Leukocyte immunophenotyping by flow cytometry in a multisite study: Standardization, quality control, and normal values in the Transfusion Safety Study

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

55,

187-220 (1998)

Leukocyte lmmunophenotyping by Flow Cytometry in a Multisite Study: Standardization, Quality Control, and Normal Values in the Transfusion Safety Study’ JOHN W. PARKER,* BERNARD ADELSBERG,’ STANLEY P. AZEN,~ DONNA BOONE,~ MARY ANN FLETCHER,* GEORGEF. GJERSET,~ JOSEPHHASSETT,’ JOSEPHKAPLAN,~ JOYCE C. NILAND,~ TAMARA ODOM-MARYON,~ EVA A. OPERSKALSKI,~HARRY PRINCE,* DIANE SCOTT,~ DANIEL P. STITES,~ JAMES W. MOSLEY,~ AND THE TRANSFUSION SAFETY STUDY GROUPS University of Southern California School of Medicine, Los Angeles, California 90032 and other participating institutions (see footnote authorship) The Transfusion Safety Study (TSS) is a multicenter, cooperative investigation of factors that may determine the occurrence and modify the expression of transfusiontransmitted infections. A flow cytometry laboratory was established in each of the six participating centers in order to avoid alterations in cell phenotypes which may be caused by shipping delays, temperature changes, and handling. As a consequence, in order to assure compatibility of results, stringent standardization, quality control, and proficiency testing procedures were developed. This paper documents (i) the effect of time from phlebotomy to specimen staining and then to analysis for the antibodies used in the study; (ii) the effects of variations in light scatter cursor location for certain antibodies; (iii) a quality control program and data management and analysis system, each specifically designed for the study; and (iv) presents extensive data on age- and sex-related reference (normal) ranges for the several individual and paired monoclonal antibodies used in the study. Problems encountered, including obtaining reliable absolute lymphocyte counts, interference by nucleated erythrocytes, and sources of variability in results, are discussed. This study is meant to serve as a reference for future TSS publications 0 1990 Academic Press. Inc.

INTRODUCTION

The Transfusion

Safety Study (TSS) is a multicenter,

cooperative

investigation

’ Supported by contracts No. NOl-HB-7003 and NOl-HB-9-7074 of the National Heart, Lung. and Blood Institute. 2 Immunology Working Group, Transfusion Safety Study. 3 Coordinating Center, Transfusion Safety Study. 4 Los Angeles: University of Southern California: J. W. Mosley, S. P. Azen, D. Boone, D. J. Bregman, M. F. Dougherty, J. Gaiennie, V. M. Edwards, C. S. Johnson, J. C. Niland, T. OdomMayron, E. A. Operskalski, J. W. Parker. D. R. Powars, D. S. Scott, M. Stuart, and E. TaylorMunoz; American Red Cross Blood Services: S. H. Kleinman and H. Prince; Cedars-Sinai Medical Center: C. Hyman; Huntington Memorial Hospital Hospital: S. L. Dietrich. Detroit: Wayne State University: J. Lusher, V. Cosgrove, J. Kaplan, and S. Samaik. Miami: University of Miami: E. R. Schiff, M. de Medina, M. A. Fletcher, E. C. Y. Lian, C. Pegelow, K. R. Reddy, J. D. Temple, and S. Toledano; American Red Cross Blood Services: B. Lenes and P. A. Tomasulo. New York: Mount Sinai Medical Center: L. M. Aledort, B. Adelsberg. T. C. Chalmers, J. Hassett, H. Sacks, and P. 187 0090-1229/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of factors that determine the occurrence and modify the expression of transfusiontransmitted infections. It has a broad concern with the immunologic and clinical consequences of the administration of blood and blood products. Its particular focus is on infection with the human immunodeficiency virus type I (HIV-l). but the effects of infection with other viruses are also considered. This paper describes our effort to assure that measurements from Row cytometry laboratories in six cities in the United States are comparable. It describes (i) the effect of time from phlebotomy to specimen staining and then to analysis for the antibody pairs used in the study; (ii) the effects of variations in light scatter cursor location for certain antibody pairs; and (iii) a quality control program and data management and analysis system, each specifically designed for the study. We also present extensive data on age- and sex-related reference (normal) ranges for several individual and paired monoclonal antibodies. Problems encountered, including the difficulty in obtaining reliable absolute lymphocyte counts, interference by nucleated erythrocytes, and sources of variability in results, are discussed. This study is meant to serve as a reference for future TSS publications. In 1983, the National Heart, Lung, and Blood Institute (NHLBI) developed a request for proposals (RFP) to study the immunologic and clinical consequences of the transfusion of blood components and plasma products. The epidemiologic pattern of the acquired immunodeficiency syndrome (AIDS) was consistent with that of a new infectious agent transmitted both sexually and by transfusion. At that time, however, immunosuppression due to known viruses (I) and other factors had not been excluded as explanations for at least some of the immunological alterations associated with AIDS. In addition, it was important to better define the immunologic consequences of transfusion itself (2-5). Because the infectious etiology of AIDS was still in question in 1983 and no specific diagnostic laboratory test was available, the NHLBI RFP stipulated that the prevalence of immunologic changes among transfused and untransfused persons in geographic areas of high and low prevalence of AIDS be compared. The intent was to determine whether there was an excess frequency of lymphocyte abnormalities that could serve as an index of “pre-AIDS” in areas of high AIDS prevalence. The TSS investigators responded with a plan meeting the RFP’s stipulations and were awarded a contract to carry out appropriate investigations. By the time that the first study subjects were enrolled in August 1985, HIV-1 had been accepted as the etiologic agent of AIDS. As a consequence of this advance, the major purpose of the immunologic studies shifted from epidemio-

Wiesen; Cornell University Medical Center: M. Hilgartner: Greater New York Blood Program: J Pindyck, C. D. Stevens, and P. E. Taylor. San Francisco: University of California at San Francisco: E. Donegan, C. Casavant, M. A. Koerper. K. Miller, D. P. Stites, P. T. C. Y. Toy, and G. N. Vyas; Irwin Memorial Blood Bank: H. A. Perkins and M. P. Busch; Alta Bates Hospital: R. A. Johnson and B. H. Lewis. Seattle: Puget Sound Blood Center: R. B. Counts and G. F. Gjerset. National Institutes of Health: NHLBI: C. G. Hollingsworth, G. J. Nemo. A. Chemoff, and J. Houk. Protocol and Data Monitoring Committee: H. J. Alter. G. J. Grady, P. V. Holland, J. Osborne. N. R. Rose, L.. B. Seeff. and J. Wittes.

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logic definition of populations with immunologic changes to documenting the consequences of HIV-l infection, particularly as they may be modified by infection with other transfusion-transmitted viruses and by allogeneic exposures. Transfusion

Safety Study Subjects

Subjects enrolled in the study include (i) persons with congenital clotting disorders or congenital anemias given blood components and/or plasma products from 1979 until the time of study enrollment; (ii) blood donors found to be antiHIV-l positive; and (iii) recipients of blood components from anti-HIV-l positive donors. Comparison groups were selected not only to represent immunologic norms for the general population, but also for those persons with comparable demographic characteristics (sex, age, ethnicity, socioeconomic status) and, in some instances, a similar disease background. They include (i) anti-HIV-l negative persons with congenital clotting disorders or congenital anemia, both treated and untreated by transfusion; (ii) anti-HIV-l negative blood donors matched for sex, age, and area of residence; (iii) recipients of blood from an anti-HIV-l negative donor; (iv) sexual contacts of recipients and persons with congenital hematologic disorders; and (v) nonsexual household contacts of subjects with congenital hematologic disorders. General

Study Procedures

Study and comparison groups are seen at intervals of 3 to 6 months (depending upon clinical status) for the following purposes: (i) To monitor transmission of HIV-l and other viruses; (ii) to follow the natural course of HIV-l infection and factors that may influence it; (iii) to observe interactions between HIV-l and other viruses that may change the pathogenicity of HIV-l; (iv) to determine if blood components and plasma products themselves cause immunologic change and affect the course of HIV-l infection; (v) to determine whether any immunophenotypic cell markers can be associated with resistance to HIV-l infection among those exposed but not infected; (vi) to assess immune changes associated with other viruses in the absence of HIV-l infection; and (vii) to establish and maintain a serum and leukocyte repository for future studies. At each visit, blood samples are obtained for a complete blood count (CBC), immunologic phenotyping, assay of serum alanine aminotransferase activity, and serological and viral testing. During the period from August 1985 to September 1988, a total of 3057 subjects had been enrolled in the study and 10,967 samples have been analyzed by flow cytometry at the time of this report (September 1988). Study Organization

The Coordinating Center, located at the University of Southern California, consists of the Director’s Office, the Biostatistics Office, and the Coordinating Center Laboratory. The Coordinating Center Laboratory receives specimens from the six clinical centers, carries out selected serologic tests, and distributes aliquots to reference laboratories for additional serologic and virologic testing. Clinical centers are located in New York, Miami, San Francisco, and Los Angeles, four cities selected because of their very high prevalence of AIDS, and

