Clinical Chemistry 45: 92-97, 1999;
(Clinical Chemistry. 1999;45:92-97.)
© 1999 American Association for Clinical Chemistry, Inc.
Whole Blood Capcellia CD4/CD8 Immunoassay for Enumeration of CD4+ and CD8+ Peripheral T Lymphocytes
Dominique Carrière1,a,
Jean Pierre Vendrell2,
Claude Fontaine1,
Aline Jansen1,
Jacques Reynes3,
Isabelle Pagès2,
Catherine Holzmann4,
Michel Laprade1 and
Bernard Pau5
1
Ligne de Recherche en Immunologie, Sanofi Recherche, 371 rue du professeur Joseph Blayac, 34184 Montpellier Cedex 04, France.
2
Laboratoire d'Immunologie des Infections
Rétrovirales, Hôpital Lapeyronie, 34295 Montpellier,
France.
3
Département des Maladies Infectieuses et
Tropicales, Hôpital Gui de Chauliac, 34295 Montpellier, France.
4
Sanofi Diagnostics Pasteur, 92430 Marnes la Coquette,
France.
5
Unité Mixte de Recherche 9921, Faculté de
Pharmacie, 34060 Montpellier, France.
a Author for correspondence. Fax 33 4 67 10 60 00; e-mail dominique. carriere{at}sanofi.com.
 |
Abstract
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We evaluated the Whole Blood Capcellia® CD4/CD8, an
immunoenzymatic method that provides absolute counts of CD4+ and CD8+ T
cells in peripheral blood. The assay is based on the separation of T
cells by use of an anti-CD2 magnetic bead suspension, followed by
reaction of the CD4 or CD8 molecules with the corresponding monoclonal
antibody coupled to peroxidase. CD4-positive monocytes were excluded
from the assay. Freeze-dried magnetic bead-T-cell complexes were used
as calibrators. Capcellia counts from HIV-1-infected patients were
compared with those obtained by flow cytometry as the comparison
method. The results by Capcellia correlated well with those by flow
cytometric analysis: r2 = 0.95; P
<0.001; (y = 0.96x - 22.1);
Sy|x = 64 for CD4; r2 =
0.81; P <0.001; (y =
1.26x - 76.4); Sy|x = 139 for
CD8; n = 76. The correlation between CD4+ T-cell counts determined
by two trained experimenters was significant
(r2 = 0.96). Our results indicate that this new
ELISA technique for lymphocyte immunophenotyping is an efficient
alternative to flow cytometry.
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Introduction
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Enumeration of CD4+ T cells constitutes an essential biological
indicator in the clinical follow up of patients infected with HIV-1.
Indeed, the number of CD4+ T cells per microliter of blood is used to
determine the classification of the disease stage (1), to
predict the progression to clinical AIDS (2)(3),
to assess antiretroviral treatment, and to initiate therapy for
opportunistic infections. Moreover, the determination of CD4+ and CD8+
T-cell counts are also recommended for patients suffering from immune
disorders or after an organ transplant or a graft. The increased use of
CD4+ and CD8+ T-lymphocyte typing and enumeration has led to the
development of flow cytometric cellular analysis and, more recently, to
alternative methodologies (4) for counting T cells.
The standard method currently used to count CD4+ and CD8+ T cells in
peripheral blood requires an automated hematology analyzer for the
determination of the total number of leukocytes and the percentage of
lymphocytes (differential blood count) and a flow cytometer to assess
the percentage of CD4+ or CD8+ T lymphocytes
(5)(6). The absolute cell count (cells per liter
of blood) of the two T-cell subsets is the product of these three
measurements. The most recent flow cytometers provide absolute count
determination by use of an internal standard such as fluorescent beads,
thus eliminating the necessity of having a hematology analyzer
(7).
