Flow cytometry PRA using lymphocyte pools ... - Wiley Online Library

Cytometry Part B (Clinical Cytometry) 72B:256–264 (2007)

Flow Cytometry PRA Using Lymphocyte Pools from Random Donors Dong Il Won,1* Hee Du Jung,1 Ok-Ju Jung,1 Seung Huh,2 and Jang Soo Suh1 1

Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Republic of Korea 2 Division of Transplantation and Vascular Surgery, Department of Surgery, Kyungpook National University School of Medicine, Daegu, Republic of Korea

Background: Pools of lymphocytes from carefully chosen donors have been used for flow cytometry (FC) panel reactive antibody (PRA) assays. We intended to devise an FC PRA assay using mixed lymphocyte pools from a large number of randomly selected donors (RD FC PRA) to accurately predict the likelihood of a positive HLA crossmatch. Methods: Lymphocyte pools were prepared from randomly selected donors (N = 120). %PRA was calculated based on the anti-IgG FITC histogram of the T cells. The proposed RD FC PRA assay was assessed in comparison with the bead FC PRA, antiglobulin-augmented CDC (AHG-CDC) PRA assay, and the expected %PRA calculated by summing up the antigen frequencies of the known specificities. Results: In 29 FC crossmatch positive sera, the positivity rate for the bead FC, RD FC, and AHG-CDC PRA was 100, 100, and 79%, and the mean %PRA was 77% 6 20%, 73% 6 21%, and 33% 6 33%, respectively. The RD FC %PRA was not significantly different from the bead FC %PRA (P 5 0.205). In 19 sensitized patients with a negative FC crossmatch, the positivity rate was 21% using the RD FC PRA and 16% using the bead FC PRA, which suggested that both assays had similar abilities to detect low levels of HLA antibodies. Conclusions: The RD FC PRA assay allows easy panel preparation, reduces cost, and naturally reflects the probabilities of a positive crossmatch in the population to which the cadaveric donor belongs. Therefore, this new assay is expected to be useful as another approach to determine the % PRA. q 2007 Clinical Cytometry Society

Key terms: HLA antibody; panel reactive antibody; flow cytometry

The detection of antibodies (Abs) to HLA antigens in sera is often necessary in various clinical situations. For example, detection and characterization of HLA class I- and II-specific Abs in the sera of potential kidney transplant recipients and exclusion of mismatches for these alloantibodies are key success factors for a good graft outcome (1). Traditionally, panel reactive anti-HLA Abs (PRA) are measured using complement-dependent lymphocytotoxicity (CDC), which screens for the presence of HLA Abs and identifies the specificities of HLA Abs. However, CDC PRA assay has many disadvantages: (1) the lymphocyte panel should be created with carefully selected HLA types, (2) the lymphocytes should be stored in a viable condition, (3) the assay procedure is both labor-intensive and time-consuming, and (4) CDC assays often result in high intra- and interlaboratory variability due to the lack of standardization in assay protocols, reagents, or reference specimen calibration, technician expertise, and subjective reading criteria (2).

q 2007 Clinical Cytometry Society

In contrast, enzyme-linked immunosorbent assay (ELISA) PRA assay is sensitive, specific, and technically less demanding (3). The specificities of Abs and the frequency of target lymphocytes that will react to a serum are determined after the analysis of positive reactions in each individual well (4). It has been recently demonstrated that the flow cytometry (FC) method can detect Abs at a more sensitive level than many other assays. Additionally, FC can easily distinguish between IgG and IgM Abs by using anti-IgG secondary Ab, and can also detect noncomplement-fixing *Correspondence to: Dong Il Won, M.D., Department of Laboratory Medicine, Kyungpook National University Hospital, 50 Samduk-Dong 2-Ga, Jung-Gu, Daegu 700-721, Republic of Korea. E-mail: [email protected] Received 29 April 2006; Revision 20 September 2006; Accepted 6 November 2006 Published online 4 January 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/cyto.b.20175