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in Detroit and Seattle, two cities with a low prevalence. The two latter cities were chosen because of populations of patients with congenital hematologic disorders already being followed. The RFP stated that a central facility should be used for as many of the laboratory procedures as feasible. This stipulation was obviously intended to reduce the variability that inevitably accompanies work in several different laboratories. Changes in immunophenotypic populations, however, have been reported by some investigators to occur with variations in temperature and prolonged storage (6-9). Therefore, we suspected that the delay in specimen handling and uncontrollable conditions during shipping would adversely affect the reliability of testing, even though others have reported no effect of low temperature on subsets within 24 hr (10). Furthermore, it was correctly anticipated that many specimens collected in the field could not be delivered even to the local clinical center laboratory until the morning after phlebotomy. Accordingly, individual flow cytometry laboratories were established as a part of each clinical center. The Immunological Reagents Laboratory (IRL) has responsibility for the acquisition, preparation, and distribution of quality control (QC) specimens. The Immunological Standardization Laboratory (ISL) develops and monitors laboratory protocols, trains immunology technicians in conjunction with technical personnel from Coulter Diagnostics, reviews laboratory data, and monitors QC results. Through June 1988, the IRL was located at the American Red Cross Regional Blood Services in Los Angeles, and the ISL at the University of Southern California School of Medicine. Since July 1988, both functions have been carried out at the Puget Sound Blood Center. The Immunology Working Group (IWG) consists of an immunologist from each clinical center and the Director of the IRL. It meets from two to four times annually to consider procedures, problems, results, and plans. Coordination is further increased by monthly telephone conference calls. The IWG, in response to the RFP, proposed to measure both the function and the phenotype of immune system cells from subjects. In the design of the study at present, however, immunologic characterization is limited to immunophenotyping by flow cytometry for three reasons: (i) Available data suggest that the time from phlebotomy to functional analysis should be no more than a few hours, a major limitation in collecting large numbers of specimens; (ii) the problems of standardization seemed likely to be even larger than those for flow cytometry; and (iii) the estimated cost of implementing functional tests under these circumstances was beyond the available resources. The IWG initially selected a panel of antibodies for phenotyping and established standardization and quality control procedures. Major steps in achieving standardization have included the following components: (i) use of the same model of flow cytometer; (ii) a common panel of monoclonal antibodies; (iii) centralized training of laboratory personnel; (iv) standard protocols for specimen handling, cell processing, staining, and instrument operation: (v) standardized calibration of the flow cytometers; (vi) a quality control program involving regularly scheduled quality control testing for interlaboratory agreement and intralaboratory reproducibility with replicate samples; and (vii) transmission of data to a centralized data bank for uniform processing, reporting, and analysis. Decisions were based

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on the experience of the investigators, the literature, and many small pilot experiments. Changes in testing protocols were made until subjects were enrolled in the study. Since then, the basic protocols have remained in place with only minor changes. The IWG now reviews and interprets data, monitors the quality control program, and guides the overall direction of the immunology component of the study. The following section describes the laboratory procedures in place as of September 1988. A brief description of early results of the study has been presented (11). LABORATORY Standardization

of Flow

METHODS

AND RESULTS

Cytometers

To reduce interlaboratory variability, the same model of flow cytometer was purchased for each laboratory. Identical single laser instruments (EPICS C, Coulter Instruments Laboratory, Hialeah, FL) were installed at each clinical center. The flow cytometers, which discriminate forward and right angle light scatter and two colors, are configured with identical filters and lenses. Standard procedures were established for instrument start-up, optimization of laser output, and optical alignment. Fullbright Grade 1 beads (EPICS Division of Coulter Immunology, Hialeah, FL) are used for calibration, maintaining peak laser output and coefficients of variation (CV) for light scatter and fluorescence of Fullbright beads within stated limits. This step enhances the consistency of measurements, as judged by the CV, and provides an overall measure of instrument function. The relative channel numbers, corresponding to size and fluorescence, provide a measure of day-to-day consistency of laser output, optics, fluidics, and photomultiplier tube (PMT) function. The stability of the instrument is further monitored by a daily recording of the PMT gains and voltages and laser power. Monoclonal

Antibodies

The same monoclonal antibodies, purchased by the Coordinating Center from Coulter Immunology (Hialeah. FL), are used in each laboratory to quantitate the specific mononuclear leukocyte populations and subpopulations listed in Table 1. One antibody in each pair is conjugated with fluorescein isothiocyanate (FITC) and the other with a phycoerythrin derivative (RD-1, Coulter). Antibody-labeled cells are quantitated by the flow cytometer and the results are recorded as both percentages and absolute numbers on Form B4 (Fig. 1). The proportions of the subsets of the major populations are CD2 + Tal + /CD2, CD4 + CD29 + /CD4, CD4 + CD45-RA + ICD4, HLA-DR + CD8 + KD8, CD20 + CD21 + KD20, HLA-DR + CD-14 + /CD14, and CD2 + CD14 + ICD2. Sample Preparation

Standard sample preparation and staining procedures are used by the six laboratories. Monoclonal antibodies (Coulter Immunology) are prepared and stored according to the manufacturer’s instructions. At the time of use each antibody pair is diluted to 200 t~,l in a 13 x loo-mm polystyrene tube. One hundred microliters of heparinized whole blood is added; the washing solution and blood

PARKER ET Al ,

192

TABLE PHENOTYPIC

DEFINITIONS

Phenotypes T-lymphocyte related CD2 + CD2 + Tal + CD2+CDllb+

OF LYMPHOCYTE,

Antibodies”

+ CD45RA + + CD45RA + + CD29+ HLA-DR +

Cytotoxicisuppressor CD8 + CD8 + HLA-DR + CD8 + HLA-DR-

Tll+ Tll-tTal+ TII+MOI+

T8+12+ T8 + 12-

Killer/natural killer cell related Tll +NKH-I+ CD2 + CD56 +

CD8 + CD56 + CD8CD56 -

TS+NKH-I+ TX-NKHI-

B-lymphocyte related CD20 t CD20 + CD2 I+ CD2O+CD21Monocyte related CDllb+ CD14+ CD14+12+

Bl+ Bl+B2+ Bl +B2-

MOl+ M02+ M02+12+

KILLER

CEL.L

SUBSETS

Total helper/inducer T cells (35-38) Induces mitogen stimulated B cell immunoglobulin synthesis and proliferation (39) Similar to CD4 t CD29 + population (391 Induces CD8 + suppressor cell functions (40) Similar to CD4 + CD45RA + population (40) Helper T cells. with HLA-DR expression

T-lymphocyte related T8+

Tl I-NKH-I

NATURAL

Total ‘1‘ cells and a subset of NK cells (3 1. 321 Activated T cells (331 Subset of cytolytic T cells, NKH- I. and/or CD8 i- (34)

T4 + 2H4T4+2H4+ T4 + 4B4T4+12+

CD2-CD56 +

AND

Associated subpopulations or functions

Helper/inducer T-lymphocyte related CD4 + T4+ CD4 + CD29 + T4+484+ CD4 CD4 CD4 CD4

I

MONOCYTE,

+

Total cytotoxicisuppressor ‘1‘ cells (41-431 and a subset of NK cells Predominant suppressor function (44-46) Predominant cytotoxic function @U-t61 Subset of large granular lymphocytes, with lymphokine activated killer function (47. 4X) Subset of large granular lymphocytes. with most effective NK activity (49-51) Large granular lymphocytes (49-511 Natural killer cytolytic cells (49-5 I) Total B cells (52) B cells of intermediate differentiation (5 1) or resting B cells (53, 541 Differentiated B lymphocytes with plasmacyttc features or pre-B cells (541 Monocytes, granulocytes, null cells. some T cells (55, 56) Monocytes and macrophages (55. 561 Most normal circulating monocytes (55, 561

” All monoclonal antibodies were obtained from Coulter Immunology (Hialeah, FL)

are mixed (vortexed) and incubated at 4°C for 45 min. Cells are washed twice with 4 ml PBS (400g for 3 mm). After aspirating the supernatants and vigorous vortexing, 1 ml of Coulter Immuno-Lyse reagent is added to each tube and immediately mixed with the cells. After 30 to 120 set at ambient temperature, 250 t.~l of Coulter fixative solution is added with immediate vortexing. Following addition of the fixative, cells are washed twice (400g for 3 min). The final supernatant is removed, the pellet resuspended in 1 ml of PBS, and the mixture is stored at 4°C until analysis.

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Counts

Because heparinized blood specimens are not optimally counted with automated hematology cell counters, a separate blood sample collected in EDTA is submitted with each heparinized specimen. These EDTA samples are used for total leukocyte counts (WBC) and differential cell counts. Because the same model of automatic hematology cell counter was not made available to each flow cytometry laboratory, the absolute lymphocyte values reported here utilize the unstandardized leukocyte (WBC) and differential counts obtained at the six clinical centers. WBC counts are all from automated hematology cell counters, as are differential counts at four centers. However, the instruments that have been used vary from center to center and in one instance within the center. The extent of interlaboratory and intralaboratory variation in leukocyte/lymphocyte counts has been monitored as part of the quality control program described below and is seen to introduce a major source of variation in absolute subpopulation cell counts. Only percentages are presented here. Specimen Management

Whole blood rather than separated mononuclear cells is used in flow cytometric analysis because the volume of blood obtained from pediatric patients is often small and there is less potential for the loss of subsets of mononuclear cells that sometimes occurs with lymphocyte separation procedures. Prior to beginning analyses for the study, it was established that whole blood could be held in heparin for at least 24 hr prior to staining and fixing without significantly altering results (12). It was also found that for most surface markers, stained and fixed cells could be stored for up to 72 hr at 4°C prior to flow cytometer analysis without apparent deterioration (12). However, during this storage period the light scatter properties of the samples change so that discrimination of lymphocytes, monocytes, and granulocytes sometimes becomes difftcult. It was initially planned that cells would be stained and fixed within 24 hr of phlebotomy and that flow cytometry would be performed within an additional 24 hr. This did not prove to be practical, however, for a significant minority of specimens at some centers: 60% were stained and fixed within 12 hr, 95% within 24 hr, and 99% within 30 hr. Similarly, specimens analyzed within 12, 24, and 30 hr after staining and fixing were 60, 80, and 96%, respectively. Therefore, the comparability of values was determined for varying intervals from phlebotomy to staining and fixing and from staining and fixing to flow cytometry as described below. Effect of Time Lapse between

Phlebotomy

and Analysis

In addition to the study by Fletcher et al. (12), at one clinical center 23 whole blood samples from healthy donors were divided into two aliquots to evaluate the effect of storage at ambient temperature on the phenotyping results. The first aliquot of whole blood was stained and fixed from 4 to 6 hr after phlebotomy. The second aliquot was stained and fixed from 22 to 27 hr after the time of phlebotomy. Flow cytometry analysis of all stained and fixed samples was performed within 6 hr after staining and fixing.

