Vol. 31, No. 11JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1993, p. 2851-28560095-1137/93/112851-06$02.00/0Copyright © 1993, American Society for Microbiology

Comparison of Quantitative Cytomegalovirus AntigenemiaAssay with Culture Methods and Correlation with

Clinical DiseaseMARIE L. LANDRY' 2* AND DAVID FERGUSON'

Clinical Virology Laboratory, Yale-New Haven Hosfital,' and Department of Laboratory Medicine,Yale University School ofMedicine, New Haven, Connecticut 06516

Received 22 April 1993/Returned for modification 1 June 1993/Accepted 29 July 1993

Blood samples, obtained predominantly from human immunodeficiency virus-infected patients and solid-organ and bone marrow transplant recipients, were submitted to the clinical laboratory for detection ofcytomegalovirus (CMV) and were processed by three methods: conventional culture, centrifugation culture,and CMV antigenemia assay with monoclonal antibodies (Clonab CMV; Biotest Diagnostic Corporation,Denville, N.J.) to CMV antigens. Of 496 blood samples tested, 107 were positive by one or more methods: 56were positive by conventional culture, 27 were positive by centrifugation culture, and 97 were positive for CMVantigen (Ag) by the antigenemia assay. Forty-seven samples were positive by the CMV antigenemia assay only;in these samples, a mean of 12 Ag-positive cells was detected per 200,000 polymorphonuclear leukocytesexamined. In contrast, samples positive by the CMV antigenemia assay and both culture methods had a meanof 193 Ag-positive cells, and samples positive by the CMV antigenemia assay and conventional culture alonehad a mean of 157 Ag-positive cells. In the antigenemia assay, paraformaldehyde fixation resulted in superiorcell morphology when compared with acetone fixation. Use of immunofluorescence staining reduced sampleprocessing time and the complexity of reagent preparation in comparison with immunoperoxidase staining.Differences in the sensitivities between the immunofluorescence and immunoperoxidase staining techniques fordetection of antigenemia were minor, with discrepant samples showing only one or two Ag-positive cells.Clinical disease was generally associated with high-level antigenemia, but exceptions were noted. The CMVantigenemia test is a rapid, quantitative assay that greatly facilitated the rapid diagnosis of CMV infection.However, quantitation of antigenemia is labor-intensive, requires processing of samples soon after collection,and does not always correlate with clinical disease in the individual patient.

Cytomegalovirus (CMV) is a serious pathogen in immu-nocompromised patients. However, infection with CMV isubiquitous; differentiation of clinically significant diseasethat requires treatment from inconsequential virus excretionis a difficult dilemma for clinicians. The available therapiesfor CMV infection include ganciclovir and foscamet, both ofwhich require intravenous administration and are associatedwith serious side effects.

Isolation of CMV from blood leukocytes (CMV viremia)has been found to correlate better with clinical disease thanCMV isolation from urine or saliva. Culture methods for thedetection of CMV viremia are slow and hampered by thetoxicity of specimens to cell cultures. Compared with con-ventional cultures, the sensitivity of centrifugation culturesfor detection of CMV viremia is low (3, 11, 15).

Recently, the CMV antigenemia test has been found to bea sensitive and rapid method for the detection of CMVantigen in peripheral blood leukocytes, predominantly in thepolymorphonuclear leukocyte (PMNL) fraction. In addition,higher numbers of CMV antigen-positive leukocytes havebeen reported to correlate with clinical symptoms and theneed for treatment (4-6, 12-14). Monoclonal antibodies tothe CMV lower matrix protein pp65 have been used inpublished studies of the antigenemia assay and are criticalfor sensitive results (5, 8, 13, 14). These antibodies are nowavailable commercially and can be purchased alone (ClonabCMV; Biotest Diagnostic Corporation, Denville, N.J.) or

* Corresponding author.

as part of a CMV antigen staining kit (CMV-vue; IncstarCorporation).The CMV antigenemia test with Clonab CMV monoclonal

antibodies was introduced into our clinical laboratory inmid-1992 and is performed quantitatively. The results ob-tained from the first 496 samples examined by the CMVantigenemia test as well as both conventional and centrifu-gation culture methods are presented in this report.