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Abs (5). Pooled panels used for the FC PRA assays have been different in laboratories reporting on their use, including: (1) a pooled panel of chronic lymphocytic leukemia cells from 12 donors (6), (2) a pooled panel of lymphocytes from 6 or 10 healthy donors (7,8), and (3) six pools of panel cells, each of which contained four HLA-typed cells (9). However, creating pools that account for all of the HLA specificities is quite a difficult task. Nowadays, the FC PRA assay using beads coated with purified HLA antigens (bead FC PRA) is being used to avoid reactions to non-HLA antigens and to separate class I Abs from class II Abs (10). Each bead is coated with different HLA class I or class II antigens. All common HLA antigens, as well as many rare HLA antigens, are represented in the pool. The percent PRA (%PRA) is represented by the percentage of beads that react positively with the serum. However, the bead FC PRA does not always show perfect diagnostic efficiency. It was reported that bead-reactive Abs were nonreactive with lymphocytes, and HLA Abs detectable on lymphocytes could not be detected by the bead FC PRA assay (9); these findings support those of another report which acknowledged that no single method, even the most sensitive bead FC PRA assay, was capable of confirming the presence of all detectable HLA Abs (11). Additionally, the bead FC %PRA may not reflect the exact likelihood of a positive crossmatch in the population to which the cadaveric donor belongs, especially in ethnic groups with distinct HLA frequencies. Lastly, the cost of the commercial kits for this assay is relatively high. Therefore, in the current study, we devised a method using mixed lymphocyte pools from randomly selected donors without HLA typing to provide an accurate likelihood of a positive crossmatch with a cadaveric donor, provided that the number of lymphocyte donors is sufficiently large. The effectiveness of the proposed PRA assay was assessed in comparison with the conventional assays. MATERIALS AND METHODS Cell and Sera Preparation The protocol of this study was approved by the Investigational Review Board of Kyungpook National University Hospital. The lymphocytes composing the lymphocyte pools were obtained from random selection of donors among the individuals who visited the Health Promotion Center at Kyungpook National University Hospital for a routine health checkup, rather than for the treatment of a known condition or disease. The selection criteria for sera used in this study are listed later. The major subject sera were obtained from 29 renal transplant candidates exhibiting a positive T cell flow cytometry crossmatch (FCXM) and regarded as HLA allosera. The minor subject sera were obtained from: (1) 19 renal transplant candidates who had undergone sensitization events, such as transfusions or deliveries, yet showed a negative T cell FCXM, and (2) males who had

Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b

not experienced any sensitization events, including seven healthy individuals and 26 dialysis patients, for whom the sera were regarded to be absent HLA alloantibodies. The negative control sera used as an indicator for the positive and negative cutoff points on the anti-IgG FITC histogram were obtained from the nontransfused healthy males (blood group AB). PRA Assays The FC PRA assay using lymphocyte pools from randomly selected donors (RD FC PRA) was investigated for the analysis of the assay variables, and was compared with the bead FC PRA and antiglobulin (AHG)-augmented CDC PRA assays. The latter method was adopted because the American Society of Histocompatibility and Immunogenetics standards continue to require CDC screening, although CDC assays alone are often inadequate due to their low sensitivity. RD FC PRA assay. Heparinized blood was obtained from 20 randomly selected donors each day. One microliter of Histopaque-1077 (Sigma-Aldrich, Louis, Mo) was aliquotted into 20 tubes, and 1 mL of whole blood was overlaid in each tube. The mononuclear cells (MNCs) were then separated after centrifugation at 700g for 30 min, followed by washing three times with 2 mL of phosphate-buffered saline (PBS) using centrifugation at 200g for 5 min. The washed lymphocytes were then randomly mixed in a single tube. The cell count in the pool was adjusted to 1.0 106/mL. Aliquots (100 mL, 1.0 105 MNCs) from the pool were resuspended in each tube with 10% dimethyl sulphoxide in Gibco RPMI medium 1640 (Invitrogen, Carlsbad, CA) for storage at 708C in a deep freezer. This protocol was repeated each day for 6 days, resulting in six pools from a total of 120 donors (i.e., 6 20 ¼ 120). Frozen aliquots were thawed immediately prior to the assay, and two aliquots were combined in one tube so that only three tubes per test serum were used, with one tube containing the mixture lymphocytes from 40 individuals (120/3 ¼ 40). The cells were washed in 2 mL of PBS. The cell suspensions were then incubated with 100 mL of the test serum in FACS tubes at 378C for 30 min, followed by four washes with 2 mL of PBS. Next, 20 mL (at a 1:40 dilution) of Fcg-specific fluorescein isothiocyanate (FITC)-conjugated goat F(ab)2 antihuman IgG (Jackson Immunoresearch Laboratories, West Grove, PA) and 20 mL of pre-titered phycoerythrin (PE)conjugated anti-CD3 (Dinona, Seoul, Korea), used for identification of T cells, were added to the resuspended cell pellet. The mixture was incubated in the dark at 48C for 30 min. The cells were washed one more time and resuspended after the addition of 130 mL of PBS, after which they were ready for FC analysis. Fluorescence was evaluated using a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA). The viable lymphocytes were gated on a forward scatter (FSC)/side scatter (SSC) plot. The dead lymphocytes were excluded