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PARKER

ET AL.

At the same clinical center, 19 stained and fixed samples were used to examine the effect of time from staining and fixing to flow cytometry analysis. These samples were all stained and fixed within 4 hr of phlebotomy. Two aliquots were prepared, the lirst analyzed by flow cytometry between 1 and 4 hr after staining and fixing and the second between 24 and 29 hr after staining and fixing. For each antibody the differences between aliquot pairs were tested for significance using the Wilcoxon signed rank test. For all tests of significance we used a Bonferroi adjusted (Ylevel of 0.002 ( = 0.05133 immune parameters), to maintain an overall x = 0.05. For the two aliquots of whole blood, only CD2 + CD1 1b + (I,, lymphocytes) and CD2+CDIlb+/CD2 (L) were significantly affected by the time lapse between phlebotomy and staining and fixing (P < 0.002). For the two time periods between staining and fixing and analysis, only CD14+HLA-DR+ (L) and CDl4+ HLA-DR+ (M, monocytes) were significantly affected. Based on these results, the decision was made to exclude from data analysis samples not stained and fixed within 30 hr postphlebotomy or not cytometrically analyzed within 30 hr of staining and fixing. Technician

Training

The initial training class in instrument operation and sample preparation was conducted at the Coulter Laboratories in Hialeah, Florida, for the technicians and/or immunologist from each clinical center. A subsequent training session for new cytometry operators was presented at the ISL in Los Angeles in conjunction with Coulter personnel. In addition, review sessions are held as needed to determine whether or not standard protocols are being followed uniformly. Deviations from protocol are identified and corrected at these meetings. Procedures

Manual

To ensure standardization of the protocol, the IWC has developed a procedures manual, which is maintained by the ISL. Each laboratory has named a key technician who is responsible for updating the procedures manual. Questions and answers regarding protocols are communicated among the technicians using electronic mail (TELEMAIL, GTE-TELENET, Vienna. VA). Alterations in laboratory procedures since institution of patient follow-up have been restricted to those which add to or help clarify existing procedures. If a change is suggested by an immunologist or technician, a draft of the proposed change is prepared and circulated by the ISL and reviewed during the monthly conference call. If the change is approved, a “protocol change sheet” is circulated and inserted into the procedures manual. Flow

Cytometric

Analysis

For each sample of blood, a forward vs log 90” light scattergram (LS) delineating the major leukocyte populations is obtained, as well as a histogram showing the distributions of these populations on the log 90” LS axis. An electronic window, designated bit map (BM), is used to “gate” the cell populations of interest. Bit maps are set for lymphocytes only and for lymphocytes plus monocytes. For each pair of monoclonal antibodies, a bivariate scatterplot is produced by the Quad Stat program (Coulter). This plot shows the distribution and number of

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cells recognized by each antibody in each of the four quadrants. Negative (nonstained) cells are defined by analysis of cells exposed to appropriate RD-1 and FITC-conjugated mouse immunoglobulin isotypes. To delineate the negative population, horizontal and vertical cursors place at least 95% of cells in the left lower quadrant (LLQ, Q3). The percentages of stained cells are recorded using the fixed settings established by the isotype controls, except for antibody pairs in which operator judgment is used to set cursors in addition to the fixed settings. Results are recorded by bit map and quadrant. For three of the antibody pairs (CD2/Tal, CD29/CD4, and CD45RA/CD4), the separation of cells into quadrants by settings dictated by the isotype controls frequently bisects populations that span two quadrants. For these three pairs, cytometer operators are permitted to reset the cursors at their discretion to optimally identify these populations. Both initial and reset values were recorded during the first 2 years of the study and were entered into the data base. However, upon statistical analysis (see below), reset values for CD2/Tal and CD45RAKD4 were found to be no different then those for isotype control-dictated settings, whereas CD29/CD4 values were different so that reset values for this pair are still used (Fig. 2). These results were confirmed in the study described below. The data set includes a range of 14 to 16 pairs of results: isotype control, 9 pairs in bit map 1 (lymphocytes), 3 pairs in bit map 2 (lymphocytes and monocytes), and one to three optional reset values. Effect of Resetting

Cursors

Interlaboratory quality control data, from the three laboratories in which technicians frequently recorded reset values, were analyzed to compare the results of resetting cursors using three paired antibodies: Tal and CD2, CD4 and CD29, CD4 and CD45RA. Because the spread of these subsets across isotype-based reset cursor settings was variable, the decision to reset the cursor positions was optional and was based on operator judgment. Consequently, the cursor positions for the same samples were not always reset in each laboratory. Based on the results described above, samples were excluded from this analysis if the time lapse from phlebotomy to staining or staining to flow cytometric analysis was greater than 30 hr. Differences between the initial and reset values from the interlaboratory quality control samples obtained from the three immunology laboratories were calculated for each laboratory. Significance was determined by the Wilcoxon signed rank test using a Bonferroi adjusted (Y level of 0.006 (= 0.05/9 immune parameters examined). For laboratory 6 (whole blood samples), the initial and reset values were significantly different for CD2 +Tal+ and CD2+Tal+/CD2 (P < 0.006). For laboratory 5 (whole blood and stained/fixed samples) and laboratory 6 (whole blood samples only), the initial and reset values for CD4 + CD29 + were significantly different. For all three laboratories (whole blood samples only for laboratory 6), the initial and reset values for CD4 + CD29 + /CD4 were significantly different (P < 0.006). Resetting the cursor for CD4 paired with CD29 (Fig. 2) decreased the amount of variability of the measure as shown by the smaller interquartile range. In addition, cursor resetting for this pair produced better agreement among laboratories. For CD2 paired with Tal, better agreement among laboratories was

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observed using the reset data; however. significance was not observed for the stained/fixed data, nor was this trend observed consistently for all the laboratories. On the basis of these results it was decided to report the reset values only for CD4 paired with CD29. The initial, fixed cursor settings were concluded to be preferable for the CD2/Tal and CD4KD45RA pairs. Data Management

and Storage

Bit maps, percentages for each quadrant, and histograms for each pair of antibodies from each subject and quality control testing specimen are recorded at

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each immunology laboratory on g-in. floppy disks. The coordinates of the points of the scattergrams and the percentages for each quadrant for each blood sample are stored for subsequent analysis on SYi-in. floppy disks. Data management and analysis are centralized in the Biostatistics Office (BSO). Summary data, consisting of the subject identification number and the percentage of cells staining in each quadrant, are transcribed by the laboratory technician onto a data entry form. These data are entered into an IBM personal computer by a data manager at each clinical center, using the SMART data management system (Innovative Software, Overland Park, KS) and are transmitted over telephone lines to the BSO. Error checking occurs both at the clinical centers and at the BSO. Data are ultimately merged with clinical and laboratory data obtained from other sources (13). Problem Codes In order to identify problems which might affect the results of the flow cytometric analyses, a set of problem codes was developed to be entered on the form recording the results of the flow cytometry (Form B4, Fig 1). These problem codes are used either to exclude the data from statistical analysis, in the case of “severe” problem codes, or to simply flag the results as potential problems which may help explain outlying values. It would also be possible to exclude any type of problem code from analysis in the future, should this be indicated. Table 2 describes each problem code and indicates whether it involves the entire sample or is specific to one tube, in addition to giving the assigned severity level. In general, criteria leading to automatic rejection of a sample were those which definitely invalidated the results of the flow cytometry and which are not reliant upon subjective judgement of the technician. Data Analysis Results presented in this paper do not include all phenotypic results recorded, but rather only those cell subsets for which functions have been reported. For all of the analyses performed, data were excluded if (i) the sum of the percentages in the four quadrants was less than 99% or more than lOl%, indicating a technical error or (ii) a rejection problem code was recorded, indicating that the results in question were due to a technical error. Because the data were generally not normally distributed, nonparametric descriptive statistics (median and the 5th and 95th percentiles) were chosen. Wilcoxon rank sum tests were used to test for differences between two independent groups. Wilcoxon signed rank tests were used to test for differences between paired samples. Kruskal-Wallis analysis of variance was used to test for differences among more than two groups. For all comparisons, significance was determined using a Bonferroni adjustment of the (Ylevel, based on the number of variables examined (i.e., a = 0.05/k, where k is the number of immune parameters examined). Interlaboratory Quality Control Interlaboratory variability is monitored by quality control testing using whole blood and stained and fixed samples from healthy blood donors. Samples are

200

PARKER

TABLE

PROBLEM Problem code

CODES USED TO EXCLUDE

AL.