MATERIALS AND METHODS

Patient specimens. Blood specimens collected in twoEDTA-treated tubes were submitted to the Yale-New HavenHospital Clinical Virology Laboratory for isolation of CMV.One tube was processed for conventional virus isolation inroller tubes and centrifugation culture in shell vials, and theother tube was processed for the CMV antigenemia assay.Samples were received from outpatients and hospitalizedpatients, from Yale-New Haven Hospital, and from otherhospitals and clinics in the New Haven area.CMV antigenemia assay. PMNLs were isolated from 3 to 5

ml of EDTA-treated venous blood by dextran sedimentation.The CMV antigen detection procedure of van der Bij et al.(12, 13) was followed initially and was modified subsequentlyaccording to Gerna et al. (5) and Boeckh et al. (1). Briefly,contaminating erythrocytes were lysed with 0.8% NH4C1.Leukocytes were washed twice in phosphate-buffered saline(PBS) and then the cells were counted in a hemacytometerand the concentration of the cells was adjusted to 106/ml. Atotal of 100 RI (100,000 cells) was applied by cytocentrifuga-

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tion (Cytospin from Shandon Incorp., Pittsburgh, Pa.) at 800rpm for 4 min onto uncoated Teflon-masked glass slides (twoslides per patient). Cells were air-dried and fixed withacetone or 5% paraformaldehyde. Paraformaldehyde-fixedcells were then permeabilized with 0.5% Nonidet P-40. Cellswere stained with 60 ,ul of Clonab CMV monoclonal anti-bodies (Biotest Diagnostic Corporation) and were incubatedat 37°C for 30 min. Cells were then washed twice in PBS for2 min each time. A total of 60 ,ul of either peroxidase-labelledgoat anti-mouse immunoglobulins (Biotest Diagnostic Cor-poration) or affinity-purified fluorescein isothiocyanate-la-belled sheep anti-mouse immunoglobulin G (FAb; OrganonTeknika-Cappel, Malvern, Pa.) was added, and the mixturewas allowed to incubate at 37°C for 30 min. Slides werewashed in PBS twice for 2 min each time. For immunoper-oxidase staining (IP), a substrate (3-amino-9-ethylcarbazole)was added and the slides were counterstained with hematox-ylin. The slides were mounted in glycerol and examined atx250 magnification. Positively stained leukocytes showedhomogeneous yellow-green nuclear fluorescence by immu-nofluorescence staining (IF) or a red-brown homogeneous orgranular nuclear staining by IP. Results were expressedquantitatively as the number of CMV antigen-positive cellsper 200,000 cells examined: fewer than 10 positive cells wasconsidered a low positive result, 10 to 49 positive cells wasconsidered an intermediate result, and 50 or more positivecells was considered a highly positive result (see reference 6,page 491).CMV culture. PMNLs were isolated from 3 to 5 ml of

EDTA-treated venous blood by dextran sedimentation. Thecell pellet was washed twice and was resuspended in 2 ml ofveal infusion broth containing 0.5% bovine serum albuminand antibiotics. For conventional culture, two roller tubescontaining confluent monolayers of MRC-5 cells (BioWhit-taker, Walkersville, Md.) were inoculated with 0.2 ml of cellsuspension per tube, and after 1 h of adsorption at 35°C,minimum essential medium with 2% fetal bovine serum wasadded. Cultures were then incubated at 35°C in a roller drumand were examined for cytopathic effects (CPEs) daily forthe first 7 days, every other day for the second week, andtwice a week for the third week. Culture medium wasreplaced 24 h after inoculation and weekly thereafter. Toxicor degenerating monolayers were passaged to fresh mono-layers as needed. For the centrifugation culture, one MRC-5and two mink lung cell monolayers (7) in shell vials wereinoculated with 0.3 ml of leukocyte suspension and werecentrifuged at 700 x g for 45 min. Monolayers were washedand refed with minimum essential medium-2% fetal bovineserum. After incubation for 1 to 2 days at 35°C, monolayerswere examined and were then fixed in acetone and stainedwith monoclonal antibody to CMV early antigen (CambridgeBiotech, Boston, Mass.). Monolayers were examined undera fluorescence microscope for positively stained nuclei.