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rescence scale, and the % positive region of the negative control was 1.7% 6 1.0% in this study. (2) In the ‘‘overlay’’ method, the channel-by-channel subtraction method was used. That is, a control histogram was overlaid on and subtracted from the test histogram. A marker was set at the intersection. The differential histogram indicated the net % positive region (Fig. 3). Bead FC PRA assay. Class I PRA was analyzed in serum samples using a FlowPRA class I Screening Test (One Lambda, Canoga Park, CA), according to the recommendations of the manufacturer. In brief, 20 mL of test serum was incubated with 5 mL of FlowPRATM I beads for 30 min in the dark at 20–258C. The mixture was washed twice, and 100 mL of anti-IgG FITC was added to the pellet, followed by 30-min incubation. The pellet was washed twice and resuspended in 0.5 mL of buffer. Positive and negative controls were used, and the %PRA was represented by the percentage of events shifted to the right of the negative control or a distinct second peak. AHG-CDC PRA assay. The AHG-CDC PRA assay was performed according to previously published protocols

FIG. 1. Analytic procedures used to obtain the %PRA in the RD FC PRA assay (up to Fig. 3). A viable lymphocyte gate was set to exclude dead lymphocytes on the FSC/SSC plot (A), and a T cell gate was set on the CD3-PE/SSC plot (B). FSC, forward scatter characteristics; SSC, side scatter characteristics. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

based on their decreased FSC (Fig. 1A). T cells were gated on a CD3-PE/SSC plot (Fig. 1B). Nearly 5,000 viable T cells were acquired per tube. The %PRA was determined based on the anti-IgG FITC histogram of the previously gated T cells as the average of three values from three tubes used per test. The positive and negative cutoff points were set in two ways: (1) in the ‘‘shoulder’’ method, the cutoff point was set at the end of the peak in each one of three tubes for the negative control serum (Fig. 2A). A marker was set up at the same cutoff point on the histogram for the test sera (Fig. 2B). The % positive region was assessed as the percentage of events shifted to the right of the cutoff point. The net % positive region was the % positive region of the test sera minus that of each negative control. The cutoff point was usually close to FL1 102 on a log fluo-

FIG. 2. Determination of the %PRA using the shoulder method on the anti-IgG FITC histogram of T cells. The %PRA (42.5%) indicates the net percentage of the positive region, obtained by subtracting control sera (1.7%, A) from test sera (44.2%, B).


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expected %PRA was 24.41.1 ¼ 23.3%. The values for the expected %PRA are listed in Table 1. The difference between the AHG-CDC %PRA and expected %PRA was not statistically significant (P ¼ 0.099). Statistical Analysis

FIG. 3. Determination of the %PRA using the overlay method on the anti-IgG FITC histogram of T cells. On the overlaid histogram of the control (blue) and test (red), a marker (M1) is set at the intersection. The differential histogram (dotted area) indicates the %PRA (54.6%). [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]