ET

2

OR FLAG

FLOWCYTOMETRY Level

Description

Automatic rejection criteria 01 Specimen clotted’ 02 Specimen held at 4°C 03 Specimen received at >2S”C 04 Quantity received is ~200 p,l 05 Lab accident/error’ Specimen problems 10 Less than 2000 cells analyzed’ 11 Insufficient cells’ 12 Unexpectedly high fluorescence subtraction required* Cytogram problems 20 Cytogram very poor; cannot set bitmaps 21 Cytogram poor; two distinct cell populations not seen 22 Cytogram poor; FALS low. lymphocytes touching X-axis 23 Divided lymphocyte population 24 Distinct lymphocyte population not seen Background problems 30 High background in both bitmaps; X and Y cursors in HI :a 20 to get *95% in Q3 and X and Y cursors in H2 > 25 to get 390% in Q3 31 High background in all tubes” 32 High background in bitmap No. 1 (see PC 30) 33 High background in bitmap No. 2 (see PC 30) 34 High fluorescent noise; green channel 63-64 > 10% 35 High fluorescent noise; green channel 63-64 > S-10% 36 Extension of Q3 population beyond negative cursor setting,’ 31 Bleeding from Ql into Q2 population Specimen analysis problems 40 Antibody pattern unusual”~’ 41 Incomplete hemolysis; RBC pellet in tube 42 Definite positive population in Q2 for Tube 48 tCM/CDXl Other problems 50 Flow cytometer malfunction” 51 NRBC’s presen? 52 Tube mislabeled on floppy disk 98 Specimen checked: no problems found 99 Other-Include comment

DATA or

mvolvement”

Severiti level”

Sample Sample Sample Sample Sample. tube

Reject Reject Keject Reject

Sample Sample, tube Sample

a&Spt Reject .Accept

Sample. tube Sample. tube Sample Sample Sample

Accept Accept .4ccept Accept

Tube Sample Tube Tube Tube Tube Tube Tube

Accep1 Accept .4ccept Accept Reject hccepf .4ccept .4ccept

Tube Tube Tube

.kUZpt Keject ?iCCCPi

hIlplt2

kJKl

Tube Tube Sample Sample, tube

Reject

K~ZJCXI

ReJecl

tkCept

Accepl Accept

” “Sample” indicates that the problem affects the entire blood sample. “Tube” indicate5 that the problem affect!, only the specific tube. ’ “Reject” indicates a severe problem. which is generally unrelated to technician judgement: resulting data arc invalid and should be excluded from all analyses. “Accept” indicates a less severe problem, and which is generally reliant upon technician judgement; data may be included in analyses. ’ To check for clots, place two applicator sticks into the blood specimen and gently move the sticks around the entire surface of the container (remain in contact with the glass). Remove the sticks from the container. If there is clotted material attached to the sticks, do not process the specimen for flow cytometry. If you see only fibrin strands (thread like) attached to the sticks, go ahead and process. ’ Includes: Damaged or lost tube; technician did not add antibody; antibody cross contaminated; no or insufficiem reagent added; lack of any definite positive population for Tll, T4, or T8: time from phlebotomy to staining >30 hr. ’ Enter the number of cells analyzed on the B4 form. ‘If ~300 cells were analyzed after running sample for 30 sec. then reject tube. If this problem occurs m three consecutive tubes, reject entire sample. r Fluorescent subtraction is >5 points more than for other samples of the day. h Based on technician judgement. ’ Negative Q3 population in the tube clearly extends beyond present negative cursor settings and when cursor I~ to include the clear Q3 population this yields > 10% change in quadrant 1, 2. or 4 population. ’ Print histogram and submit with B4 form. Describe pattern under PC 99. k Sum of Tll, Bl. and M02% in bitmap No. 1 <80%.

A MULTISITE

FLOW

CYTOMETRY

STUDY

201

distributed weekly by the IRL to the six laboratories. Every other week, the assessment is focused on instrument and operator variability by using already stained and fixed samples. Heparinized blood from a healthy donor is collected, and the lymphocytes are stained and fixed at the IRL. An aliquot of the stained and fixed cells is transmitted to each of the six laboratories by overnight delivery. On arrival, the cells are analyzed within 30 hr of the staining and fixation procedure . On the alternate weeks, all aspects of flow cytometry are evaluated, including the variability introduced by cell preparation, staining, and fixing. Two tubes of blood (one collected in heparin and one in EDTA) from a single healthy donor are mailed to each laboratory the same day that they are collected. They are received by the six laboratories on the next day so that analysis is usually within 48 hr of phlebotomy. The EDTA-stored blood is used for a WBC and differential, while the heparinized blood is stained with antibodies and analyzed according to protocol using the full TSS antibody panel. The results of this quality control testing are analyzed and shared with the six laboratories on a regular basis. Outlier results are identified and recommendations for correction are made. Percentile plots. Although values for all antibodies are obtained on each quality control specimen, only those for CD4 (paired with CDS) and CD4 + CD45RNCD4 are presented here as examples of the best and worst agreement. Figures 3 and 4 show the percentage of cells staining with CD4 and the percentage of CD4 + ICD45RA + cells in CD4. Horizontal reference lines denoting the 5th, 50th, and 95th percentiles are based on the normal ranges presented in Table 3. For CD4 paired with CD8 the results tended to cluster closely together (Fig. 3). Although some samples were “high” or “low normal,” no center consistently produced high or low values. On the other hand, for the subsets CD4 + CD45RA + /CD4 and CD4 + CD29 + /CD4 the sample determinations as well as the normal ranges were more variable (Fig. 4). Average dispersion from the median. To compare the overall performance of each center to the other centers for all antibody pairs simultaneously, the average dispersion from the median was used. The average dispersion for a sample was calculated as follows: (i) for each quadrant of each antibody pair the median across all six centers was determined; (ii) for each center and each quadrant of each antibody pair, the absolute difference between its value and the median was calculated; and (iii) for each center the absolute difference for each of the 44 quadrants of the 12 antibody pairs was summed and averaged. Figure 5 (A and B) shows the average dispersion from the median for whole blood samples and for stained and fixed samples. More intercenter variability occurred at the beginning of the study (i.e., wider spread in median dispersion). Except for individual outliers, the centers tended to cluster together as the study progressed. Although one might expect greater variability in the whole blood samples (which may be altered by shipping delays, differences in shipping conditions, and differences in staining and fixing in each lab), comparison with the samples that were stained and fixed by the ISL prior to shipping demonstrated no significant difference from the whole blood samples.

PARKER ET AL.

OC

I

I 10

I

I I I 20 30 SAMPLE NUMBER

I

I 40

Le.--.... 50

F-f00 r

~60 5 2 40 ~--.

.-.-

~ --_

I 1 -AL-_ -i-L-----l 20 30 50 40 SAMPLE NUMBER FIG. 3. Interlaboratory quality control for CD4 [CD81 (percentage of cells stained). (A) Whole blood samples (mean CV, 8.9; range, 1.7-20.5); (B) stained/fixed samples (mean CV. 7.7: range. 0.0-29.4). Note: Horizontal lines represent the 5th. 50th. and 95th percentiles based on the reference ranges in Table 3. Sample numbers on the horizontal axis for whole blood and stained/fixed samples do not represent the same specimens. Centers are represented by the numbers I-6. OCI

Intralaboratory

I

10

Precision Quality Control

To determine the variability of results (precision) within each laboratory, replicate blood samples are collected from participating TSS study subjects in each city or from personnel at the clinical centers. “Dummy” identification numbers, indistinguishable from true study numbers, are assigned to the replicate samples

A MULTISITE

FLOW

CYTOMETRY

203

STUDY

p95

p50

p5

10 700

20 SAMPLE

30 NUMBER

40

50

p95

p50

p5

10

20 SAMPLE

30 NUMBER

40

50

FIG. 4. Interlaboratory quality control for CD4 + CD45RA + /CD4 (percentage of cells stained). (A) Whole blood samples (mean CV, 38.8; range, 5.3-104.2); (B) stained/fixed samples (mean CV, 40.4; range, 3.5-104.9). Note: Horizontal lines represent the 5th, 5Oth, and 95th percentiles based on reference ranges in Table 3. Sample numbers on the horizontal axis for whole blood and stained/fixed samples do not represent the same specimens. Centers are represented by the numbers 1-6.

and the aliquots are submitted to the laboratories at different times during the same day. These “blind” replicates are processed along with the routine study samples received by each laboratory. The results from this replicate testing are placed into a separate database for analysis. Results from replicate samples for intralaboratory quality controls are periodically transmitted to the laboratories. Figures 6 and 7 show the absolute difference between 363 pairs of replicate samples for CD4 [CD81 and CD4 +CD29+CD4. Because some samples were tested in duplicate and others in triplicate, two of the three values of the triplicate

204

PARKER

ET

TABLE

AGE- AND SEX-SPECIFIC DESIGNATION

NORMAL

RANGES

AL.. 3

(1) FOR ANTIBODY

i?!.%l&

[PAIRED WITH?

ANTIBDDYIZ) [PAIRED WITH1

CD4

~CDSI

74

CT81

CD4

CCDZI11

14

14841

AGE GRDUP

N

o-5 6-9 10-19 20-D 30-39 40-49 so-59 69% TOTAL

23 II 20 54 85 44 24 e 276

9 42 24 40 21 45 33 44 34 47 34 50 31 55 4.4.Y 31 47 t 0.0001

57 55 57 56 60 66 72 L? 61

LO-19 20-29 30-39 40-49 SO-59

72 55 90 46 26

20 27 27 30 22

57 52 57 65 66

TOTAL

291

E22

P-“alW(3,

cl.?

Jl

!-5%-E

42 39 42 47 46

27 11 35 62 102

23 33 30 34 38

44 50 45 49 50

57 5e 58 62 64

17

30

50

63

‘“$:zz 370 0.000:

( 0.0001 CDdtCD2Pt

T4+484t

CD4+CD29+,CD4

O-5 6-9 lo-19 20-29

24 17 22 55

2 2 8 10

8 10 16 18

33 16 27 27

40-49 SO-59 faQi TDTAL

46 26 ll 291

14 12

23 25

ii 36

7

20

34

40-49 50-59 m TCTAL

46 26 ll 291

*9 51 33 60 20.u 17 48 c 0.000,

78 87 91 75

CCD45RAl

o-5 6-9 LO-19 20-29 30-39 40-49 50-59

23 16 23 56 91 47 26

m TOTAL

u 293

16 37 25 37 6 39 26 39 26 42 25 45 20 48 a846 25 41 c 0.0001

57 51 55 53 58 65 64 is 58

T4

CTZH41

o-5 6-9 IO-19 20-29 30-39 40-49 so-59 f& TOTAL

23 16 23 56 91 47 26 ll 293

O-5 6-9 10-19 20-29 30-39 40-49 SO-59 f& TOTAL

23 16 23 56 91 41 26 11 293

PW”.3l”C! CD4tCD45RAt

113 7,

14 II

24 24

17 i!

