Article

Clinical Evaluation of the ZstatFlu-II Test: a Chemiluminescent Rapid Diagnostic Test for Influenza Virus

University of Missouri - Kansas City, Kansas City, Missouri, United States
Journal of Clinical Microbiology (Impact Factor: 3.99). 08/2002; 40(7):2331-4. DOI: 10.1128/JCM.40.7.2331-2334.2002
Source: PubMed
ABSTRACT
Exploiting the high sensitivity of the chemiluminescence phenomenon, an accurate and sensitive point-of-care test, called the ZstatFlu-II test (ZymeTx, Inc., Oklahoma City, Okla.), was developed to detect influenza virus infections. The ZstatFlu-II test takes 20 min and requires approximately 2 min of "hands-on" time for operational steps. The ZstatFlu-II test does not distinguish between infections with influenza virus types A and B. ZstatFlu-II test results are printed on Polaroid High-Speed Detector Film, allowing test results to be archived. A prototype version of the ZstatFlu-II test was evaluated during the 2000-to-2001 flu season with 300 nasal aspirate specimens from children at a pediatric hospital. Compared to culture, the ZstatFlu-II test had 88% sensitivity and 92% specificity. The Directigen test had a sensitivity of 75% and a specificity of 93%. The sensitivity of the ZstatFlu-II test was significantly higher than that of the Directigen test (P < 0.0574).

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JOURNAL OF CLINICAL MICROBIOLOGY, July 2002, p. 2331–2334 Vol. 40, No. 7
0095-1137/02/$04.000 DOI: 10.1128/JCM.40.7.2331–2334.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Clinical Evaluation of the ZstatFlu-II Test: a Chemiluminescent Rapid
Diagnostic Test for Influenza Virus
Marilyn S. Hamilton,
1,2
* David M. Abel,
2
Yolanda J. Ballam,
2
Mary K. Otto,
2
Angela F. Nickell,
2
Lisa M. Pence,
3
James R. Appleman,
3
Craig D. Shimasaki,
3
and Komandoor E. Achyuthan
3
University of Missouri—Kansas City School of Medicine
1
and The Children’s Mercy Hospital,
2
Kansas City, Missouri,
and ZymeTx, Inc., Oklahoma City, Oklahoma
3
Received 15 January 2002/Returned for modification 1 March 2002/Accepted 5 April 2002
Exploiting the high sensitivity of the chemiluminescence phenomenon, an accurate and sensitive point-of-
care test, called the ZstatFlu-II test (ZymeTx, Inc., Oklahoma City, Okla.), was developed to detect influenza
virus infections. The ZstatFlu-II test takes 20 min and requires approximately 2 min of “hands-on” time for
operational steps. The ZstatFlu-II test does not distinguish between infections with influenza virus types A and
B. ZstatFlu-II test results are printed on Polaroid High-Speed Detector Film, allowing test results to be
archived. A prototype version of the ZstatFlu-II test was evaluated during the 2000-to-2001 flu season with 300
nasal aspirate specimens from children at a pediatric hospital. Compared to culture, the ZstatFlu-II test had
88% sensitivity and 92% specificity. The Directigen test had a sensitivity of 75% and a specificity of 93%. The
sensitivity of the ZstatFlu-II test was significantly higher than that of the Directigen test (P < 0.0574).
The annual economic cost of influenza disease in the United
States has been estimated at $3 billion to $5 billion (22). The
Centers for Disease Control and Prevention reported in 2001
that influenza was associated with about 20,000 deaths nation-
wide and more than 100,000 hospitalizations (http://www.cdc
.gov/ncidod/diseases/flu/fluinfo.htm). Worldwide estimates
were considerably higher (16, 17, 24). Sensitive, specific, and
rapid tests for influenza will greatly improve patient health
care and reduce costs so that only “flu-positive” patients re-
ceive the recently approved antiviral treatments (9, 21). Rapid
diagnostics for influenza will also prevent the misuse of anti-
biotics to “treat” the flu.
Influenza disease is caused by influenza virus types A and B.
Influenza virus types A and B are Orthomyxoviridae, charac-
terized by the presence of an envelope penetrated by glyco-
protein spikes with hemagglutinating and neuraminidase
activities. Influenza virus also contains matrix protein, nu-
cleoprotein, and three proteins with polymerase activity and a
segmented negative-strand RNA genome (6, 19). These viruses
are responsible for winter epidemics of respiratory illness in
which the rates of infection are highest among children (5).