(12). In brief, 2 mL of patient serum was incubated with 2,000 target lymphocytes from 36 individual donors with known HLA class I phenotypes. After incubation for 1 h, the cells were washed and flicked three times, and 1 mL of a 1:100 dilution of goat anti-human IgGk (Helena Laboratories, Beaumont, TX) was added for 2 min. This was followed by the addition of 5 mL of pre-titered rabbit complement. After 2 h of incubation, 5 mL of eosin Y was added. The reactions were read on an inverted phase contrast microscope and considered to be positive with a score of 4 or more. The specificities of the Abs were determined by an analysis of the reaction patterns of the lymphocyte panel. Expected %PRA. Our intention in using this parameter was to provide a more accurate probability of a positive crossmatch in the population to which the cadaveric donor belongs. This was calculated by summing the antigen frequencies of the specificities identified by the AHG-CDC PRA assay. In addition, the frequencies of cross-reactive group (CREG) antigens, which appear very frequently according to Professor W. Mayr, Aachen, 1991, WHO Report, 11th workshop (13), were also included, as the AHG-CDC and FC techniques also detect Abs specific for CREG antigens (14). The antigen frequencies adopted were those of the Korean population (15). In the case of more than two specificities, all probabilities showing two antigens simultaneously (the multiplication of two antigen frequencies) were subtracted from the sum to obtain the final expected %PRA. For example, the expected %PRA for an HLA alloserum with specificities of B51 and B52 was calculated as follows. The very frequent CREG antigens for B51 and B52 were B5, B5102, and B53, including their split. Their antigen frequencies in the Korean population are 18.6, 5.1, 0.1, 0.5, and 0.1%, respectively. The sum of the probabilities showing two antigens simultaneously was 1.1%. The


The statistical analyses were performed using Cellquest (BD Biosciences) and SPSS software (SPSS, Chicago, IL). The comparison between the two methods was carried out using the Pearson’s correlation coefficients, paired/ unpaired t test, or Fisher’s exact test. The false negativity rate of an assay was calculated by the likelihood that the assay showed a negative test result in the HLA allosera. A positive test result in the sera of nonsensitized persons was regarded as being falsely positive. Statistical significance was defined as P < 0.05. Data are expressed as mean 6 standard deviation (SD). RESULTS Estimation of the Appropriate Number of Donors The appropriate number of donors for mixed lymphocyte pools required to avoid missing a certain HLA antigen can be assumed using the following probability calculation. The probability, P, for a certain HLA antigen to be omitted in a panel of randomly selected donors is: P ¼ ð1 AFÞN where AF is the HLA antigen frequency and N is the number of donors. Based on this formula, the probability for each HLA antigen according to its frequency was determined and

Table 1 Expected %PRA of HLA Allosera According to Their Specificities

Specificity

%PRA Measureda

Expected

100 Polyspecific 100, 83, 100, 100b A30, 31 9, 28 17 A3, 26, 30, 33 þ extra 65 57 A3, 11; B51, 54 þ extra 39 48 A26, B40, B27 42 43 A26, B37, B40 31 36 A26 11 13 A24 þ extra 42 41 b 41 A24 39, 12 A2 48 51 B7, 13, 48 6 50 B62, 51, 35, 52, 75 55 46 B60, B61 22 25 B51, 52 16 23 B35, 46, 51, 58, 60, 61 67 54 B35 11 32 Cw5 4 2 Mean 6 SD 42.2 6 31.8 47.1 6 29.2 Extra: extra definable specificities. a The measured %PRA was based on the AHG-CDC PRA assay. b Multiple cases for the same specificity are collectively recorded in a cell for convenience.

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Comparison Between the Shoulder and Overlay Method

Table 2 Probability for a Certain HLA Antigen to be Omitted in a Panel of Randomly Selected Donors

Number of donors

1

40 80 120 160

66.9 44.8 29.9 20.0

Antigen frequency (%) 2 3 4 44.6 19.9 8.9 3.9

29.6 8.7 2.6 0.8

19.5 3.8 0.7 0.1

5 12.9 1.7 0.2 0.0

The two analytic methods for the setting of cutoff points on the histogram were compared in 29 FCXM positive sera. The overlay method showed significantly higher values than the shoulder method (P < 0.05; Fig. 5). The statistic data for comparison are presented in the following sections. %PRA of Nonsensitized Individuals

shown in Table 2. The number of donors need for less than 1% probability of omitting an HLA antigen with a 5% antigen frequency was found to be 120. Meanwhile, 459 donors would be necessary to include (i.e., to make the probability of being omitted less than 1%) an HLA antigen with an antigen frequency of more than 1%, as in the traditional creation of a panel for the AHG-CDC PRA assay.