270

7

21

36

30 12 38 68

5 9 : 7

25 12 19 11

55 43 40 33

? 14 ( 0.0001

36

T4+484+/T4

W”.Sl”CS CO4

T4+2H4+

0 3 0 2 2 1

21 13 14 11 10 10

52 36 30 23 32 43

365

P-value

P-value

(1) (2) (3) (4) (5) (6)

PAIKS (PERCENJ AGE)

1 60 7 33 1 40 6 32 5 26 3 19 4 17 42184 4 28 c 0.0003

95 71 83 52 69 78 73 72

365

5 ’

31

71

Data based on HIV(-) comparison groups. All monoclonal antibodies were obtained from Coulter immunology (Hialcah. FLi Comparison of age groups within sex using Kruskal-Wallis analysis of variance iu Comparison of males YS females using Wilcoxon rank sum test (11 =. 0.001). L. lymphocyte bit map. M. mononuclear bit map. In pages 2-5 of Table 3 12 represents HLA-DR.

Note.

5 5

ZR82 ?”

0.0001

t~.ooli.

7:

NC

A MULTISITE

FLOW

CYTOMETRY

3-Continued

TABLE

mEi

DESIGNATION [PAIRED WITH1

ANtIBCOY(2) [PAIRED WITH1

CD8

,a

[CD41

AGE GROUP

CT41

D-5 6-9

10-19 20-29 30-39 PO-49 50-59 6!2? TOTAL P-value CD8

[I21

TB

Cl21

6Qt

24 17 23 57 90 47 26

u

11 12 18 14 15 11 9

TOTAL

295

::::

P-value *

o-5 6-9 IO-19 20-29 30-39 40-49 50-59

24 17 23 57 90 47 26

0 0 1 1 1 0 1

602

ll

129

TOTAL

295

O-5

24 17 23 57 90 47 26 ll 295

TOTAL

I

TB

IM(H11

L(5)

O-5

6at TOTAL

TB+N(Hl+

21 25 25 27 24 23 25

40 36 35 44 41 41 4%

31 13 39

M

MHI

CT61

o-5 6-9 10-19 20-29 30-39 40-49 50-59 Mt

M(6)

O-5 6-9 10-19 20-29 30-39 40-49

SO-59 fiat TOTAL P-value

14

za 55 116 167 114 36

LB

P-VALUEII)

14 i6 16 16 15 13 11

27 ii 25 25 25 24 26

41 3; 3% 40 39 37 45

14

e

2lz2 25

39

2 2 3 2 2 2 3

:i 42

55 30 62 127 205 125

14 11 18 13 15 12

23 22 26 26 25 24

35 37 39 42 40 42

ii 39

2 668

i 14

:s 25

41

13 13

55 30 62 127 205 125

0 0 1 1 1 I

2 2 3 2 3 3

10 11 16 19 12 14

il 373

z:: 14

e 10 1% 20 12 11 34

31 13 39 70 115 7%

0 0 1 1 I

2 2 2 2 3

1

3

f

2 NS

14

25 NS

:: 12 15 17 1

14

0 0 4 3 2 0 5

IO 8 IO IO 11 9 14

26 43 54 64 57 41 73

31 13 39 69 115 78

0 0 3 3 3 3

12 9 10 IO 11 11

40 34

if

i! 372

!a 3

ii ID

2E 13

11

u

23 16 21

:: 41

2 NS

23 16 23 57 91 47 26 294

23 21 26 25 26 25

2

i

NS

I4

20 12 15 IO

22 24 26 25 23 21

9

13 0 0 0

34

1 1 0 2 1 0

ii 293

i

3”: 40 46

23

0 0 :

27 38 61 50 50 48

3 2

ii 48

ii 667

: 3

YE IO 46

52 is 61 127 206 125 43

13 14 17 12 15 13 9

NS

22 22 26 25 26 24

37 32 37 37 39 40

x

:z 14

25

ii 37

78

22 23 26 25 25 23 22

36 35 37 40 39 40 45 NS

NS

29 53 70 115 78

6 4 7 5

21 14 22 25 1s

IO 4

:i 28 21

NS

E,” 50 53

IO e 10 10 11 10

12 16 13 13 15 14

370

1 2 3

6

NS

55 30 62 126 205 125

29 13 38 70 115

370 23 16 23 57 91 46

NS

NS

NS L

5”

604

E

:75 78

TOTAL

CC081

14p21 25

3% 26 3% 40 38 40 43

:: 12 15 14

P-Value CD56

:: 14 14 14

24 22 25 25 26 24 2%

0

P-"al"e L

N

14 1%

NS

6-9 10-19 20-29 30-39 40-49 50-59

CD%+CD56+

27 11 35 62 102 70 12 P 32%

i 373

P-value [CD561

43 33 35 42 40 34 46

NS

6-Y 10-19 20-29 30-39 40-49 50-59 m

CD8

20 24 25 26 25 23 25

NS O-5

TB+HLA-DR

I!JLl.

N

14 15 14 15 15 11 9

(3)

6-Y 10-19 20-29 30-39 40-49 50-59

Cm-"LA-OR

EEl!!&u

N

23 17 20 54 85 44 24 e 276

205

STUDY

0 1: 1 1 1 1

3 : 3

3 5 9 a 10 12

52 29 61 127 206 125

0 0 1 1 1 1

1 : 3 3 3

5 5 9 9 IO 12

1 3 c 0.0001

NS

30 ij

0 0

4 7

12 is

53 29

0

3% 70 116 7%

2 1 1 1

6 5 7 5

20 20 20 23

61 127 207 124 43

2

1

ii 372

: 1

ii 6 NS

il 20

1

1 0 1

4 5 6 5 6 5 9

1s 13 21 20 20 23 30 NS

206

PARKER

ET

TABLE

c DESIGNATIclN [PAIREO WITH1 CD2

CTAll

Tll

AGE GROUP

CTAll

Tll+TAl+

N

F

o-5 6-9 10-19 20-29 30-39 40-49 W-59

23 17 21 5% 90 47 26

45 4% 38 64 47 55 53

O-5 6-9 10-19 m-29 30-39 40-49 SO-59 M.t TOTAL

23 17 21 58 90 47

o-5 6-9 io-19 20-29 30-39 40-49 50-59

23 17 21 58 90 47

Mi

ii 293

2 293

P-value CD2tTAltlCDZ

,lltTA1,T11

TOTAL P-value CCDllbl

3-Continued

f44Ei ANTIBOOY(2) [PAIRED WITH1

CDZ+TAl+

CD2

L

Tll

CM011

Ll51

O-5 6-9 10-19 20-29 30-39 40-49 so-59 !ioi TOTAL

22 16 23 57 91 46

95

N

95

N

7

50

95

31 13 38 69 115 7% 17

44 58 63 52 64 47 55

73 75 77 7% 82 7% 77

89 57 5% 94 92 93 90

54

47

72

57

3 15 6 15 6 19 4 17 6 27 6lR 4 17 0.0002

50 42 47 52

67 42 62 57 60 65

3, 13 38 69 115 78

4 II 10 11 13 10

24 20 32 25 33 32

59 51 60 62 71 65

ii

: 371

13Pii2 10

30 NS

46

87 87

30 I2

61 6%

15 77

8% 58

5 6 13 11 12 9 11

32 33 28 2% 29 22 44

30 12 38 69 112 I% 17

7 i 6 3 4 4 4

9 10 14 9 !7 12 12

2% 27 32 27 26 29 29

10 NS

28

1 I.9 3 13 7 20 5 20 7 28 6 26 9 36 826 6 25 0.0002 41 49 60 68 66 68 59

ii 292

Et+?

22 16 23 57 91 46 26

2 1 2 3 3 4 4

f&

ll

ILIz

TOTAL

292

3

72 75 79 77 78 78 80

Tll+MOI+,Tll

ii

L D-5 6-9 LO-19 20-29 30-39 40-49

TOTAL

MO1

CT111

P-value

Ml61

22 16 23 57 91 46

22 16 23 57 91 45

h!& TOTAL

ii 291

366

30 12

ii 292

O-5 6-9 IO-19 20-29 30-39 40-49 50-59

Lp

11 9 12 17 13 14 18 2416 14

24 26 30 36 34 34 35 34 NS

$Ulg

12

o.oow

F-value N

F%-

NS

50-59 Ii!&

LCD21

lQI&

58 54 56 57 56 90 8%

P-V.,"8 CDZtCDllb,CDZ

EfpldlEs

70 7s 74 76 76 72 78

P-"al"e

CDllb

AL..