The shared cardinal sign of fever without localization makes
differentiation of influenza from sepsis necessary for proper
patient management. Delay in this differentiation could result
in unnecessary laboratory testing and treatment for possible
bacterial “sepsis” with unnecessary antibiotics (25).
Three diagnostic methods for respiratory secretions are in
common use. Culture, both shell vial and tube, takes several
days. Direct or indirect immunofluorescence assays on exfoli-
ated nasal pharyngeal cells could be done in a few hours but
require a high level of expertise (10). Finally, rapid, point-of-
care tests are available to detect the influenza virus (3, 12, 20,
23). All of these tests use an enzyme immunoassay directed at
antigens of the viruses (3, 12, 23), with the exception of the
ZstatFlu test, which detects influenza virus neuraminidase en-
zymatic activity (18, 20). A new approach utilized in the pro-
totype ZstatFlu-II test by ZymeTx, Inc. (Oklahoma City,
Okla.), detects, but does not distinguish, influenza virus types
A and B, using chemiluminescence resulting from the hydro-
lysis of a synthetic chemiluminescent substrate by the action of
influenza virus neuraminidase. This approach for influenza
virus detection is similar to that of the previously reported
ZstatFlu test, which employed a chromogenic substrate specific
for influenza virus neuraminidase (18, 20). In chemistry ana-
lyzers, chemiluminescence is generally accepted as being more
sensitive than enzyme immunoassays (14). Chemiluminescence
provides an opportunity for increasing the sensitivity of rapid
tests (1, 4).
The purpose of this study was to describe the performance
characteristics of the prototype test, the ZstatFlu-II test, com-
pared to those of culture and the Directigen Flu A B test
(Becton Dickinson, Cockeysville, Md.).
MATERIALS AND METHODS
Patient samples. This study was determined to be exempt from review by the
University of Missouri—Kansas City Health Sciences Pediatric Institutional Re-
view Board. During the 2000-to-2001 flu season, between January 19, 2001 and
March 1, 2001, 300 nasal aspirate specimens from children were tested in a
pediatric hospital laboratory. The clinician had requested rapid influenza testing,
and Becton Dickinson’s Directigen Flu A B test was performed in a rapid-
turnaround laboratory. Residual specimen was used for culture and the Zstat-
Flu-II test. These additional tests were completed within 24 h of collection, with
storage of patient specimens at 4°C until test time.
Laboratory methods. (i) Rapid tests. Two rapid tests were used. The reported
result was based on Becton Dickinson’s Directigen Flu A B test, performed
according to the manufacturer’s instructions. Briefly, the influenza virus A or B
antigens were extracted from 200 l of the respiratory specimen and the extract
was expelled through a filter assembly into the test wells of the test device.
* Corresponding author. Mailing address: The Children’s Mercy
Hospital, Department of Pathology, 2401 Gillham Rd., Kansas City,
MO 64108. Phone: (816)-234-3234. Fax: (816) 802-1492. E-mail:
mhamilton@cmh.edu.
† Present address: National Center for Toxicological Research, Jef-
ferson, AR 72079.
‡ Present address: Anadys Pharmaceuticals, Inc., San Diego, CA
92121.
2331
Page 1
Antigen captured on the device membrane was visualized with an enzyme-
conjugated monoclonal antibody chromogen system. Positive and negative con-
trols are built into each test device.
The experimental test was the ZstatFlu-II test (ZymeTx, Inc.). In this proto-
type test, 0.9 ml of the respiratory specimen was added to a prelter tube
containing salts and detergent, which created an optimal milieu for inuenza
virus neuraminidase activity (2, 4,18). Clarication of the specimen occurred at
the prelter tip as the specimen was added to the bottom half of a proprietary
two-chamber reaction vessel containing an inuenza virus neuraminidase-spe-
cic substrate and two light enhancers. The top half of the reaction vessel
contained a sodium hydroxide solution. A 15-min test period at room temper-
ature (25°C) allowed the inuenza virus neuraminidase to act on the specic
substrate molecule and released the nascent chemiluminescent reporter groups.