The theoretical cutoff for qualitative determination is 1/40 ¼ 2.5% when the T cells from only one donor are reactive in one tube of 40 donors, or 1/120 ¼ 0.8% with a total of 120 donors in three tubes. Yet, for the total sample of nonsensitized persons, the measured mean/cutoff %PRA was 1.9%/10.0% (overlay) or 0.4%/2.8% (shoulder) (Table 3). The cutoff value was calculated by the mean þ 2SD. There was no significant difference between the healthy individuals and the transplantation candidates using either of the analytic methods (P > 0.05).

Increasing the Number of Donors

Detection of HLA Abs Against Rare HLA Antigens

The effect of increasing the number of donors was evaluated by observing the correlation between the RD FC %PRA using the shoulder method and the expected %PRA in 29 FCXM positive sera. The shoulder method was adopted because of its similarity to the expected %PRA compared with the overlay method. The greater the number of donors included in the mixed lymphocyte pools (40, 80, and up to 120 donors), the higher the correlation observed with the expected %PRA (r ¼ 0.794, 0.808, and 0.812, respectively; P ¼ 1.1 105, 5.4 106, and 4.5 106, respectively) (Fig. 4). Therefore, the remaining RD FC PRA assays were performed with a 120-donor pool.

We investigated whether the numbers of donors used (40) and analyzed lymphocytes (5,000) in each tube were appropriate for the detection of Abs against rare HLA antigens. For example, in the case of the minimal %PRA (14%) using the shoulder method in 29 FCXM positive sera, there was no distinct second peak or shoulder in the positive region on the anti-IgG FITC histogram. Instead, there was a small right shift of the whole peak. The % positive region for the three tubes was 25, 6, and 11%, respectively, and the coefficiency of variance was 72% (Fig. 6B). Conversely, in the case of a %PRA of nearly 100%, there was a substantial shifting of the whole peak (Fig. 6C).

FIG. 4. Increasing the number of donors. The RD FC %PRA (shoulder) approached the expected %PRA as the number of donors increased. The diagonal reference line (y ¼ x) is plotted as a dashed line. The closer %PRA is to this line, the more similar it is to the expected %PRA. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

FIG. 5. Comparison between the shoulder and overlay analytic method for the setting of cutoff points in the RD FC PRA assay. The overlay method showed significantly higher values than the shoulder method (73% 6 21% and 55% 6 28%, respectively; P < 0.05). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]


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Table 3 %PRA of Nonsensitized Persons: Mean and Cutoff (Mean þ 2SD) for Qualitative Determination of the RD FC PRA Assay Mean 6 SD Overlay method Shoulder method Dialysis patients (N ¼ 26) Healthy individuals (N ¼ 7) Total a (N ¼ 33)

2.6 6 3.2 0.5 6 5.9 1.9 6 4.0

0.2 6 0.6 0.2 6 2.4 0.4 6 1.2

Cutoff Overlay method

Shoulder method

9.1 11.3 10.0

1.5 4.5 2.8

There were no significant differences between the patients and healthy individuals (P > 0.05).

a

The coefficiency of variance of the individual % positive regions obtained from the three tubes used per test tended to increase in inverse proportion to the %PRA (Fig. 7), suggesting that the %PRA of Abs against rare HLA antigens was affected by the distribution of the HLA antigens of a lymphocyte pool in a tube. Comparison of the %PRA Measured by Three Assays In 29 FCXM positive sera, the positivity rates of the bead FC, RD FC (overlay), RD FC (shoulder), and AHGCDC PRA were 100, 100, 97, and 79%, respectively. The

mean of %PRA in these subjects was 77% 6 20%, 73% 6 21%, 55% 6 28%, and 33% 6 33%, respectively (Fig. 8). The RD FC %PRA (overlay) was not significantly different from the bead FC %PRA (P ¼ 0.205), and all of the remaining comparisons between the four methods showed statistically significant differences on each paired t test (P < 0.05). This suggests that the overlay method was more sensitive than the shoulder method in the RD FC PRA, and was comparable to the bead FC %PRA. As expected, the AHG-CDC PRA assay was less sensitive than all of the FC PRA assays.