Alternative noncytofluorometric methods for the enumeration of
T-lymphocyte subpopulations have been developed for applications in
developing countries (4). These methods, like flow
cytometry, involve immunolabeling of cell surface molecules by
monoclonal antibodies (MAbs), but they do not require the use of
expensive instruments. They are based on an enzyme immunoassay (ELISA)
(8), an immunofluorescence assay, or a microscopic method
involving the identification of labeled cells (9). Earlier,
we developed a Capcellia® CD4/CD8 immunoenzymatic assay to
determine CD4 and CD8 concentrations and the corresponding numbers of
CD4+ or CD8+ T lymphocytes in peripheral blood mononuclear cells
separated from blood by centrifugation on a Ficoll gradient
(10)(11). We describe here a new generation
Capcellia CD4/CD8 immunoassay method (Whole Blood Capcellia CD4/CD8)
that provides a direct determination of CD4+ and CD8+ T cells in whole
blood without requiring the preparation of peripheral blood mononuclear
cell suspensions. Briefly, EDTA-anticoagulated whole blood is mixed
with a suspension of anti-pan T MAb-coated magnetic beads, allowing the
specific trapping and separation of T cells in wells of a microtiter
plate fitted with magnets. CD4+ or CD8+ T lymphocytes are then labeled
with an anti-CD4- or anti-CD8-specific MAb coupled to peroxidase. We
include the description of this new enzyme immunoassay, its performance
characteristics (precision, accuracy, and reproducibility), and the
results of the determination of CD4+ and CD8+ T-cell counts in blood
from healthy adults and HIV-1-infected patients. Capcellia counts were
compared with those obtained by direct flow cytometry, a procedure that
bypasses the hematology measurements (7).
|
Materials and Methods
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blood samples and patients
Peripheral blood specimens were collected in EDTA liquid
anticoagulant Vacutainer Tubes (Becton-Dickinson) and analyzed within
24 h. EDTA helps to retain cellular integrity well and is used for
lymphocyte immunophenotyping (12). Twelve HIV-1-seronegative
subjects served as controls, and 64 HIV-1-infected patients (18
patients with <200 T CD4+, 37 patients with 200–500 T CD4+, and 9
patients with >500 T CD4+) were selected at the Department of
Infectious Diseases of Gui de Chauliac Hospital, Montpellier, France.
The procedures followed were in accordance with the ethics standards of
the responsible hospital committees.
flow cytometry analysis
Four-color panels were prepared, using 50 µL of blood added to 5
µL of Cyto-Stat Tetrachrome (CD45-fluorescein
isothiocyanate/CD4-phycoerythrin-Texas
red/CD8-ECD/CD3-phycoerythrin-cyanins; Coulter) to allow up to four
antigens to be measured in one sample tube. After samples were
incubated for 15 min, sample preparation was performed using the
Coulter MultiQ-Prep Workstation with the Coulter Immunoprep Epic
STM Leukocyte Preparation System. Absolute counts were
assessed using 50 µL of Flow-Count Fluorospheres (Coulter) per
sample. The use of Flow-Count Fluorospheres eliminated the necessity of
having a hematology analyzer available to report absolute counts
(7). The flow cytometry system used for this study was the
Coulter Epics XL-MCL with System IITM Software and
tetraONETM System Software. To control the efficiency of
T-cell separation in the first step of the Capcellia assay, the
percentages of T-cell subsets and monocytes were determined by flow
cytometry analysis on residual blood, previously mixed with
anti-CD2-coated magnetic beads, and then placed for 2 min in
magnet-associated microwells. Monocytes were labeled with fluorescein
isothiocyanate-conjugated anti-CD14 Leu 3M.
MAbs and conjugates
Magnetic beads (Estapor; Rhône-Poulenc) were coated with MAb
F92-3A11 (anti-CD2) by covalent binding, using the manufacturer's
protocol, washed, and kept at 4 °C until use. T-lymphocyte subsets
were labeled with MAbs F101-69 (anti-CD4) and F101-87 (anti-CD8)
conjugated to horseradish peroxidase (Boehringer Mannheim) by the
sodium periodate method (13). The specificity of the
MAbs was confirmed at the Third International Conference on Human
Leukocytes Differentiation Antigens (Oxford, UK, 1986) (14).
description of the assay
The assay is based on T-cell separation from whole blood by use of
an anti-CD2 magnetic bead suspension followed by reaction of the CD4 or
CD8 molecules with the corresponding MAb coupled to peroxidase. In the
first step of the assay, 100 µL of blood sample or human plasma taken
as control was mixed with 500 µL of anti-CD2 MAb-coated magnetic
beads in 5-mL tubes, and then shaken for 2 min manually or on an
orbital rotator. The blood-bead mixture (100 µL for CD4 or 50 µL
for CD8) and the human plasma-bead control mixture were then placed in
the microwells of a microtiter plate (Maxisorp Nunc) fitted with
magnets, which separate the T cells coated with the magnetic beads from
uncoated cells. After 2 min, the residual whole blood containing
untrapped cells was rapidly removed from the microwells by aspiration
(for 1 s) with an eight-channel manifold for microplates connected
to an electric or water-driven aspiration device. In the second step of
the assay, the magnets were separated from the microwells, and a
solution (100 µL) containing the anti-CD4- or anti-CD8-MAb-peroxidase
conjugate was added to the assay wells and the control wells
(bead-immunoconjugate blanks). The plates were allowed to stand for 20
min at room temperature, after which the plate and magnets were once
again juxtaposed (2 min), and excess conjugate was removed by multiple
washings with the wash buffer included in the kit (5 x 200
µL/well). The peroxidase activity in the microwells was measured by
addition of 100 µL of 3,3',5,5'-tetramethylbenzidine (Boehringer
Mannheim) to each well. The plates were allowed to stand 20 min in the
dark, and then 50 µL of 0.75 mol/L H2SO4
was added to stop the reaction. The absorbance was measured on a
microtiter plate reader (LP400; Sanofi Diagnostics Pasteur) at 450 nm.
