AuswertRhParis10072001.doc             Version 2.0
Rh flow cytometry - Coordinator's report
Subtitle: Rhesus index and antigen density: An analysis of the reproducibility of flow cytometric determination
Running Title: Rh flow cytometry - Coordinator's report
Willy A. Flegel1*, Vladka Čurin-Šerbec 2, Maryvonne Delamaire 3, Béatrice Donvito 4, Hisami Ikeda 5,
Jan Jørgensen 6, Belinda Kumpel 7, Pierre-Yves Le Pennec 8, Martin Písačka 9, Yoshihiko Tani10,
Makoto Uchikawa11, Silvano Wendel12, and Franz F. Wagner 1 *
1	From the Abteilung Transfusionsmedizin, Universitätsklinikum Ulm and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm, Ulm, Germany;
2	Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia;
3	Immuno-Hematology, EFS Bretagne, Rennes, France;
4	Laboratoire d'immunologie, Hôpital Robert Debré, EA 3309, Reims, France;
5	Hokkaido Red Cross Blood Center, Sapporo, Japan;
6	Dept. Clinical Immunology, Aarhus University Hospital, Skejby Sygehus, Aarhus, Denmark;
7	International Blood Group Reference Laboratory, Bristol, UK;
8	Centre National de Reference, INTS, Paris, France;
9	Institute of Hematology and Blood Transfusion, Praha, Czech Republic;
10	Osaka Red Cross Blood Center, Osaka, Japan;
11	Central Blood Center, Japanese Red Cross, Tokyo, Japan; and 
12	Instituto de Hemoterapia, Hospital Sírio Libanês, São Paulo, Brasil.
* WAF and FFW contributed equally to the work. The other authors are listed in alphabetical order and were the investigators of the participating laboratories.
Correspondence to: Willy A. Flegel, Priv.-Doz. Dr. med.; Abteilung Transfusionsmedizin, Universitätsklinikum Ulm, Helmholtzstrasse 10; D-89081 Ulm, Germany; Phone: +49-731-150-600, FAX: +49-731-150-602, email: waf@ucsd.edu
This study was supported by the DRK-Blutspendedienst Baden-Württemberg, Stuttgart; the Universitätsklinikum Ulm (Forschungsförderungsprojekte P.531); and the Deutsche Gesellschaft für Transfusionsmedizin und Immunhämatologie (DGTI/fle/00-01).

Summary

Fifty seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which twelve laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H+L) as a secondary antibody were recommended but not mandatory. An CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal "standard RBC" throughout all experiments. An RBC panel comprising 2 partial D and 4 weak D types was supplied, too. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the twelve participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

Key words: Rhesus; Rh; RHD; RHCE; anti-D; flow cytometry; monoclonal antibody;

Introduction

This is the second Rh flow cytometry section to be an integral part of this series of International Workshops and a follow-up to the previous Rh flow cytometry section of the 3rd Workshop in Nantes 1996 [1, 2]. The aim of the current section was to evaluate several technical features and objectives for their utility in analyzing aberrant RhD phenotypes (Table 1).
Table 1. Evaluation of technical features and objectives for their utility in analyzing aberrant RhD phenotypes
•	use of "standard RBC" as an internal standard
•	determination of antigen D density* for aberrant RhD
•	assessment of assay variability among participating laboratories
•	assessment of the robustness of an Rhesus D similarity index*
*	Epitope density profile and Rhesus D similarity index as described previously [3], see Methods.
The variability of flow cytometry results among laboratories was well recognized [1,4] and an obstacle for the broader use of flow cytometry in the characterization of the molecular polymorphism of both Rhesus proteins [5 - 7]. Part of this variation may be attributed to differences in staining protocols and reagents [1]. In the previous Workshop section 1996 a standard protocol, fluorescent beads and a secondary antibody were mandatory and evaluated to overcome these problems. The supplied secondary antibody was a Fab fragment anti-human IgG (H+L) and was anticipated to reduce the amount of agglutination produced when staining sensitized red blood cells (RBC). Fluorescent beads were supplied, too, and used for calibration in 1996.
In the current Workshop section, controls and data evaluation differed from the previous section (Table 2). Less emphasis was put on protocol and reagent standardization. A secondary antibody was recommended but not supplied; no fluorescent beads were used. A staining protocol as published previously [8,9] was recommended; however, the participants could apply any staining procedure including their "in house" or the 3rd Workshop's staining protocol. Specific instructions for setting up analysis regions were given; however, as a major difference to the previous Workshop section, the analysis regions should include all events. No attempts were made to restrict the analysis region to "positive events". To evaluate the utility of a different layer of standardization, "standard RBC" were provided and to be used in all tests [10]. The calculation of epitope densities was based on the relative fluorescence detected with "test RBC" compared to the "standard RBC" by applying a published algorithm [3] for which a spreadsheet calculation program was provided.