FIG. 6. Anti-IgG FITC histograms using the shoulder method with three tubes per test, showing (A) negative, (B) minimal %PRA, and (C) maximal %PRA. The net % positive region is indicated in each histogram.


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FIG. 7. Coefficiency of variance of % positive regions of three tubes in the RD FC %PRA assay (overlay).

lic specificities of HLA antigens. Rodey et al. (16) reported that 80–90% of the HLA alloantibodies formed by kidney recipients following transplantation are directed to CREG determinants. Moreover, Sutton et al. (6) reported that even Abs against low frequency antigens can be detected by other cells with CREG antigens, even though a lymphocyte pool may not contain cells with the relevant antigens. As shown in Table 2, the probability of omitting an HLA antigen with a frequency of 1% in a panel of randomly selected donors is 29.9% with 120 donors, and the statistically necessary sample size of donors is 459 in order to secure the likelihood of including almost all HLA antigens. This sample size, however, is too large to create a panel. Fortunately, the practical number of donors needed to reach an appropriate diagnostic sensitivity is considered to be far smaller due to the presence of surrogate donor cells with the corresponding CREG antigens.

Comparison of the Measured %PRA Regarding the Expected %PRA as Reference In 29 FCXM positive sera, the %PRA measured using the bead FC, RD FC (overlay), RD FC (shoulder), or AHG-CDC PRA was compared with the expected %PRA reflecting the HLA antigen frequencies in the Korean population. The correlation coefficients, r (P) for the four methods with the expected %PRA were 0.617 (2.2 103), 0.690 (3.8 104), 0.805 (6.1 106), and 0.909 (4.8 109), respectively (Fig. 8B). The P values of each paired t test were 5.6 107, 9.1 107, 0.002, and 0.099, respectively. These indicate that the RD FC %PRA (shoulder) showed higher correlation than the bead FC %PRA or RD FC %PRA (overlay). Comparison with the Bead FC PRA in the Sensitized Patients with a Negative FCXM This comparison was performed in the 19 transplantation candidates potentially possessing HLA Abs, who had histories of transfusions or pregnancy and were donorspecific crossmatch negative. The numbers of the positive cases of the bead FC, RD FC (overlay), and RD FC (shoulder) were 3 (16%), 4 (21%), and 0 (0%), respectively (Table 4). Discrepant results between the bead FC and RD FC (overlay) were observed in three cases: 8.1%/ 10.0% (bead/RD FC), 0.5%/14.1%, and 1.0%/24.4%, respectively. In general, however, the two assays were not significantly different (x2 ¼ 4.46, P ¼ 0.097). This suggests that both assays had nearly the same ability to detect low levels of HLA Abs or low %PRA. DISCUSSION The RD FC PRA positive cases of the relatively small %PRA tended to show a small right shift or skew of the whole peak on the anti-IgG FITC histogram, rather than a distinct second peak or shoulder in the positive region. This may have been because the Abs reacted to the pub-

FIG. 8. The %PRA comparison of the three assays, using the bead FC PRA (A) or expected %PRA (B) as reference. The diagonal reference line (y ¼ x) is plotted as a dashed line. A closer %PRA is to this line, the more similar it is to the reference %PRA. The RD FC PRA (overlay) was not significantly different from the bead FC PRA (P ¼ 0.205). In the comparison with the expected %PRA, the RD FC %PRA (shoulder) showed higher correlation than the bead FC %PRA or RD FC %PRA (overlay). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]


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Table 4 Comparison Between the Bead FC PRA and RD FC PRA Assay (Overlay) in Sensitized Patients with a Negative FCXM a Bead FC PRA assay Positive Negative Total RD FC PRA assay (overlay)