RESULTS

Correlation of antigenemia with culture methods. FromJune 1992 to February 1993, 496 blood samples from 308patients were processed by all three methods and are thesubject of this report. Two hundred ninety-two samples(59%) were submitted from patients infected with humanimmunodeficiency virus (HIV), 142 (29%) were from solid-organ and 31 (6%) were from bone marrow transplantrecipients, and 31 (6%) were from other patients (includingnormal hosts with mononucleosis or hepatitis, cancer pa-tients, and infants) with suspected CMV infection. Of 496

TABLE 1. Comparison of three test methods for detection ofCMV in peripheral blood PMNLs

No. of specimens with con-CMV Centrifugation ventional culture result of:

antigenemia culture result Positive NegativeassayresultPoiie Ngtv

(n = 56) (n = 440)

+ + 21 3+ - 26 47

+ 2 1-- 7 389

blood samples processed (Table 1), 107 were positive by oneor more tests and 389 were negative by all methods. Of the107 positive samples, only 21 were positive by all three tests.Virus was isolated in conventional culture from 56 (11%) of496 samples tested. Of these 56, 23 (41%) were also positiveby centrifugation culture and 47 (84%) were positive forCMV antigen. An additional three samples negative byconventional culture were positive by both centrifugationculture and antigen assay, and one sample was positive bycentrifugation culture only. CMV antigenemia was detectedin 47 (11%) of the 436 samples negative by both culturemethods. Thus, of 107 samples positive by at least one of thetest methods, 56 (52%) were detected by conventional cul-ture, 27 (25%) were positive by centrifugation culture, and 97(91%) were detected by the CMV antigenemia assay. Thedifferences in sensitivity between the three tests for detec-tion of CMV infection were significant (P < 0.0001; chi-square test). If conventional culture is taken as the "goldstandard" and the samples positive by the CMV antigenemiaassay only are interpreted as false positives, the antigenemiaassay had a sensitivity of 84% and a specificity of 89%,whereas centrifugation culture had a sensitivity of 41% and aspecificity of 99%.

(i) Impact of ganciclovir treatment on culture results. Ofnote, 19 of 107 CMV-positive samples were obtained frompatients on ganciclovir; only 6 of these (32%) were positiveby culture methods. In contrast, 54 of 88 samples (61%) frompatients not on ganciclovir were culture positive.

(ii) Samples positive by culture methods only. Of the 10samples positive by culture methods only, analysis of anti-genemia test results revealed that 3 samples were noted tohave one or two questionable cells, one sample was noted tohave poor cellular morphology, and three samples were leftovernight before processing.

(iii) Samples positive by CMV antigenemia assay only. Atotal of 47 samples were positive by CMV antigenemia assayonly. For the majority of these (32, or 68%), fewer than 10positive cells were detected.To evaluate the specificity of these results, information

was obtained from the patients' laboratory and medicalrecords. Thirty-five of 43 patients had CMV infection docu-mented by isolation of CMV from other cultures (includingblood, bronchoalveolar lavage, and urine), the presence ofCMV antibody, or previous, current, or subsequent CMVdisease. Eleven patients had active CMV disease, and nineof these were receiving ganciclovir therapy. For eight pa-tients with HIV infection, no other samples were submittedfor testing.Samples from two culture-negative patients had >50 pos-

itive cells in the CMV antigenemia assay (patients 15 and 24;see Table 4) and are discussed below.