Table 2. Comparison of technical features of the Rh flow cytometry sections 1996 and 2001
Feature	3rd Workshop 1996 (Nantes)	4th Workshop 2001 (current)	
Staining protocol	Mandatory and supplied	Recommended, but not mandatory	
Secondary antibody *	Mandatory and supplied	Recommended, but not supplied	
Fluorescence beads	Mandatory and supplied	Not used	
"standard RBC" **	Not used	Mandatory and supplied	
"Negative events"	Excluded from analysis	Included in flow cytometric analysis	
*	Fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H+L).
**	For internal control.
IgM monoclonal antibodies were generally spared from analysis to avoid problems caused by direct agglutination [9, see Fig. 2d]. There were 66 IgG anti-Rh monoclonal antibodies available in at least one laboratory, of which 60 were anti-D representing 56 different anti-D clones. The testers in the twelve participating laboratories of this Workshop section elected to evaluate between 22 to 55 different anti-D; including two IgM anti-D (BS223 and BS225) tested in one lab. The aggregate analysis of their results is presented in this coordinator's report. The complete set of original data is available online (see Methods). In addition, seven groups of testers detailed their individual results, which were presented as separate reports [11 - 17]. Kumpel and Jackson [14] provided a detailed study with discussion of the literature on the influence of the sensitization procedure on the quantification of IgG bound to RBC.

Materials and Methods
Testers and antibodies
Like in the Nantes 1996 Workshop section, twelve laboratories participated in the flow cytometry evaluation (Table 3). The monoclonal antibodies were supplied as culture supernatant, most of which with 0.1% sodium azide added as preservative. Although it might have been feasible to use some of the monoclonal antibodies in diluted form (particularly when evaluating RBC with strong antigens), results were to be provided for the undiluted antibody to ensure that near saturating conditions were used. Two monoclonal antibodies were supplied more than once: Fog-1 and H1121G6; the two preparations most often used by the laboratories were chosen (1-54 and 1-95), whilst the other preparations were excluded from all analyses (1-17; 1-18; 1-19; and 1-67). The set of monoclonal anti-D used differed much among the laboratories. However, twelve antibodies (1-23, 1-24, 1-25, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-72, 1-73, 1-75) had been investigated by all laboratories.
Table 3. Evaluating laboratories and their instrumentation

Laboratory	
Manufacturer	
Model	
Software	Log
Scale	No. of 
Channels	Voltage
(V) 1	
JHM Cohen	Becton Dickinson	FACSTAR plus	CELLQuest	4	1,024	575	
V Curin-Serbec	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	798	
WA Flegel	Becton Dickinson	FACSort	CELLQuest	4	1,024	632	
T Ikeda	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	-	
J Jorgensen	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	590	
B Kumpel	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	700	
PY Le Pennec	Becton Dickinson	FACScan	CELLQuest	4	256	600	
A Martin	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	- 	
M Pisacka	Coulter	Epics XL	Coulter Analysis II 2	4	1,024	- 	
Y Tani	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	745	
M Uchikawa	Ortho	CYTORON
ABSOLUTE	ImmunoCount 2.2	3	256	- 	
S Wendel	Becton Dickinson	FACS Calibur	CELLQuest	4	1,024	- 	
1	Reporting of PMT (photomultiplier tube) voltage was optional; -, not reported.
2	In conjunction with WinMDI (Windows Multiple Document Interface for Flow Cytometry) Version 2.8, see <http://facs.scripps.edu/software.html>.

In addition to the 60 anti-D, there were one IgG anti-C, one anti-c; three anti-E; one anti-CD; one anti-D/G and one anti-G. No anti-e of IgG class was submitted to the Workshop. Data for anti-Rh other than anti-D and for the two scFv reagents were not analyzed in this report, because the limited number of antibodies tested and the limited number of testing laboratories did not allow a thorough analysis.