The number of CD4+ or CD8+ T cells/L in the blood samples was
determined from the calibration curves. The absorbance values of the
bead-immunoconjugate blanks were not subtracted from the values of the
blood samples.
To analyze blood from children, which presents a higher lymphocyte
count than blood from adults (15), we mixed only 50 µL of
the blood sample instead of 100 µL (as in the case of adults) with
550 µL of the magnetic bead suspension; the number of T cells
determined from the calibration curve was then multiplied by a factor
of two.
preparation of calibrators
An Ichikawa human CD4+/CD8+ T-cell line (16) was
cultured in RPMI 1640 (Life Technologies) containing 100 mL/L
heat-inactivated (56 °C for 30 min) fetal calf serum. Calibrators
were prepared by addition of the anti-CD2 MAb magnetic beads to these
cells (2.5 x 108/L); the suspension was then mixed
for 15 min at room temperature. For each calibration curve (CD4 and
CD8), four different volumes of the mixture were transferred to a 3-mL
vial and freeze-dried. Before use, the freeze-dried cells were
rehydrated by addition of 0.9 mL of wash buffer and 0.1 mL of human
plasma; under these conditions, the calibrators were stable for 4 weeks
at 4 °C. To assign values to the calibrators, we analyzed 10 blood
samples, including samples from healthy donors and HIV-1-infected
patients. CD4 and CD8 calibration curves were prepared using the data
collected from the Capcellia assay (absorbance values) and the flow
cytometry analyzer (number of T cells per microliter of blood) for the
10 blood samples. The equivalent CD4+ or CD8+ human T-lymphocyte
content of the calibrators assayed by Capcellia could then be
determined.
magnetic frame
Forty-eight circular magnets (6 mm in diameter and 2 mm thick)
with an intense magnetic field, placed in front of an equal number of
microwells, efficiently attracted T-cell-bead complexes, allowing the
safe removal of wash buffer and uncoated cells by aspiration. The
magnet-supporting frame was juxtaposed with or removed from the
microtiter plate and remained associated with the plate while the
absorbance was measured in the microtiter plate reader.
statistical analysis
The imprecision of the flow cytometry analysis and the Whole Blood
Capcellia CD4/CD8 assay was expressed as CVs for 10 replicate results
for blood samples from healthy subjects and HIV-1-infected patients.
The Student t-test was used to determine the significance
between the controls and the assays for the determination of the lower
detection limits. T-cell counts obtained using the new Capcellia assay
vs those values determined by flow cytometry were compared by linear
regression analysis, and the correlation coefficients
(r2) were determined.
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Results
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calibration curves
The calibration curves were prepared using the four calibrator
preparations with known CD4+ or CD8+ T- lymphocyte contents and the
bead-immunoconjugate blank (Fig. 1
). There was a linear relationship between the absorbances
measured at 450 nm by the Capcellia assay and the corresponding numbers
of T cells (r2 
0.99 for CD4 and CD8). The
limits of detection were 14 x 106 to 1250 x
106 CD4 T cells/L and 15 x 106 to
1600 x 106 CD8 T cells/L.
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Figure 1. Calibration curves for determination of CD4+
(A) and CD8+ (B) T-cell counts. (A),
r2 = 0.99; regression line, y =
0.002x + 0.04; (B),
r2 = 0.99; regression line, y =
0.002x + 0.06.
|
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immunoassay validation
Separation of T lymphocytes from whole blood by MAb-coated magnetic
particles.
To control the efficiency of the T-cell immunocapture
by the anti-CD2 MAb-coated magnetic particles, 100 µL of blood was
untreated (control) or mixed for 2 min with the suspension of magnetic
beads before transfer to the magnetized microwells of the microtiter
plate. The percentage of residual T cell subsets was then determined by
flow cytometry analysis. The CD4+ and CD8+ T lymphocytes decreased by
~99% and 94%, respectively, in the samples mixed with the magnetic
beads and transferred to microwells, compared with controls. In
contrast, the percentage of CD14+ monocytes was practically unchanged
in the control and the treated samples, indicating that these CD2- and
CD4+ cells were not captured by the magnetic phase (Table 1
). The Capcellia assays of CD4+ and CD8+ T cells in the controls
and the samples mixed with the magnetic beads also indicated that
practically all (96%) the CD4+ and CD8+ T cells had been removed
from the samples, confirming the efficiency of T-lymphocyte capture by
the magnetic phase.