63 50 51 53 56 55 I2 55

3 10

11 14

41 36

F-VALUE(4)

A MULTISITE

FLOW

CYTOMETRY

TABLE

3-Continued

ns.Es LlESIGNATION [PAIRED WITH1

CD21

ANTIBCOY~Z, [PAIRED WITH,

[CD201

82

AGE GRrnJP

[HI

P-value CD20

LCD211

Bl

[!&?I

o-5 6-9 10-19 20-29 30-39 40-49 SO-59 Mt TOTAL

23 17 23 57 91 47

3 1 1 1 1 0

10 6 7 5 6 6

29 14 21 I2 16 17

31 13 38 70 115 76

2 295

;

z 6 NS

;

ii 372

o-5 6-9 IO-19 20-29 30-39 40-49 SO-59 it!?3 TOTAL

23

7 6 6 6 7 6 4

15 13 15 12 13 13 13

32 23 23 22 23 24 22

:: 57 91 4, I(; 295

2::

23

o-5 6-9 IO-19 20-29 30-39 40-49 SO-59

23 17 23 57 91 4, 26

fL!a TOTAL

ll 295

:: 57 91 4, 26 ll 295

CCD81

12

[TBI

o-5 6-9 10-19 20-29 30-39 40-49 50-59 f&Ii TOTAL

24 17 23 57 90 4, 26 ll 295

LCD141

I

12

tMD21

115)

o-5

23

6-9 IO-19 TO-.?9 30-39 40-49 SO-59

:: 56 91 47 26

f&J2

ll

TOTAL

292

[I21

I

MCJZ Cl21

L

P-"al"e

o-5 6-9 10-19 20-29 30-39 40-49 SO-59 Liw TOTAL

23 15 23 56 91 47 26 ll 292

ii 66,

14 1, 14 II 11 11

:: 25 16 19 22

54 30 61 12, 206 125

9 7 6 5 6 5

16 14 14 12 12 12

31 31 23 21 23 22

: ii 5 12 c 0.0001

ii 22

ii 667

: 6

Eii 12

23

11 10 7 5 5 4

22 22 18 10 11 I3

54 30 61 127 206 125

3 2 1 1 1 1 1

11 9 7 5 6 4 5

24 20 18 11 13 15 1,

1 1

: 1

i 6 NS

E 16

: 372

: ii 1 5 < 0.0001

iI7 15

ii 66,

25 9 11 0 7 2 17 u3lpz 9

73 50 63 44 48 47 47

96 62 95 79 83 04 90

31 13 38 70 114 70

23 29 15 11 20 8

60 71 59 50 50 30

93 85 89 65 78 72

54 30 61 127 205 125

50 NS

85

zz 13 50 0.0001

ii 85

ii 666

21 16 19 17 16 16 16

3.5 38 30 42 33 30 52 2l 33

9 3 8 5 7 7 6 1u 7

10 2 B 5 7 6 4 hlz 7

P-value co14

ii 15

31 13 36 70 115 78

1, NS 19 16 17 14 15 14 14 15 NS

3 4 2 1 2 1

s 31 13 39 70 115 76

6 9 7 6 7 6

21 16 16 I3 14 15

35 32 52 28 27 30

55 30 62 127 205 125

ii 373

: 7

:: 14

:: 30

31 28

3 2 2 1 1 1

P-“AL”E(4)

NS

25 15 14 11 10 7

2% 61 60 63 50 50 43

0.0003

NS

94 85 91 63 82 76

NS

10 6 8 5 7

21 16 17 14 14

34 35 32 34 32

: 3. 7

15 fi22 15

36 30

i.i 666

23 21 23

53 2, 60 122 201 123

10 5 5 5 6 6

19 16 16 13 13 14

33 26 27 24 26 25

!c.z 6 13 0.0001

24

ii 650

i 6

ii:: 14

27

3 2 3 2 3 3

26 16 18 9 12 15

53 27 60 122 201 123

: 3

121 15

ii 650

0 0 1 0 1 1 0 1 1

32

NS

0.0004 34 27 28 29 37 2.8 3.6 al 30

30 12 3, 66 110 76

8 10 4 5 6 6

El 356 c

1 0 0 1 0 0

3 3 4 3 3 2

10 13 12 13 10 11

30 12 37 66 110 16

0 0 1 0 1 1

1

: 3 NS

ii 10

ii 356

1 1

i

24 20 18 11 13 15

: i 1 5 < 0.0001

79 6 5 6 5

ii 371

11 9 7 5 6 4

54 30 61 127 206 125

29 I4 21 I1 15 17

P-value I2

ii 372

95

22 22 18 10 11 13

31 13 36 70 115 7.8

z

50

I1 10 7 5 5 4

10 6 7 5 6 6

P-value 12

3 4 2 1 2 1

3 1 1 1 1 0

P-value B2+Bl,Bl

N

NS o-5 6-9 IO-19 *o-z9 30-39 40-49 SO-59 6Qk TOTAL

.J.!mL

N

P-value

CDZl+CoZO+,COZO

-

N

(3)

207

STUDY

19 16 15 12 I2 13

NS

21

3 2 3 29 3 3 3 1J.R 3

0.0002

18 16 13 10 13 12 12

NS

PARKER

ET AL.

TABLE

P-value Mm+iZ+

L(6)

6-9 10-19 20-29 30-39 40-49 50-59 fu TOTAL (3,

15 23 56 90 47 26 ll 291

4 4 0 3 4 3 il.5 4

O-5 6-9 10-19 n-29 30-39 40-49 so-59 fiQi TOTAL

23 15 23 56 91 47 26 u 292

01 0 0 0 0 0 0 2 0

O-5 6-9 10-19 20-29 30-39 40-49 50-59

23 15 23 56 90 47 26

2 4 4 5 3 4 2

P-“al”C? MO*+I*+

M

9 12 14 13 13 13 13 NS

2 3 2 2 2 2 1 2 NS 0 0 11 11 10 9 11

3-Continued

20 24 34 26 26 30 42 27

8 12 10 8 * 9 13 ll 0

16 17 19 24 21 1s 28

30 12 37 67 114 75

3 5 3

i!i 362

ii 4

30 12 37 66 110 76

0 0

2 i

18 4

2

4

15

0 24 14

10 75 74

100 100 100

30 12 37 6, 114 75

23 43 51 38 4, 25

05 92 65 00 03 02

100 100 100 100 100 100

60 123 204 121

60 36 39 39

05 02 83 83

100 100 100 100

: 362

~~% 40

83 w

100

ii 653

2 40

ixi 83

100

52 28 59 123

0.1 0.2 0.4 0.6

0.4 0.0 I.0 1.6

11 13 i3

25 27 29 G 77

12 NS

:: 35.8

30 12 31 67 114 75 z 362

56 91 47 h

0 0 20 17

292

FE%

00 75 75 05

100 100 100 100

30 12 37 66 110 76 z 358

NS 23 15 :i

,“-l-3 20-m 30-39 40-49 50-59

90 47 26

611t

ll

TOTAL

291

38 50 61 30 36 47 51

P-“.I”* CO4+CD29t/CO4tCD45RA T4+484+,T4+2H4

T4*4B4+,T8+12+

P-value

100 100 100 100 100 100 100

N5 o-5 6-9 IO-19 m-29 30-39 40-49 50-w !m TOTAL

22 16 21 55 00 46 26 11 285

0.2 0.5 0.2 0.9 0.4 I.1 0.6 1.6 0.6 2.0 0.5 2.9 0.7 3.6 eh2.&l.LR 0.4 1.7 c 0.0001

o-5 6-9 IO-19 20-29 30-39 40-49 50-59

20 14 22 53 85 43 26

0.7 1.0 0.1 0.6 1.4 1.3 0.7

h!u

ll29lu25!2

TOTAL

274

P-value C04+C029t,CD8+I2+

00 09 85 86 83 86 85

3.7 3.0 14.3 10.2 8.3 25.6 11.7

3” 12 38 68 111 73 2

11.4

359

3.0 6.8 7.4 9.0 8.0 10.5 10.2

19.0 15.0 15.6 23.9 27.4 29.6 26.6

79 12 39 68 110 16 16

I.0 0.0 c 0.0009

25.0

360

22

0.1 O.? 0.3 0.5 0.4 I.0 0.9 2.0 0.7 1.9 0.6 2.5 0.6 2.5 ulAl.9 0.3 1.6 c 0.0001 0.4 1.3 1.2 1.3 1.0 1.6 2.0

10.0 21.0 21.5 28.0 20.5 33.1 31.0

.

A$+

“.OOOl

1 2.: 3.2 8.0

10.3

3.5 7.0 5.5 9.2 0.5 8.5 11.8

“lf$ 1.4 (

4.1 1.9 6.7 17.3 0.9 17.5 12.0

49 26 61 121 1% 119

G.1 I.0 1.2 0.9 1.7 1.8 I.3

3.3 6.0 6.0 9.0 0.0 9.5 10.6

Il.0 10.9 20.9 25.9 20.0 31.0 27.4

0.0

25.3

NS

A MULTISITE

OC

I

I 10

1

FLOW

CYTOMETRY

I I I 20 30 SAMPLE NUMBER

209

STUDY

I

I 40

1

I 50

I

I 50

B14-

10

20

30

40

SAMPLE NUMBER FIG. 5. Average median dispersion plots. (A) Whole blood samples. (B) Stained/fixed samples. Sample numbers on the horizontal axis for whole blood and stained/fixed samples do not represent the same specimens. Centers are represented by the numbers l-6. The dotted line connects the mean value for sample.

were chosen at random for analysis. In general, for CD4 [CDS] (Fig. 6) the median absolute differences were approximately equal to 3, with most absolute differences ranging between 0 and 12. There were few values outside of this range. For the subset CD4+CD29+/CD4 (Fig. 7), the median absolute differences were approximately equal to 4, with most absolute differences ranging from 0 to 20. For this data set specimens and individual tubes were ;t2& excluded when problem codes were recorded.

210

PARKER ET AL. 40. g +

. 30-

! z

-

g 200 w + 2

. . . m zl

lo-

5

o-

t . -==

=

A

t 1

I 2

.

. : . --_ T

_= -= -

.

--- = TzI 3

CLINICAL

L-d---.-. 4

:

. =.

m -v-

a

--e

AL

5

6

CENTER

FIG. 6. Precision of intralaboratory replicate testing for CD4 [CDS]. The absolute differences for replicate pairs are plotted for each of the six centers. The horizontal line represents the median of the absolute differences.

Development of Reference (Normal) Ranges for Control Groups

To establish age- and sex-specific ranges for antibody pairs, baseline data from healthy TSS subjects (320 males and 401 females) were obtained between September 1985 and April 1988 Table 3. These healthy subjects included anti-HIV-l negative blood donors and anti-HIV-l negative, nonsexual household contacts of congenital clotting disorder or anemia patients. The differences among age groups were compared within gender. The differences between male and female data

.