Positive Negative Total

2 (11)b 1 (5) 3 (16)

2 (11) 14 (74) 16 (84)

4 (21) 15 (79) 19 (100)

x ¼ 4.46, P ¼ 0.097. Values given indicate numbers (percentages).

a 2 b

As reported in another study, the overlay analytic method was useful to obtain a more similar value to the bead FC PRA or to increase sensitivity compared with the shoulder method (17). The overlay method was also more sensitive than the shoulder method even though its cutoff %PRA (10.0%) was higher than that of the latter (2.8%). This may have been because the intersection point of the overlay method maximized the % positive region of the differential histogram. Among the subjects with a positive FCXM, neither bead FC PRA nor RD FC PRA (overlay) showed false negativity. The false negativity rates for the RD FC PRA (shoulder) and AHG-CDC PRA were 3 and 18%, respectively. Among the FCXM negative subjects with histories of transfusion or pregnancy, the positivity rate was found to be 16% by the bead FC PRA, and 21% by the RD FC PRA (overlay). Therefore, in regard to diagnostic sensitivity, the RD FC PRA (overlay) was comparable to the bead FC PRA. There were no false positive cases detected by the RD FC PRA in this study. To investigate the differences among the bead FC, RD FC, and AHG-CDC PRA, a serum showing a remarkable difference between the two FC PRA assays was selected (bead FC, 82%; RD FC (overlay), 46%; RD FC (shoulder), 15%; AHG-CDC PRA, 11%). Then, individual donorspecific FCXMs from 13 randomly selected lymphocyte donors were performed. The resulting positivity rate was 85% using a cutoff mean fluorescence intensity ratio of 2.0 (18). Regarding the %PRA, this rate was similar to the bead FC of 82%. The RD FC %PRA (shoulder) was closer to the AHG-CDC %PRA than was the bead FC. When considering that an FCXM that is too sensitive can deprive candidates of the chance of a transplant of cadaveric organs, despite a subclinical level of HLA Abs, the shoulder analytic method may also offer subsidiary information for the %PRA closer to the probability of a positive AHG-CDC crossmatch, rather than that of a positive FCXM. In this study, no HLA class II Abs were detected, as only the reactions to T cells were analyzed. However, it is expected that both class I and class II Abs can be detected if the reactions to B cells are analyzed using a B cell marker. However, in this case, the sera (18) or lymphocytes (19) must be pretreated to decrease nonspecific binding, as B cells have a high tendency to bind non-HLA Abs.