Cell panels
A panel of seven different RBC suspensions was distributed to all testers. The RBC were fresh (not frozen/thawed) and mailed to the testers in September 2000 within two weeks after being drawn. The RBC comprised 4 samples of weak D (2 weak D type 1, 1 weak D type 2 and 1 weak D type 3), 2 samples of partial D (D category VI type III and D category VII) and 1 control of CcDEe phenotype ("standard RBC").
It was recommended to aliquot and to freeze all RBC samples immediately upon receipt. Any analyses with reconstituted RBC were to be performed within 5 days after thawing and reconstitution. Otherwise, the RBC were to be kept at +4°C and analyzed within 3 weeks after receipt. These tight time schedules should have avoided any potential bias, as it was known that antigen densities may be diminished or increased after prolonged storage.
For the six aberrant RHD distributed by the Workshop, antigen density and Rhesus index (RI) had previously been determined in the coordinator's laboratory using a different set of monoclonal anti-D and different samples [3, see Table 5]. For DVI type III, results were an antigen density of 14,502 antigens/cell and an RI of 0; for DVII 8,398 antigens/cell, RI 0.48; for weak D type 1 1,285 antigens/cell, RI 0.57; for weak D type 2 489 antigens/cell, RI 0.68; for weak D type 3 1,932 antigens/cell, RI 0.77. The weak D type 2 sample of the Workshop cell panel represented inadvertently the sample with the highest antigen density of all 24 weak D type 2 donors tested and thus likely carried an higher antigen density than most weak D type 2 samples.
The testers were encouraged to include additional RBC samples in their testing. For each monoclonal antibody, those RBC samples should be analyzed along with the RBC samples provided by the Workshop in the same experiment (during the same flow cytometer session). Results for additional RBC are presented in separate reports [13,15 - 17].
"Standard RBC" as internal standard
One sample of CcDEe phenotype ("standard RBC") that was shown to be RHD homozygous by molecular methods [18] was provided as positive control. The antigen density of this CcDEe control sample (no. 63 86 148) was determined to be 27,500 antigen D per RBC by comparing to our previous standard RBC [3,19]. These standard RBC were to be prepared twice independently; they were to be used in all tests as internal standard and tested twice during each flow cytometer session (at the beginning and at the end of each flow cytometer session).
The results of both testings should not deviate from each other by more than 15%. It was recommended to repeat the whole flow cytometer session if both test result differ by more than 15%; however, it remained at the tester's discretion to submit the results of such a session without repetition.

RBC as negative control
One sample of ccddee phenotype was required as negative control ("background") but not provided by the Workshop. In difference to the previous Workshop, a pool of ccddee samples should not be used as negative control.
Staining protocol
A staining protocol was recommended (Table 4) but testers were free to choose any staining procedure that they were used to and most familiar with ("in house" protocol) or the 3rd Workshop's staining protocol [1]. As secondary antibody an Fab fragment antibody, preferably an Fab fragment anti-human IgG (H+L) antibody, fluorescence-labeled was mandatory but not provided by the Workshop.
Table 4. Recommended staining procedure *
1.	Prepare 2% suspensions of sample RBC in, for example, LISS or PBS **.
2.	Prepare 2% suspensions of control RBC in, for example, LISS or PBS.
3.	Mix 50 L of RBC suspension with 50 L of primary antibody (usually undiluted).
4.	Incubate at 37°C for 30 min. 
5.	Wash twice by hand.
6.	Add 50 L of diluted secondary antibody (e.g., FITC-labeled Fab fragment anti-human IgG (H+L) antibody 1:120).
7.	Incubate at 20°C for 30 min.
8.	Wash twice by hand.
9.	To achieve disaggregation of potential red cell agglutinates, aspirate the suspensions twice through a 23 G needle.
10.	Immediately before flow cytometric analysis, re-suspend the samples by vortexing.
11.	Measure 20,000 RBC by flow cytometry.
*	See <http://www.uni-ulm.de/~wflegel/RH/RBCmethods/fluostain.htm> and [8].
**	LISS - low ionic strength solution; PBS - phosphate buffered saline.
Flow cytometric analysis
Alike the staining protocol, a protocol for flow cytometric analysis was recommended (Table 5 and Figure 1) but testers were free to choose their own protocol.
Table 5. Flow cytometric analysis
1.	Collect the data on 20,000 events for each sample. 
2.	The "Analysis Region" or "Marker" should be set to count all events observed in the analysis (see Figure 1). This Analysis Region may include Channel 0 or the lowest channel of the flow cytometer. For most flow cytometers it is appropriate to select "All".
3.	Important: Do not try to exclude events representative for the negative control.
4.	Use median result (not mean or geometric mean) for spreadsheet calculation.
Figure 1. Example for marker setting. The analysis regions include all events ("All" or "M1"); no attempts are made to restrict the analysis region to "positive events" ("M2"). Median fluorescence data were used for spreadsheet calculations.