-1

A--i---i--L.1

2

3 CLINICAL

-- -.4

5

6

CENTER

FIG. 7. Precision of intralaboratory replicate testing for CD4 + CD29 + /CD4. The absolute differences for each replicate pair are plotted for each of the six centers. The horizontal line represents the median of the absolute differences.

A MULTISITE

FLOW

CYTOMETRY

STUDY

211

were compared over all age groups. For all comparisons, significance was determined using an CYlevel of 0.001 (=0.05/35 variables). In summary, the following significant age and sex differences were observed: (i) Six phenotypes or ratios increased with age for both sexes (CD4 [CD8], CD4 [CD29], CD4 + CD29 + , CD4 + CD29 + /CD4, CD4[CD45RA], and CD4 + CD29 + /CD8 + HLA-DR + ); (ii) four phenotypes increased with age in females only (CD8 + CD56 + [L], CD2 [Tall, CD2 [CD1 lb] [L], and CD2 + CD1 lb + [L]; (iii) three phenotypes or ratios increased with age, but then decreased at age group 60 + (CD2 +Tal+ , males only; CD2 + Tal+ /CD2, males only; CD4 + CD29 + /CD4 + CD45RA + , both sexes); (iv) three phenotypes decreased with age in females only (CD20 [CD21], HLA-DR [CD8], and HLA-DR [CD14]); and (v) live phenotypes or ratios decreased with age, but then increased at age group 60 + (CD4 + CD45RA + , CD21 [CD20], CD21 + CD20 + , and CD21 + CD20 + /CD20, females only; CD4 + CD45RA + /CD4, both sexes). All other markers were not significantly related to age or sex. DISCUSSION

Immunophenotyping of peripheral blood leukocytes has been used to assess the immunological status of patients with a range of diseases. Not only do the diseases themselves influence immunophenotypes, but a variety of biological and technical factors may also affect results. Biological factors include age and sex (l&22), viral infections (l), stress (23, 24), medications (25-27), cigarette smoking (28), and endocrine changes (29). In addition to these factors, which can only be controlled for by history and selection of appropriate control subjects, there are technical factors relating to specimen preparation, instrumentation, procedures, and reagents which influence results. All of these must be addressed in any immunophenotype study of patients involving cross-sectional or longitudinal evaluations. We have addressed as many of these factors as was practical in this study. Standardization

of Flow Cytometry

across Centers

The TSS is broadly concerned with the immunological consequences of the administration of blood components and plasma products. Because of the prolonged incubation period of HIV-l infection, extensive, long-term follow-up of patients enrolled in the study is required. In order to observe subtle longitudinal changes in our cohorts, a high level of comparability in the immunological determinations cross-sectionally and over long-term follow-up must be maintained. To this end, the TSS IWG has utilized common instruments and reagents, standard protocols, standardized instrument calibration, and a rigorous quality control program. In the process of standardizing procedures across centers we have encountered many intra- and interlaboratory factors which can produce variable results. Frequent communication among investigators and technicians through electronic mail, conference calls, training and review sessions, and meetings of the investigators was found to be critical to the standardization of the study. Without these

212

PARKER

ET

AL.

reinforcements, uniformity of instruments, reagents, and procedures would insufficient to maintain protocol adherence and the integrity of the project.

be

Quality Control Procedures

An important mechanism of the quality control program established by the IWG is the regular review by the ISL of the histograms returned by the laboratories for each proficiency testing sample. This review focuses on standardized gating and cursor settings-major sources of potential variability. The ISL reviews the histograms and numerical results, and recommendations for technical corrections are returned to the laboratories within 2 weeks for review by the immunologists and technicians. The average dispersion plots provide another feedback mechanism which permits a global assessment of interlaboratory variability. Large median dispersion values for a given center indicate deviation relative to the other centers; inconsistent median dispersion values over time within a center reflect variability in sample preparation, reagents, and/or flow cytometric technique at a given center. As shown in Figs. SA and SB, except for a few outlying values, the overall interlaboratory QC improved over time, as indicated by decreasing median dispersion values. When a laboratory is identified as being deviant, an attempt is made to identify the problem and recommend corrective action. For example, early in the study, one laboratory had values consistently higher than the others. This was found to be due to improper instrument settings. Appropriate adjustments were made and subsequent values from that laboratory were well within the clusters for all laboratories. Another important QC concern in immunological determinations over time is that of laboratory drift. The average median dispersion plots give an indication of laboratory drift for an individual center. However, should all centers experience laboratory drift due to a common cause, a different indicator is needed to detect it. Measures of laboratory drift for the overall study used by the TSS are: fi) testing of normal proficiency samples over time and inspection for any consistently increasing or decreasing patterns relative to our established normal ranges (see Figs. 3 and 4) and (ii) the statistical comparison of the reference ranges of our study controls at entry and at subsequent follow-up intervals. Should significant changes in study control values be seen longitudinally, laboratory drift among all centers would be suspected and would require corrective action. In Fig. 3, the results from four different samples of whole blood showed that all of the laboratories fell at or above the 95th percentile (reference range). Either the donors were not healthy at the time of phlebotomy or the sample was not properly prepared. Intralaboratory

Quality Control

The ability of each laboratory to produce the same values for blind replicates received on the same day is another important QC exercise. Replicate agreement for CD4, CD& and CD4+CD45RA+/CD4 was generally good (CVs: 3.7 to 8.9), whereas CVs were significantly higher for CD4+CD29+/CD4 (CVS:9.9-19.0),

A MULTISITE

supporting our other observations nificant variability. Sources

FLOW

CYTOMETRY

that CD29 antibody

STUDY

213

results are subject to sig-

of Variability

An important consideration for both single institution and multicenter flow cytometric studies is the impact of sample handling on agreement of results. In particular, the TSS has evaluated the effects of length of time from phlebotomy to sample staining and fixing and from stain/fix to cytometric analysis. Heparinized samples can be stored for approximately 30 hr before stain/fix without causing significant changes in the cytometric determinations for any of the antibodies used. A previous study which used some of the antibodies in the TSS panel demonstrated that cells could be stained at any time within 24 hr; however, by 48 hr significant changes in the results had occurred (12). Based on these two studies, we chose a maximum time of 30 hr from phlebotomy to stain/fix for sample acceptability. In a similar fashion, we demonstrated little effect of holding the samples for an additional 30 hr after stain/fix until analysis, whereas Fletcher et al. (12) showed that for a subset of our antibody panel, samples could be held as long as 72 hr after fixation. On the basis of both studies, we chose to use the more conservative 30 hr derived from our current study. This decision was also compatible with the practical considerations of scheduling patients for phlebotomy and flow cytometric analysis. An additional source of variability can arise from the pairing of two antibodies, each labeled with a different fluorochrome. Examples of this occur with CD4 and CDS. In the TSS panel, CD4 and CD8 are measured three times, using different combinations of dyes and antibody pairs. We have examined these data and have seen significant variations in results, but have no explanation for this phenomenon. Is it due to fluorochrome or antibody interference or bleed? In the absence of an identified cause and no apparent way of selecting the “correct” value, we the values. These algorithms were developed several algorithms to “smooth” compared and on the basis of this study the median of the three determinations for CD4 will be used for longitudinal studies (J. Niland, manuscript in preparation). Another procedure implemented by the TSS to reduce variability and to facilitate the analysis of data across centers was the development of a set of problem codes by the IWG (Table 2). Flow cytometric analysis of certain blood samples can involve inherent difficulties (e.g., the presence of nucleated erythrocytes) or technical errors (e.g., failure to add the proper reagent) which would invalidate the results. A major consideration in the development of the problem codes was to eliminate subjective decisions by the technicians which might influence cytometric results. Consequently, this list of codes permitted objective evaluation of the samples at the time of data analysis and prevented the use of biased exclusionary criteria. Study Control

(Reference)

Ranges

This study provides the first published data on age-sex specific reference ranges for many of these antibodies (Table 3). Age- and sex-specific “normal

214

PARKER

ET

AL.

ranges” emphasize the necessity for making cross-sectional comparison by age and sex. These ranges are currently being used in the TSS for population comparisons to identify patterns which may be associated with clinical status and progression of HIV-related disease, as well as to provide information about the immunological consequences of transfusion with HIV- 1 negative blood and blood products. To assist in hypothesis generation and clinical assessment on a caseby-case basis, a mechanism has been implemented to routinely report flow cytometric results relative to the age-sex specific normal ranges for an individual subject (Fig. 8). This report (R3), produced on a monthly basis, shows the cumulative immunological results to date for all subjects with new tests over the previous month. Continuing Problems and Studies

Multicenter immunophenotyping studies using flow cytometry are complex. We have identified many factors which can affect results and have resolved some, but others remain. One, of major concern for all flow cytometry studies, is the accurate determination of the total WBC and differential leukocyte counts which are used to calculate absolute numbers of subsets. Methodologies under assessment by TSS include traditional slide differentials, automated WBC and differentials, and the use of the flow cytometer itself as a particle counter and discriminator. These studies are in progress and will be the focus of subsequent reports. In the interim, variations in WBCs and lymphocyte counts in multicenter flow cytometry studies remain a fundamental flaw. Because nucleated erythrocytes may fall within the lymphocyte bit map and because some of the patients (e.g., congenital hemolytic anemias) may have large numbers of these cells, the IWG is currently addressing direct and indirect methods for the identification and removal of these cells from the total lymphocyte count (30). Another problem relates to unannounced changes in concentration of antibody reagents by manufacturers. Preliminary results have indicated that some antibodies in our panel cannot be significantly diluted without a substantial change in the percentage of cells stained with these antibodies. This change undoubtedly reflects differences in the degree of expression of lymphocyte surface antigens and emphasizes the desirability of using single lots of reagents in longitudinal studies. However, because of the numbers of patients in the TSS and the long follow-up period, use of a single lot has not been possible. Single lots of each antibody were purchased early in the study but were insufficient for the total period of study. As an alternate solution, TSS is now attempting to purchase a single lot of reagents for use over an extended period and, prior to distribution to laboratories. compare titers with those of earlier lots in the IX. Future Direction