At the end of 15 min, the entire reaction vessel was placed into position inside
a turret within a specially designed Polaroid (Waltham, Mass.) imaging device.
The cover of the imaging device was then closed. The closing action ushed the
sodium hydroxide solution from the top chamber into the bottom chamber.
Contact with sodium hydroxide terminated the virus neuraminidase reaction
accompanied by chemiluminescence. Light from the reaction mixture strikes a
Polaroid High-Speed Detector instant”film housed inside the imaging device.
The lm was exposed to the chemiluminescence for 5 min. An inuenza virus-
positive specimen was detected by observation of a white image shaped like a
plus sign against the black background of the Polaroid High-Speed Detector
Film, which also provided a permanent record of the results. Positive and neg-
ative quality controls, which came with the prototype kit, were run daily.
(ii) Culture. Viral cultures were performed in the Virology Laboratory of
Childrens Mercy Hospital. Specically, 0.1 to 0.2 ml of specimen was inoculated
into tube cultures of human embryonic lung broblasts (MRC-5), human lung
carcinoma cells (A-549), and rhesus monkey kidney (RMK) cells (Viromed,
Minneapolis, Minn.), maintained in a roller drum apparatus at 37°C, and then
observed for cytopathic effect for 14 days. Shell vial cultures of A-549 and RMK
(Viromed) were screened on days 4 and 7 with a Chemicon (Temecula, Calif.)
Respiratory Viral Screen. Virus-positive cultures were identied with a Viral
Respiratory Kit from Bartel (Issaquah, Wash.).
(iii) RT-PCR. Reverse transcriptase PCR (RT-PCR) was performed by
Prodesse, Inc. (Milwaukee, Wis.), on available specimens that were negative in
culture but positive by both rapid tests and therefore were considered possible
positive specimens with discrepant results. Briey, the Hexaplex RT-PCR assay
(Prodesse, Inc.) involved extraction of viral genomic RNA from the test speci-
men followed by PCR amplication using the primers to seven upper-respirato-
ry-tract viruses including inuenza virus types A and B. PCR products were
detected by using microplate wells coated with a streptavidin system as described
previously (13). Specimens were coded and then sent for RT-PCR analyses.
Investigators at Prodesse, Inc., were never informed of the results of the rapid
tests and viral culture, either before or subsequent to RT-PCR analyses of the
specimens.
Statistical analysis. The individual rapid tests were compared to culture and
to consensus. In consensus, a specimen was considered positive if it was
positive either in culture or in both rapid tests. Sensitivities, specicities, positive
predictive values, and negative predictive values were calculated as follows:
sensitivity number of true positives/(number of true positives number of
false negatives); specicity number of true negatives/(number of true negatives
number of false positives); positive predictive value number of true posi-
tives/(number of true positives number of false positives); and negative pre-
dictive value number of true negatives/(number of true negatives number of
false negatives). Statistical analyses of the data were performed as described by
Galen (8). Statistical signicance was determined by McNemars test for paired
binary data (7).
RESULTS
Patient characteristics. The patient age range was 12 days to
19 years. The mean, median, and mode ages of the tested
patients were 43, 20, and 2 months, respectively. There were
136 females and 164 males.
Test results and performance characteristics. Representa-
tive examples of three different patients test results are shown
in Fig. 1. These are, respectively, a strong positive test result, a
weak positive test result, and a negative test result. A strong
positive test result producing a bright image was easily discern-
ible, whereas a weak positive test result was considerably less
so. It should be noted that the clinical study resulted in positive
test results over a range of brightness of images and that the
examples shown in Fig. 1 are merely illustrative. The data
underscore the importance of having an archival record of test
results as shown on these Polaroid High-Speed Detector Film
images.
Table 1 shows the results of the two rapid detection tests and
viral culture compared to viral culture and the consensus. Ta-
ble 2 shows the performance characteristics of the two rapid
tests and viral culture compared to culture and the consensus.
Compared to culture, the sensitivity of the ZstatFlu-II test was
FIG. 1. Photographic recording of patient results in the ZstatFlu-II test. These photographs are representative examples of a strong positive
test result (left), a weak positive test result (center) and a negative test result (right), obtained by administering the ZstatFlu-II test to three
different patients. In order to protect condentiality, the patient specimens were identied only serially in the photographs, by the order in which
the various specimens were obtained or tested (115, 88, and 3, respectively). The margins of the Polaroid photographs provided ample surface to
record such additional test-related information as test date and time, test result, and investigator identication, as shown in this gure. The initials
that appear on these photographs belong to the investigators who performed the tests, Y.J.B. and M.K.O. (both coauthors of this work).
