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 ,
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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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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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” “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 =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
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21
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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
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.
188
PARKER ET AL..
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
A MULTISITE
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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.
194
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Tube Specific Problem Codes
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I
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>
196
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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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197
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
198
PARKER ET AL..
‘I_ ,. ‘)’ ilkhi1 2 ? ;
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1s 2 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 =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
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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