(iv) Correlation of test results with number ofCMV antigen-

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QUANTITATIVE CMV ANTIGENEMIA 2853

TABLE 2. Correlation of test results with number of CMVantigen-positive cells and day of onset of CPE

No. of Day CPE ob-

Test result antigen-positive served by con-cells/200,000 ventional cul-No. of PMNLs turesamples

Conven- Centrif- CMV anti-tional ugation genemia Mean Range Mean Rangeculture culture assay

21 + + + 193 1-1,683 7.6 4-1626 + - + 157 1-1,898 8.8 5-1647 - - + 12 1-1007 + - - 10.3 7-17

positive cells and time to CPE. When the number of antigen-positive cells and day to the onset of CPE was correlatedwith test results (Table 2), those samples positive by theCMV antigenemia assay and both culture methods had amean of 193 antigen-positive cells and 7.6 days to the onsetof CPE. Those samples positive by the CMV antigenemiaassay and conventional culture only had 157 antigen-positivecells and 8.8 days to detection of CPE. The differencesbetween these two groups were not statistically significant (P< 0.15; Wilcoxon test). Samples positive only by the CMVantigenemia assay had a mean of 12 antigen-positive cells,which was statistically different from 193 and 157 antigen-positive cells (P < 0.0001 and P < 0.05, respectively;Wilcoxon test). The time to CPE was 10.3 days for samplesin which CMV was detected by the conventional culturemethod only. Thus, there was a clear correlation betweenthe number of antigen-positive cells and the days to isolationof CMV in conventional cultures.Comparison of staining methods. Samples fixed in acetone

were examined by the IF and IP techniques; subsequently,samples fixed in paraformaldehyde were examined by IF andIP. Results are shown in Table 3. Of 52 samples fixed inacetone, 9 were positive by both IP and IF and 5 werepositive by IF only. Of 53 samples fixed in paraformalde-hyde, 7 were positive by both IP and IF and 2 were positiveby IP only. The differences in sensitivity between IP and IFwere not significant (Fisher exact test). All of the discrepantsamples had only one or two positive cells. In contrast to thereport by Gerna et al. (5), paraformaldehyde fixation ap-peared to diminish rather than enhance staining intensity inour hands. However, the morphologies of the positive cellswere much better than those obtained with acetone fixation,

TABLE 3. Comparison of IP and IF methods with twodifferent fixatives

No. of samples showing theFixative IP following result by IF:

resultPositive Negative Total

Acetone + 9 0 ga

-5 38 43Total 14a 38 52

Paraformaldehyde + 7 2" 9c0 44 44

Total 7c 46 53a The difference was not significant (P < 0.35; Fisher exact test).b Discrepant samples had only one or two positive cells.c The difference was not significant (P < 0.80; Fisher exact test).

resulting in an improved ability to distinguish specific stain-ing from artifacts. In agreement with the report by Gernaet al. (5), IF detected a higher mean number of positivecells than IP, whether by acetone (8.9 versus 6.9 cells) orparaformaldehyde (12.4 versus 8.4 cells) fixation. Thesedifferences were not significant (P < 0.55 and P < 0.15,respectively; paired t test).

Correlation of quantitative CMV antigenemia with clinicaldisease. Of 96 antigen-positive samples for which quantita-tive results were available, 49 (51%) had fewer than 10positive cells per 200,000 cells examined (low level), 26samples (27%) had 10 to 49 positive cells (intermediatelevel), and 21 samples (22%) had 50 or more positive cells(high level). The antigenemia levels of 25 patients whoseblood was submitted for testing within 2 weeks of developingnew CMV disease, who had received ganciclovir for lessthan 24 h at the time blood was sampled, and/or who hadhigh CMV antigenemia levels during the study period arepresented in Table 4.