To summarize, the advantages of the RD FC PRA assay we documented are: (1) easy preparation of a lymphocyte panel using randomly selected donors without any HLA typing, (2) cost-effective panel preparation, (3) a %PRA that naturally reflects the population to which the donor actually belongs, (4) function as an alternative assay for ethnic groups, especially those with distinct HLA frequencies from those of commercial beads, and (5) the estimation of closer probabilities to a positive AHG-CDC crossmatch using the shoulder method. Meanwhile, the disadvantages are: (1) the assay procedure is more complex than the bead FC PRA, (2) random donor selection can miss rare antigens unless a sufficient number of donors is secured, (3) lymphocyte pools can become biologically hazardous material if a few donors have infectious diseases, (4) non-HLA-specific Abs can react to lymphocytes, making it difficult to distinguish HLA from non-HLA Abs, and (5) class II Abs cannot be clearly defined using B cells if the serum contains both class I and class II Abs. In conclusion, the PRA assay using lymphocyte pools from randomly selected donors allows easy panel preparation, reduces cost, and naturally reflects the probabilities of a positive crossmatch in the population to which the cadaveric donor belongs. Therefore, this new assay is expected to be useful as another approach used to determine the %PRA. LITERATURE CITED 1. Schonemann C, Groth J, Leverenz S, May G. HLA class I and class II antibodies: Monitoring before and after kidney transplantation and their clinical relevance. Transplantation 1998;65:1519–1523. 2. Duquesnoy RJ, Marrari M. Multilaboratory evaluation of serum analysis for HLA antibody and crossmatch reactivity by lymphocytotoxicity methods. Arch Pathol Lab Med 2003;127:149–156. 3. Kao KJ, Scornik JC, Small SJ. Enzyme-linked immunoassay for antiHLA antibodies—An alternative to panel studies by lymphocytotoxicity. Transplantation 1993;55:192–196. 4. Monteiro F, Buelow R, Mineiro C, Rodrigues H, Kalil J. Identification of patients at high risk of graft loss by pre- and posttransplant monitoring of anti-HLA class I IgG antibodies by enzyme-linked immunosorbent assay. Transplantation 1997;63:542–546. 5. Bray RA, Lebeck LL, Gebel NP, Ting A, Morris PJ. The flow cytometric crossmatch. Transplantation 1985;48:834–840. 6. Sutton PM, Harmer AH, Bayne AM, Welsh KI. The flow cytometric detection of alloantibodies in screening for renal transplantation. Transpl Int 1995;8:360–365. 7. Shroyer TW, Deierhoi MH, Mink CA, Cagle LR, Hudson SL, Rhea SD, Diethelm AG. A rapid flow cytometry assay for HLA antibody detection using a pooled cell panel covering 14 serological crossreacting groups. Transplantation 1995;59:626–630. 8. Cicciarelli J, Helstab K, Mendez R. Flow cytometry PRA, a new test that is highly correlated with graft survival. Clin Transplant 1992;6:159– 164. 9. Gebel HM, Harris SB, Zibari G, Bray RA. Conundrums with FlowPRA beads. Clin Transplant 2002;16 (Suppl 7):24–29. 10. Rebibou JM, Chabod J, Bittencourt MC, Thevenin C, Chalopin JM, Herve P, Tiberghien P. Flow-PRA evaluation for antibody screening in patients awaiting kidney transplantation. Transpl Immunol 2000; 8:125–128. 11. Magee B, Martin J, Middleton D. The repercussions of implementing flow cytometry as a single HLA antibody screening technique in prospective renal transplant recipients. Transpl Int 2006;19:105–109. 12. Fuller TC, Phelan D, Gebel HM, Rodey GE. Antigenic specificity of antibody reactive in the antiglobulin-augmented lymphocytotoxicity test. Transplantation 1982;34:24–29. 13. Johnson AH, Hurley CK, Hartzman RJ, Alper CA, Yunis EJ. HLA: The major histocompatibility complex of man. In: Henry JB, editor. Clinical Diagnosis and Management by Laboratory Methods, 18th ed. Philadelphia: WB Saunders; 1991. pp 761–794.

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WON ET AL.

14. Johnson AH, Hurley CK, Hartzman RJ, Wade JA. Human leukocyte antigen: The major histocompatibility complex of man. In: Henry JB, editor. Clinical Diagnosis and Management by Laboratory Methods, 20th ed. Philadelphia: WB Saunders; 2001. pp 927–948. 15. Kim HS, Hwang YS, Park MH. The distribution of HLA antigens and haplotypes in Koreans. Korean J Clin Pathol 1997;17:1109–1123. 16. Rodey GE, Neylan JF, Whelchel JD, Revels KW, Bray RA. Epitope specificity of HLA class I alloantibodies. I. Frequency analysis of antibodies to private versus public specificities in potential transplant recipients. Hum Immunol 1994;39:272–280. 17. Mentula P, Kylanpaa-Back ML, Kemppainen E, Takala A, Jansson SE, Kautiainen H, Puolakkainen P, Haapiainen R, Repo H. Decreased

HLA (human leucocyte antigen)-DR expression on peripheral blood monocytes predicts the development of organ failure in patients with acute pancreatitis. Clin Sci (Lond) 2003;105:409– 417. 18. Ta M, Scornik JC. Improved flow cytometric detection of donor-specific HLA class II antibodies by heat inactivation. Transplantation 2002;73:1611–1614. 19. Vaidya S, Cooper TY, Avandsalehi J, Barnes T, Brooks K, Hymel P, Noor M, Sellers R, Thomas A, Stewart D, Daller J, Fish JC, Gugliuzza KK, Bray RA. Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation. Transplantation 2001;71:422–428.