2332 HAMILTON ET AL. J. C
LIN.MICROBIOL.
Page 2
signicantly (P 0.0574) higher than that of Becton Dickin-
sons Directigen test. By comparison to consensus, there was
no statistical difference. The specicities and positive and neg-
ative predictive values of the two rapid tests were not signi-
cantly different.
Of the 300 specimens tested, the results of 255 (85%) were
in complete agreement, with identical results in all three tests,
namely, both rapid tests and culture. Of these, 210 specimens
were negative and 45 were positive for inuenza virus, with 13
positive for inuenza virus type A and 32 positive for inuenza
virus type B. In all cases, identications of inuenza virus type
A versus type B by culture and by Becton Dickinsons Direc-
tigen test were in total agreement. Although the ZstatFlu-II
test did not distinguish between inuenza virus types A and B,
the test identied both types of viruses with a sensitivity and a
specicity comparable to those of culture (Table 2). Here, the
identication of the virus types was based on the results from
either viral culture or Becton Dickinsons Directigen test.
Of the 45 specimens without complete agreement, 20 were
positive in culture and negative in rapid testing, 4 in both rapid
tests, 4 in the ZstatFlu-II test alone, and 12 in the Becton
Dickinson Directigen test alone. Eleven specimens were posi-
tive in both rapid tests but negative in culture. Of these 11
specimens, 8 were tested by RT-PCR and 7 were positive by
this technique. Eight specimens were positive only by the
ZstatFlu-II test, and six specimens were positive only in the
Becton Dickinson Directigen test.
Culture isolation of other respiratory viruses. No specimen
was positive for both inuenza virus types A and B. Fifty-six
specimens were positive for respiratory viruses other than in-
uenza virus type A or B alone: 43 specimens were positive for
respiratory syncytial virus (RSV), 5 were positive for adenovi-
rus, 3 were positive for parainuenza-3 virus, and 1 specimen
each was positive for parainuenza-2 virus, parainuenza-4
virus, adenovirus plus parainuenza-3 virus, inuenza virus
type A plus RSV, and inuenza virus type B plus adenovirus.
DISCUSSION
We found that the ZstatFlu-II test, compared to culture, was
more sensitive than Becton Dickinsons Directigen test. This
could be due to the following two differences in the tests or to
a combination of the differences. The ZstatFlu-II test is based
on the functional activity of the inuenza virus neuraminidase.
Becton Dickinsons Directigen test, on the other hand, is de-
pendent on monoclonal antibodies binding to extracted viral
antigens. The ZstatFlu-II test detects chemiluminescence us-
ing Polaroid High-Speed Detector Film. Becton Dickinsons
Directigen test is based on visual detection of a chromogen
that yields a purple signal on a white background. It is not
possible to distinguish the relative contributions of these two
parameters to the increased sensitivity of the ZstatFlu-II test.
However, it is commonly accepted by chemistry analyzers that
chemiluminescence is more sensitive than enzyme immunoas-
says (14). This suggests that the detection method contributed
to the increased sensitivity of the ZstatFlu-II test (1, 4).
None of the rapid inuenza diagnostic tests currently on the
market provide archiving capabilities for the test results. The
ZstatFlu-II test described here has the added advantage of
providing a permanent record of the test result as a Polaroid
picture.This could be reviewed as necessary if the results are
judged questionable. The Polaroid photograph containing the
patient test result could also be included in the patients
records for documentation. The margins of the Polaroid pho-
tograph provide ample surface on which to record such addi-
tional test-related information as test date and time, reported
result, and patient identication (Fig. 1).
The ZstatFlu-II test did not detect several other upper re-
spiratory disease-causing viruses that were present in patient
specimens such as the parainuenza viruses, adenovirus, and
RSV. Some of these related viruses, such as parainuenza
viruses, also express signicant neuraminidase activity (2). In
our testing, the chemiluminescent substrate molecule, which is
at the core of the ZstatFlu-II test technology, was specic for
the inuenza virus neuraminidases. The data are consistent
with our earlier results and those of others obtained from using
a highly specic chromogenic substrate molecule for inuenza
virus neuraminidase (18, 20).