(i) Low-level antigenemia. Patient 1 was negative by theCMV antigenemia assay and culture methods at the time thatCMV retinitis was diagnosed. He had received ganciclovirtreatment for less than 24 h, and in addition, laboratory notesindicated that the stained slide preparation contained lownumbers of cells. The low result for patient 2 is unexplained.Significantly, patients 3 and 4 were not immunosuppressed.Their first blood sample was not obtained until 2 weeks afterthe onset of symptoms, and the antigenemia result was thefirst indication that CMV was responsible for their illnesses.CMV was subsequently isolated from other sites from bothpatients, and CMV IgM antibody was also detected.

(ii) Intermediate-level antigenemia. Intermediate-level an-tigenemia was detected in three patients at the time ofdiagnosis of CMV disease. Patient 5 had an antigenemialevel of close to 50. The sample from patient 6 was heldovernight before processing. The sample from patient 7 hada very low leukocyte count, and the cell smear was noted tobe thin.

(iii) High-level antigenemia. During the study period, 21blood samples from 18 patients with antigen levels of 50 orgreater were processed by all three test methods. Additionalsamples were assayed for antigenemia only. Sixteen of the18 patients were documented to have CMV-related disease.Patient 15 was positive by the CMV antigenemia assay onlyand had two subsequent culture-negative, antigenemia-pos-itive samples over the ensuing 6 weeks before presentingwith visual loss and CMV retinitis. Two patients did nothave CMV disease. CMV was isolated from the bronchoal-veolar lavage fluid of patient 24, who had 100 CMV antigen-positive PMNLs. However, no CMV inclusions were notedin the transbronchial biopsy specimen and she was treatedfor Pneumocystis carinii pneumoniae alone, with resolutionof cough and pulmonary infiltrates. A subsequent bloodsample obtained 2 months later revealed only two CMVantigen-positive PMNLs. Ten months posttransplant, pa-tient 25 demonstrated a transient rise in CMV antigenemia,which peaked at 107 positive PMNLs. However, his clinicalsymptom was congestive heart failure that responded todigitalis and diuretics. His antigenemia resolved withouttreatment.

DISCUSSION

The CMV antigenemia test is a rapid and sensitive assay,and when performed quantitatively, it can help to identifyclinically significant viral infections. As opposed to the

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TABLE 4. Correlation of quantitative CMV antigenemia assay with culture methods and clinical disease

Antigenemia and Underlying disease Age (yr), Type of CMV No. of CMV Conventional Centrifuga- Ganciclovir

patient no. sexdiseaseUnderlyindisease A d antigen-positive culture tion cul- treatmentcells (peak) result ture result (duration)

Low-level antigenemia1 HIV positive 32, M Retinitis 0 - - Yes (<24 h)2 HIV positive 36, M Retinitis 2 + - No3 Crohn's disease 22, M Mononucleosis lb- No4 None 15, F Mononucleosis, 2b + - No

pneumoniaIntermediate-level antigenemia

5 HIV positive 32, F Retinitis 45 + + No6 HIV positive 41, M Retinitis 22 + - No7 HIV positive 24, F Retinitis 32 - - Yes (<24 h)

High-level antigenemia8 HIV positive 32, M Gastrointestinal 58 + + No

disease9 HIV positive 34, F Retinitis 50 + - No10 HIV positive 57, F Fevers, sweats, 91 + + No

retinitis11 HIV positive 53, M Retinitis 61 + - No12 HIV positive 41, M Gastrointestinal 53 + + No

disease13 HIV positive 39, M Retinitis 68 + + No14 HIV positive 33, M Pneumonia 51 + - No15 HIV positive 37, M Retinitis 60 - - No16 Cardiac bypass 58, M Hepatitis >100 - + No17 Heart transplant 51, F Pneumonia 185 + + Yes (<24 h)18 Bone marrow transplant 41, F Hepatitis 396 +C + No19 HIV positive 38, M Retinitis, neu- 601 +C + No