Based on our experience and questions raised by results to date i 11). we have altered the antibody panel for the remainder of the study. Four different panels will focus on specific groups of patients: (i) a screening panel, CDS/I2 and CD2/ CD4; (ii) a basic panel, CD4/CD29, CD4/CD45RA, CD8/12, CDS/CD56, CD21

INCLUDES INCLUDES

INCLUOES

“SEROCONVERSION” “NEGATIVE”

~UNCLASSIFIABLE”

EVAL:

INCLUDES

HIV

41 25 32 43 37 47

19 31 52

FOR

“POSITIVE”

OVERALL

ADJUSTED 4300 6150 llOD0

NORMAL RANGES 5tH 12 6 133000 50TH 14 8 244ooO 95TH 16 7 425000

5

8

9 3

AND 32 44 62

43 27 30 30 32 26 SEX 474 900 1945

882 398 490 503 604 550 (PERCENTILES 11 163 23 469 38 958 1.0 2.0 4.9

RATIO ___-_ I.8 0.8 0.7 0.6 0.9 0.5

CD4/CD8

(III)

FLOW DATE 22OCTtlS 060CT67 17NoV87 00APR80 040CT88 03APR89

y. ABS ---+---24 492 33 407 46 751 47 708 37 698 56 1184

CD.9

(I!)

DRAW NO. 1 1 2 f 1 1

FOR:

1579 I121 1404 I375 I321 1840

ABS 6 6 6 5 6 2

123 89 98 84 1 I3 42

% ABS ---+----

COZ+TAl+ SUBSET

(VI

AGE= 40 - 49 VR 52 @I7 5 90 72 1406 17 396 89 26134 52 1370

% ---+---77 76 86 82 70 87

CD2

(IV)

0 3 15

.

0 56 335

MALE

FIG. 8. Patient report form: Hematologic data (R3 report).

(VII!)

INTERVAL (DAYS) 0 346 42 144 178 182

,

SAMPLE 30 52 77

TS

AND

3 23 69

SIZE-

55

CD20

7 13 24

REMAINS

I

)

(IX)

Y. ---*---19 14 I3 8 13 27

I2 17 23 29

0 11

CUMULATIVE (~~ON~HS

co4+ CD4+ CDW29+ C045R+ PROPORTION OF CD4+ OF CO4+ ________------44 19 41 36 54 23 50 23 45 47 42 46

(VI!)

CBC DATE 22DCT86 05OCT87 16NOV87 OBAPRBB 03OCT88 03APR89

X At3S ---+---3 62 II 162 13 212 13 219 II 200 22 465

CD8+!2+ SUBSET

(VI)

DRAW NC. 1 1 2 1 1 1

HAS AIDS. >=2 PIE(+) RESULTS AT LEAST 3 MONTHS APART. ~!a(+) AND RIP(+) ON SAME VISIT, OR “,Z,T ON ALL V!S!TS(EVEN IF ONLY 1 To DATE) _ - . -. HIV!+) VISIT HIV CHANGES FROM (-) T O (t) AN0 REMAINS (+) ON 2 SUBSEO. v!s! VISIT-HIV (-1 ON ALL VISITS (EVEN ! F ONLY I VISIT T O DATE). OR IF-1 VISIT HIV (I). AT LEAST 2(-1’S, 1 FOLLOWING THE (I) ALL OTHER POS~!EI!L!T!ES NOT GIVEN ABOVE(!NCL. TRANSIENT POSITIVITY)

AGE 1241 2090 3642

2050 1475 1632 1677 1087 2115

% ABS ---+----

13 I4 14.4 13 14.2 13

X ABS ---+----

1 I 2 I 2 2 3 1 4 1 5 1

WBC _____ 5000 5900 5100 3900 5100 4500

HGE

PLT ______ 187000 193ooo 197000 188000 2 19000 189000

co4

LVMPHS

CUMULAT!VE (MONTHS) 0 II 11 17 23 29

VR I A SW NN 0.0.

INTERVAL (DAYS) 0 340 0 186 I78 182 (I)

VISIT DATE 22OCT86 05OCT87 05DCT87 06APRB8 03DCT88 03APR69

D

_--_-_-----

2 2 3 4 5

VISIT NO.

!+I

I17 268 858

390 207 I31 I34 245 57 1

ABS

--II=II======_IEr=EI========~=======~~==~== CATEGORY. CLOTTING OISOROER. TREATED SCHEDULE: 6 MOS FOLLOW-UP. LA DATE: N/A ANTI-HIV EVAL: SEROCONVERSION (SEE FOOTNOTE

STUDY

SUBJECT FOLLOW-UP CITY OF AIDS OX OVERALL

SAFETY

INTERVAL CUMULATIVE (MONTHS) (OAVS) 0 0 349 ii 42 I2 143 I7 179 23 181 29 ______- - ___________

TRANSFUSION

i=2===I===II==ISEII=_.=========.I..C=I=5=~~~~~===-===~==~~~~=======~~===~~~~~~=~=-=-STUDY IO: HOUSEHOCO CODE: ALPHA IO: RELATIONSHIP TO 1ST INTERVIEW. IO/Z?/86 PROBANO: SELF AGE AT 1ST AIDS STATUS: NO SEX: MALE INTERVIEW fVRS): 46 )

1 2 9

50 294

11

21

74 33 34 94 1

2

82 5 2 2 5

w G

s *

3

z

2

8 x

2

;I ABS

--

% ---+--4

CO14

(X)

z 4 6

>

216

PARKER ET AL.

CD20, and KC56/CD14; (iii) a T suppressoricytotoxic panel (Ts/c), CDSiTAl. CDS/CDllb, CD8/CD25, and CD8/CD28; and (iv) a T helper/inducer panel (‘Th), CD4/Tal, CD4/12, and CD4/CD25. HIV seropositive subjects with CD4 cell counts <200/mm3 and CD8 <800/mm3, or with AIDS, will be phenotyped with the screening panel at 6-month intervals. HIV + subjects with CD4 >200, CD4 <:200, and CD8 >800 and HIV( - ) subjects who are anti-HTLV I/II + will receive the basic, Ts/c, and Th panels at 6-month intervals with control subjects being studied with these at 12-month intervals. Treated HIV{- ) congenital clotting disorder (CCD) patients will be phenotyped with the basic panel at 6-month intervals and the Ts/c and Th panels at 12-month intervals. The basic panel in addition to providing overall 1‘ and B cell quantitation will assess CD4 + /CD29 + cells, the subset which preferentially falls in HIV infection (I l), and its approximate reciprocal subset (CD4 + /CD45RA + ). In addition, subsets of CD8 shown to rise (HLA-DR + ) and fall (CD56 + ) will also be measured. The basic panel will be applied to HIV + contacts. who are usually early in the course of infection, to establish early changes that predate immune deficiency. The same panel will also be applied to low-risk and high-risk subgroups of HIV( -) contacts, both for control purposes and to establish baselines in the event of later seroconversion. The Th panel will be applied more selectively to some control subsets and to HIV+ patients with relatively normal phenotypes. Because activated T cells are more susceptible to HIV infection in vitro (57) and activated T cells are more frequent in HIV-infected individuals (58, 59), the proportion of CD4 + cells with activation markers (TA 1, 12, CD25) will be followed. The Ts/c panel will measure CD8 subsets (cytotoxic, CD28 (60) and suppressor, CD1 lb) as indicators of early progression to immune deficiency. The screening panel will be used on all subsets because it provides isotypic antibody controls, CD4/CD8 for setting fluorescence subtraction and cursors, and KC56/CD14 to set bitmaps. CONCLUSIONS

Our experience in this six center study involving a large number of patients (3057) and repeated samples (10,967) has demonstrated that. with attention to the potential variables in such a study, immunophenotypic results are statistically comparable among centers and are valid in a longitudinal study. Standardization of instruments, reagents, procedures, and technician training has been achieved. but some specific inherent problems remain to be solved. ACKNOWLEDGMENTS We express our appreciation to the technical flow cytometry staff at each center for their dedication and excellent work in the planning and implementation of this study. They include Susan Autio and Nancy Lewis Fine (Detroit); Alma Aguirre, Philippe Bishop. Zahida Haddad, Diane Scott, and Urduja Trinidad (Los Angeles); Nuzhat Ahmed, Rosa Melendez, Margarita Ashman, and Mirian Yaniz (Miami); Maureen Blair, Sharon Ellis. italis George, Mayra Romero, and Elise Smith (New York); Theresa Kadlecek, Margaret Sharp, and Christopher Vandevert (San Francisco); and Kelli Carlson. Donna Larry, and Susan Van Benthem (Seattle).

A MULTISITE

FLOW

CYTOMETRY

STUDY

217

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1985.

Jorizzo. J. J., Sams, W. M., Jegasothy. and Olansky, A. J.. Cimetidine as an immunomoduiator: Chronic mucocutaneous candidasis as a model. Ann. Intern. Med. 92, 192-195, 1980. 26. Griswold, D. E., Alessi, S., Badger, A. M., Poste, G., and Hanna, N., Inhibition of T suppressor cell expression by histamine type 2 (H21 receptor antagonists. J. Immunol. 132, 3054-3057. 1984. 27. Meuleman, J., and Katz, The immunologic effects, kinetics, and use of glucocorticosteroids. Med. 25.

C/in.

North

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69, 805-816,

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Received April 18. 1989; accepted with revision August 14, 1989