Experienced technologists found the prototype ZstatFlu-II
test awkward but identied simple improvements that have
been implemented in later models. Unlike Becton Dickinsons
Directigen test, the ZstatFlu-II test does not have internal
positive and negative controls. If a test has internal positive
and negative controls, the College of American Pathologists
Laboratory Accreditation Program Microbiology Checklist,
question MIC.61385, requires that external controls be run
only on each shipment of each lot of tests. Becton Dickinsons
Directigen test meets this requirement. On the other hand,
Checklist question MIC.61400 requires that external controls
TABLE 1. Results of rapid testing compared to
culture and consensus
Gold
standard
No. of specimens with the indicated result by:
ZstatFlu-II test Directigen test Culture
Positive Negative Positive Negative Positive Negative
Culture
Positive 57 8 49 16
Negative 19 216 17 218
Consensus
Positive
a
68 860 1665 11
Negative 8 216 6 218 0 224
a
Positive either by culture or by both rapid tests.
TABLE 2. Performance characteristics of rapid testing
compared to culture and consensus
Gold
standard
Rapid test
Sensitivity
(%)
Specicity
(%)
Predictive
value (%)
Positive Negative
Culture ZstatFlu-II test 88
a
92 75 96
Directigen test 75 93 74 93
Consensus
b
ZstatFlu-II test 89 96 89 96
Directigen test 79 97 91 93
Culture 86 100 100 95
a
P 0.0574 compared to the Directigen test.
b
A specimen positive in culture or in both rapid tests is considered positive by
consensus.
VOL. 40, 2002 ZstatFlu-II TEST FOR INFLUENZA VIRUS 2333
Page 3
be run daily if the test does not have internal controls. The
ZstatFlu-II test is in this category. The added requirement for
controls could be a burden in some situations. It should also be
emphasized that as of this writing, the ZstatFlu-II test has not
gone through a 510(k) ling process with the Food and Drug
Administration (FDA) and is therefore not an FDA-approved
test for inuenza.
The study reported here was based on nasal aspirate speci-
mens. This is the best specimen available for both of the rapid
tests (11, 15). Becton Dickinsons Directigen test does permit
the use of throat swabs (11, 15). Testing based on throat swabs
was less sensitive (11, 15). How the ZstatFlu-II test would
function with a nasal swab or a throat swab specimen is not
known at this time.
The inability of the ZstatFlu-II test to distinguish inuenza
virus type A from type B may or may not be a problem.
Amantadine will treat only inuenza virus type A, but the
newer treatments are effective for both types of inuenza vi-
ruses (9, 21). In the latter case, the lack of information regard-
ing the precise type of inuenza virus infection is not critical
for treatment purposes. Treatment decisions are based more
on symptomology than on virus type identication but must be
initiated early in the disease course to be effective. From a
treatment standpoint, a rapid test that could identify the dis-
ease earliest after onset would be the best. This aspect was not
determined in this study.
In summary, the ZstatFlu-II test provides an alternative
rapid test technology for inuenza virus types A and B which,
compared to culture, is more sensitive than Becton Dickinsons
Directigen test and has the added advantage of providing a
permanent record of the test result. Chemiluminescence tech-
nology has the potential to increase the sensitivity of detection
systems used in rapid tests.
ACKNOWLEDGMENTS
The clinical trial cost was defrayed by a grant from ZymeTx, Inc.
Development of the ZstatFlu-II test was funded in part by grants from
the Oklahoma Center for the Advancement of Science and Technol-
ogy (OCAST) awarded to C. D. Shimasaki (AR982-026) and to K. E.
Achyuthan (HN5-039 and AR021-004).
We gratefully acknowledge Don Mauchan, Paul Nangeroni, Dan
Mantell, Ed Gaffey, Paul Graham, and Leonard Aberbach (Polaroid
Corporation) for supplying the imaging devices and the Polaroid High-
Speed Detector Film. Stephen Simon (Childrens Mercy Hospital,
Kansas City, Mo.) helped with statistical analyses. Roberta Allen and
Robyn Cannedy provided technical assistance during the development
phase of the ZstatFlu-II test.