rologic dis-ease

20 HIV positive 54, M Weight loss, 680 + - Noweakness,neurologicdisease

21 HIV positive 35, M Retinitis 898 + - No22 HIV positive 34, M Fever, pneumo- 1,124 + + No

nia23 HIV positive 33, M Retinitis, poly- 1,898 + + Yesd (5 mo)

radiculopathy24 HIV positive 37, F None (PCP") 100 - - No25 Kidney transplant 47, M None (CHF) 107 + + No

a M, male; F, female.b Samples were obtained 2 weeks after the onset of clinical disease.c Cultures were not performed when peak antigen levels were detected, but were performed several days earlier, when antigen levels were 31 and 237 for

patients 18 and 19, respectively.d Developed CMV polyradiculopathy while receiving ganciclovir for retinitis.e PCP, P. carinii pneumonia; CMV was also isolated from bronchoalveolar lavage fluid, but no CMV inclusions were detected.f CHF, congestive heart failure.

polymerase chain reaction and nucleic acid hybridizationtechniques, the antigenemia test uses a methodology that isfamiliar to workers in routine diagnostic laboratories. Thereare two published reports of this assay used in studies donein clinical laboratories. The first study (9) used Clonab CMVantibodies and the method of van der Bij et al. (12, 13) andtested blood samples from 27 transplant recipients; however,no antigenemia was demonstrated. More than half of thesamples were held overnight before processing, and fixationof cells may have been suboptimal (14). The second study (3)used the CMV-vue kit on 280 blood specimens from solid-organ transplant recipients. The CMV antigenemia assaywas found to be significantly more sensitive than shell vialculture techniques. However, quantitation was not reported,and except for samples positive by the CMV antigenemiaassay only, a clinical correlation was not mentioned.Most published reports (3, 5, 6, 9, 12, 13) have focused on

solid-organ transplant recipients, with few exceptions (1, 4).Our patient population was predominantly infected withHIV and included solid-organ and bone marrow transplantrecipients, other immunosuppressed groups of people, in-fants, and immunocompetent patients as well. In our studygroup, the sensitivity of the CMV antigenemia assay farsurpassed that of the culture method. Overall, 11% of bloodspecimens were positive by conventional culture, and anadditional 11% of culture-negative samples were positive bythe CMV antigenemia assay. This is similar to the sensitivityreported by van der Bij et al. (13) and exceeds that reportedby Erice et al. (3). This difference may be explained by theCMV-vue kit procedure used in the latter study, whichspecifies examination of only 100,000 instead of 200,000 cellsper sample. In the present study, 32 (33%) of CMV antigen-emia assay-positive samples had only one or two positivecells. Many of these would have been missed if only half the

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QUANTITATIVE CMV ANTIGENEMIA 2855

number of cells had been examined. Although detection oflow-level antigenemia provided the first evidence of CMVinfection in several severely ill patients, most patients withlow-level positive antigenemia in our study were asymptom-atic individuals or patients on therapy, and thus, the anti-genemia was not clinically important.

In many cases, blood samples with <10 antigen-positivecells were culture negative, but some samples with interme-diate- to high-level antigenemia were also culture negative. Ithas been reported that a minimum of 4 x 106 leukocytesmust be cultured for sensitive results (2). In the presentstudy, culture methods were not quantitative, and thus,cultures were inoculated with varying amounts of cells.Furthermore, cell cultures vary in their susceptibilities tovirus, and the toxicities of specimens to cell monolayers canimpair sensitivity.