Determination of the lower detection limits.
CD4 and CD8
absorbance values were determined by the Whole Blood Capcellia assay on
successive dilutions of a blood sample that had been analyzed
previously by flow cytometry. The linearity of the responses confirmed
the accuracy of the method over the range of cell densities tested
(data not shown). The absorbance values were significant for a
suspension containing 14 x 106 CD4+ T cells/L whole
blood as shown by the following results: assay absorbance, 0.116
± 0.006 (n = 11); nonspecific absorbance, 0.085 ± 0.007
(n = 11); P <0.0001. Assays of CD8+ T lymphocytes were
significant for a suspension of 15 x 106 cells/L
whole blood [assay absorbance, 0.101 ± 0.008 (n = 12);
nonspecific absorbance, 0.074 ± 0.014 (n = 12); P
<0.0001]. These lower limits of detection corresponded to as few as
of 230 CD4+ or 125 CD8+ T cells in the assay wells.
Specificity of CD4+ and CD8+ T-lymphocyte assays.
The
specificity of the absorbance values obtained from the assay of the
calibrators and blood samples was demonstrated by mixing conjugated
anti-CD4 or anti-CD8 antibodies coupled to peroxidase and a 100-fold
excess of unlabeled antibody of the same specificity. Under these
conditions, the excess of unlabeled antibody reduced absorbance values
by >98%. In contrast, an anti-CD5 MAb used under the same conditions
did not affect the absorbance values.
Imprecision (CV) of flow cytometric analysis and Capcellia.
To
determine the CV of the flow cytometric analysis, the lymphocytes from
one healthy control and from one HIV-1-infected patient were counted 10
times. For the Capcellia assay, the CV was calculated from 10
determinations of three healthy controls and three HIV-1 infected
patients. The CV for different measurements was 2.8–5.6% for flow
cytometry and 3.2–7.8% for the Capcellia assay (Table 2
). We also assessed the reproducibility of the Capcellia assay
by comparison of CD4 T cell counts determined by two trained
technicians (Fig. 2
). The correlation coefficient (r2) was
0.96 (n = 76).
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Figure 2. Comparison of CD4+ T-cell counts determined by two trained
technicians.
r2 = 0.96; regression line, y =
1.00x + 0.48; 95% confidence interval for the slope,
0.96–1.05; 95% confidence interval for the intercept, -22.04 to
23.01; Sy|x = 54.
|
|
T-lymphocyte subset counts with the Whole Blood Capcellia
assay.
Comparisons were made between CD4+ and CD8+ T-lymphocyte
subset counts determined by the Capcellia assay and those determined by
flow cytometry as the comparison method. The evaluation of CD4+ T
lymphocytes in 76 blood samples from healthy volunteers (n = 12)
and HIV-1-infected patients (n = 64) showed an
r2 of 0.95 between the flow cytometry method and
the Whole Blood Capcellia CD4/CD8 assay (Fig. 3
A). Analysis of CD8+ T lymphocytes showed an
r2 of 0.81 between the flow cytometry method and
the Whole Blood Capcellia CD4/CD8 assay (Fig. 3B
). Finally, the CD4+ T
cell counts determined by flow cytometry and Capcellia on blood samples
from 21 HIV-1-infected patients receiving antiretroviral treatment
(zidovudine + didanosine) over an 18-week period were similar. Before
treatment, there were 448 ± 105 x 106/L and
463 ± 118 x 106 CD4+ T cells/L for flow
cytometry and Capcellia, respectively; after treatment we found
579 ± 172 x 106 CD4+ T cells/L and 564 ±
154 x 106 CD4+ T cells/L for flow cytometry and
Capcellia, respectively.
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Figure 3. Comparison of CD4 T-cell counts (A) and CD8
T-cell counts (B) determined by Whole Blood Capcellia and
flow cytometry.
(A), r2 = 0.95; P <0.001;
regression line, y = 0.96x - 22.08;
95% confidence interval for the slope, 0.91–1.01; 95% confidence
interval for the intercept, -48.06 to 3.89; Sy|x
= 64. (B), r2 = 0.81; P
<0.001; regression line, y = 1.26x -
76.48; 95% confidence interval for the slope, 1.12–1.39; 95%
confidence interval for the intercept, -171.95 to 18.99;
Sy|x = 139.
