154.[Abstract/Free Full Text]
  8. Kamihira S, Nakashima S, Fukahori Y, Toriya K, Kanamura M, Ohta T. Serological evaluation of human T lymphotropic virus type-I infection in routine hospital samples [in Japanese]. Rinsho Byori 1990;38:783-788.[Medline] [Order article via Infotrieve]
  9. Shinkai E, Motoi S. Measurement principle of the automated immunochemical analyzer PAMIA.. [in Japanese]Sysmex J 1997;20:77-86.
  10. Niederau C, Reinauer H. Pamia-30, a new counting immunoassay system. Eur J Lab Med 1994;2:117-124.



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