In the present study, the usefulness of quantitation and itscorrelation with clinical disease were also evaluated. Ingeneral, high-level antigenemia correlated with clinical dis-ease, whereas low-level antigenemia was usually asymptom-atic. However, it was clear, as reported previously (12, 13),that exceptions occur. Antigenemia levels were guidelinesonly; low or negative antigenemia did not exclude thepossibility of CMV disease. Whether low-level antigenemiain the face of clinical disease is due to technical error, timedelay in processing, low-level antigen expression, or lack ofreactivity of the monoclonal antibody in some cases is notknown. The timing of sample collection in patients withdisease, especially immunocompetent patients, is critical.As the immune response develops, antigenemia ceases (14).In severely immunosuppressed patients, antigenemia usuallypersists for a longer period because of a delayed or inade-quate immune response. Quantitative antigenemia was alsouseful in monitoring the response to antiviral therapy in anumber of patients (data not shown), as reported previously(1, 4, 6).

Despite the inconvenience of paraformaldehyde fixation,the enhanced cellular morphology it provided helped thespecific identification of CMV-infected cells. This is espe-cially important in clinical laboratories, where many individ-uals may be responsible for reading the slides. IF providedtime and cost savings (reagent cost per test, $18 for IF versus$21 for IP) and is widely used in clinical virology laborato-ries. IP, on the other hand, was advantageous for training ofpersonnel, since slides are permanent and can be readilystored and reexamined. The difference in the sensitivities ofIF and IP was not significant; therefore, the choice offixative and staining methods can be made on the basis of thepreference of the laboratory.

In the present study, the centrifugation culture detectedonly 41% of CMV-positive samples detected by conven-tional culture, despite the use of three shell vials (10) whichincluded mink lung monolayers to circumvent toxicity prob-lems (7). Although some shell vials were unreadable becauseof toxicity, at least two shell vials were examined for allspecimens included in the study. Others have also reporteda suboptimal sensitivity of the shell vial assay for bloodspecimens. In the report by Erice et al. (3), the shell vialassay detected 69% of culture-positive blood samples. Ra-bella and Drew (11) reported that the shell vial assaydetected CMV in 6 of 12 culture-positive blood samples aswell as in 3 additional culture-negative blood samples (11). Insome studies in which the shell vial assay performed well,the culture method used for comparison consisted of oneroller tube kept for 2 weeks (10), whereas the use of two

roller tubes observed for 3 to 4 weeks is routine practice inmost diagnostic laboratories.

Several practical problems in routinely performing quan-titation in the clinical laboratory were also recognized duringthe study period. Samples may arrive too late for same-dayprocessing. When the time to processing is delayed, thenumber of antigen-positive cells can drop significantly.Thus, performing quantitation on these samples may givefalse security that antigen levels are falling. In addition,collection times are often not recorded; thus, the time toprocessing cannot be accurately determined.The CMV antigenemia assay is a labor-intensive test.

Substantial technical time is spent counting cells and adjust-ing cell concentrations prior to cytocentrifugation. For ac-curate quantitation, reading of stained slides requires ap-proximately 5 min for a negative slide and 10 min for apositive slide. Alternatively, slides can be read and resultscan be reported qualitatively, as positive or negative (3), asis the routine for centrifugation cultures. In addition, report-ing of quantitative antigenemia results may not be appropri-ate unless sample processing has been accomplished withinthe accepted time frame.

In order to accommodate the addition of this test to thelaboratory, the elimination of other CMV tests that weresuperfluous or of dubious clinical utility was strongly recom-mended to clinicians. Furthermore, once personnel weretrained and test results were validated, antigenemia levelsalone without culture confirmation were determined forselected specimens.

In our experience, despite the significant investment intime and resources, the quantitative CMV antigenemia assayhas proved to be a very valuable addition to the clinicallaboratory for the rapid diagnosis of CMV in blood speci-mens and is of benefit to patient management.

ACKNOWLEDGMENTS

We thank Sandra Cohen, Karyn Huber, Lisa Langan, CarolynFalconer, and Joan Wlochowski for excellent technical assistance;Helena Brett-Smith for providing clinical information on her pa-tients; Ruth Cooper and Ruth Ferro for helping to compile the data;and Emmanuel Lerner for providing statistical analysis.

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