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|
Discussion
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We have developed a Whole Blood Capcellia CD4/CD8 assay for the
determination of human CD4 and CD8 T-cell counts. This method uses a
suspension of magnetic beads for the rapid separation of T cells from
whole blood, followed by addition of an anti-CD4 or anti-CD8 MAb
coupled to peroxidase for T-cell subset detection. When the method was
assessed on a large number of blood samples, the new Capcellia gave
lymphocyte counts that correlated closely with those obtained using the
comparison method, i.e., a flow cytometer that used Flow-Count
Fluorospheres (7) (r2 = 0.95 and 0.81
for CD4+ and CD8+ T lymphocytes, respectively). Moreover, the positive
effects of combined antiretroviral therapy on CD4+ T-cell numbers were
observed with both methods. A significant correlation was also observed
between CD4+ T-cell counts determined by two technicians
(r2 = 0.96).
Separation of T cells from whole blood, followed by their
immobilization in the microwells of microtiter plates are essential
steps for the complete assay of T-cell subsets. The first generation of
the Capcellia assay described previously (10) imposed a
centrifugation step on the Ficoll-Paque gradient to separate the
mononuclear cells from whole blood and another centrifugation step to
place the cells in close contact with the adsorbed anti-CD2 MAb to
immobilize them. We developed an assay that could be performed easily
without the centrifugation steps, by use of a suspension of magnetic
beads with anti-CD2 specificity and a magnetic frame associated with
the microtiter plate. Magnets placed close to the assay microwells
allow rapid attraction of the magnetic bead-coated T cells, without
interference with the CD4+, CD2- monocytes.
The Capcellia assay can evaluate blood samples containing as few as
15 x 106 CD4+ or CD8+ T lymphocytes/L; this low
limit of detection allows the follow up of HIV-1-infected patients
until their AIDS reaches an advanced stage. The use of high affinity,
enzyme-conjugated anti-CD4 or anti-CD8 antibodies
(Ka >1010 L/mol, data not shown)
allowed us to develop a procedure for rapid immunological T-cell
detection in as short a time as 20 min. The assay ranges are 15 x
106 to 1250 x 106 cells/L whole blood for
CD4+ T lymphocytes and 15 x 106 to 1600 x
106 cells/L whole blood for CD8+ T lymphocytes. For
pediatric samples, which contain higher T-cell concentrations, the
protocols can be modified to extend the measurement range by use of
less than the usual 100 µL (e.g., 50 µL) of blood and multiplying
the final result by the appropriate factor.
The new Capcellia assay uses the microtiter plate format, which allows
simultaneous evaluation of batched specimens, e.g., 3 samples with a
single strip of wells and up to 19 samples with five strips on one
microtiter plate. The assay is facilitated by use of a manual
eight-channel pipettor to place reagents and washing liquid in the
microwells. Undesired cells and washing solution are removed from the
microwells by aspiration using a manual washing manifold connected to
an electric vacuum pump or water-driven aspiration. For this operation,
the tips of the manual washing manifold must not touch the side of the
wells where the magnetic bead-T-cell complexes are held by the magnets.
Up to 19 samples can be processed for CD4+ and CD8+ T-cell
determination in ~1.5 h. Blood drawn into EDTA-containing tubes can
be used for up to 24 h if kept at room temperature, allowing
assays to be grouped.
In conclusion, assessment of the Whole Blood Capcellia CD4/CD8
assay on samples from healthy volunteers and from HIV-1-infected
patients has shown that this new ELISA for cell markers may represent
an efficient alternative to flow cytometry. This method offers the
following advantages: (a) accuracy and good reproducibility
of T-lymphocyte counts; (b) high specificity of the
evaluation of the CD4 T-lymphocyte population, excluding contamination
by CD4 monocytes; (c) absence of technical problems linked
to incomplete lysis of red blood cells, especially encountered in blood
samples from certain patients; (d) internal standardization
by means of a freeze-dried CD4+ or CD8+ T-cell preparation, which
permits comparison of counts obtained from different laboratories; and
(e) rapid and easy performance in all laboratories without
the need for expensive equipment.
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Acknowledgments
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We thank Pierre Gros for helpful discussions and advice and Sharon
Lynn Salhi for critical comments and help in preparing the manuscript.
We also thank Bernadette Buser and Stéphane Lameynardie for
performing the Capcellia assays, Alain Rouaud and Luc Beck for the
electronic cell counts, and Luc Essermeant and Guy Mathieu for the
statistical analyses.
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Abstract
Introduction