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    • "Although we did not observe a statistically significant difference in test sensitivity in children versus adults, the relatively high sensitivity in children is consistent with previously published results (Landry et al., 2004; Weinberg and Walker, 2005; Fader, 2005; Hurt et al., 2007). A decrease in sensitivity in adult patients has also been observed with other enzyme immunoassay rapid tests for detecting influenza infections (Yamazaki et al., 2000; Chan et al., 2002; Reina et al., 2002; Cazacu et al., 2003; Hamilton et al., 2002; Ruest et al., 2003; Cazacu et al., 2004; Landry et al., 2004; Alexander et al., 2005). Higher viral excretion by children has been suggested as a possible mechanism for higher sensitivity in children (Landry et al., 2000). "
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    Full-text · Article · Dec 2007 · Diagnostic Microbiology and Infectious Disease
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    • "However, the sensitivity estimates generated by other studies do not support this hypothesis (Yamazaki et al., 2000; Chan et al., 2002; Reina et al., 2002; Landry and Ferguson, 2003; Ruest et al., 2003; Landry et al., 2004; Alexander et al., 2005; Grondahl et al., 2005; Weinberg and Walker, 2005; Drinka 2006). Sensitivity estimates also did not vary with the type of gold standard (culture versus RT-PCR) (Yamazaki et al., 2000; Chan et al., 2002; Cazacu et al., 2003; Dunn et al., 2003; Ruest et al., 2003; Hamilton et al., 2002; Cazacu et al., 2004; Alexander et al., 2005; Grondahl et al., 2005; Walker, 2005 Drinka, 2006). One factor that might contribute to low sensitivity of the EIA is that the manufacturers of the EIA test specify submitting the swab specimen in 1 to 2 mL of transport medium. "
    [Show abstract] [Hide abstract] ABSTRACT: Early diagnosis of influenza infection is needed to optimize the benefit of prescribing antiviral drugs. However, the accuracy of rapid tests is highly variable. This study evaluated the performance of Directigen flu A+B enzyme immunoassay (EIA) and direct fluorescent assay (DFA) during the 2004-2005 influenza season. Participants with medically attended acute respiratory illness were identified through an active surveillance. Consenting patients (n=818) were enrolled and cultured for influenza. Physicians ordered a rapid antigen test (EIA or DFA) according to their clinical judgment. Physicians ordered rapid tests with EIA (n=109), DFA (n=86), or both (n=9) in 204 patients with acute respiratory illness who were also cultured for influenza. The EIA detected 18 of 43 influenza infections (sensitivity, 42%; 95% confidence interval [CI], 28-57%), whereas DFA detected 26 of 38 influenza infections (sensitivity, 68%; 95% CI, 53-81%). Compared with culture, specificity of both EIA and DFA was 96%. During the 2004-2005 influenza season, both the EIA and DFA had low sensitivity and failed to detect influenza in many patients.
    Full-text · Article · Sep 2007 · Diagnostic Microbiology and Infectious Disease
  • [Show abstract] [Hide abstract] ABSTRACT: We describe the engineering and product development of the chemiluminescent ZstatFlu-II Test kit for influenza diagnostics. The reaction vessel is a chemical implementation device with a polystyrene bottom chamber and a polypropylene top chamber that screw together. The patient's specimen is dispersed in a proprietary diluent and mixed inside the bottom chamber with the influenza viral neuraminidase-specific substrate, 1,2-dioxetane-4,7-dimethoxy-Neu5Ac. Neuraminidase catalysis releases the dioxetane. The top chamber contains 40% NaOH and is sealed at the top with an ABS plastic plug-crush pin assembly. The top chamber floor is 85% thinner at the centre, forming a frangible flap. An automated imaging device serves as an incubator for the chemical implementation devices and also facilitates the piercing of the flap by the crush pin. This action results in NaOH flushing into the bottom chamber, initiating chemiluminescence. The imaging device also exposes the Polaroid high-speed detector film to chemiluminescence. At the end of exposure, the film is automatically processed and ejected. Chemiluminescence from an influenza virus-positive specimen produces a "+"-shaped white image, archiving the diagnostic outcome. The modular ZstatFlu-II test kit components are easily adaptable for the chemiluminescent detection of a wide range of analytes.
    No preview · Article · Mar 2003 · Luminescence
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