Reporting of results and evaluation software
A Microsoft EXCEL spreadsheet version 1.0 was provided for the documentation of fluorescence data. The spreadsheet provided entries for the two CcDEe control tests to be performed with the "standard RBC", for the six variant RBC samples provided by the Workshop and for up to three additional RBC samples of the testers own choice. Median fluorescence as depicted in Figure 1 had to be entered. Additional data, like mean, geometric mean, peak channel and coefficient of variation, could be entered and might be used to resolve possible discrepancies. The calculation of epitope densities was based on the median fluorescence detected with "test RBC" relative to the "standard RBC". All calculations of this study were done using the spreadsheet version 1.1. A blank spreadsheet may be retrieved at: <http://www.uni-ulm.de/~wflegel/RH/Paris2001/>.

Calculation of antigen density on RBC
Details of the algorithm were described previously [3, 19]. The antigen density (antigens per cell) of a RBC sample was estimated as the median of the epitope densities detected with all antibodies that resulted in epitope densities above a cutoff. The cutoff was defined as 0.1 of the 90 percentile of the epitope densities detected with all monoclonal anti-D used [3].
Using this algorithm the relative antigen densities were automatically computed from the ratios of the median fluorescence data that were entered into the spreadsheet. Based on the relative antigen density, the absolute antigen densities were calculated by the spreadsheet applying the known antigen density of the standard RBC sample (27,500 antigens/cell) that was utilized as internal control in all experiments.
Determination of the Rhesus D similarity index
A Rhesus index was calculated as the ratio of the 10 percentile to the 90 percentile of the epitope densities detected with all anti-D [3]. The Rhesus index was derived as a measure of the variation of the epitopes detected by different antibodies under the assumption of an approximately log-normal distribution. The Rhesus index may range from 1 in normal D carrying all D epitopes (epD) to 0 in partial D that was lacking most epD.

Results and Discussion
Utility of using "standard RBC" as an internal standard
Twelve antibodies were used by all laboratories. These antibodies were tested with the six RBC samples of the cell panel. Whilst the results are expected to differ depending on anti-D and RBC sample, for each pair of anti-D/RBC the result among the twelve laboratory should be identical under ideal conditions. Then, the coefficient of variation (CV) of the results among the twelve laboratories would be 0. Larger CV implies inter-laboratory variability, which of course always occurs and is of practical importance.
We evaluated the utility of "standard RBC" as a new technical feature of this Workshop flow cytometry section to reduce this inter-laboratory variability, which may be gauged by an "inter-laboratory CV". For each RBC sample, median and range of the inter-laboratory CV obtained with the twelve different anti-D were calculated. This calculation was done for the (non-standardized) fluorescence detected and the (standardized) epitope density (Table 6).
Table 6. Decrease of the coefficients of variation (CV) achieved by using an RBC sample as internal standard
	"inter-laboratory CV" for the results of twelve laboratories 1	
	Fluorescence detected		Standardized epitope density 2	
Aberrant RhD	Median	Range		Median	Range	
Weak D type 2 3	48%	43% - 59%		27%	19% - 56%	
D category VII	57%	50% - 80%		27%	20% - 58%	
D category VI	40%	34% - 67%		113% 4	26% - 157%	
1	Median and range of the CV determined with the twelve anti-D that were tested by all laboratories.
2	Standardized by using the "standard RBC" sample provided by the Workshop section expressing 27,500 antigens/cell and as described in the Methods.
3	Representative results for the 4 samples of weak D.
4	CV was 26% for both antibodies with appreciable binding.
Generally, the standardized epitope densities had lower coefficients of variation (CV) than the fluorescence detected (non-standardized) for all six partial D and weak D samples (three of which are not shown). The single exception was D category VI type III. Ten anti-D that did not bind had larger CVs, which was explained by the almost zero epitope densities obtained after subtraction of background fluorescence. For the two anti-D with appreciable binding to DVI, the CVs were reduced to 26% and thus in a similar range like the CV for weak D samples. We conclude that the use of standard RBC reduced the variability of the data among the laboratories. Moreover, the fluorescence data could be converted into epitope densities and represented as antigens/cell (see below).
Determination of antigen D density for aberrant RhD
The antigen densities were determined for each laboratory and aberrant RhD sample (Table 7). Median and range of these results were calculated (Table 8). The highest median antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest median antigen density was determined for weak D type 1 sample A.
The results obtained in this Workshop section correlated with previous experiments [3] that utilized a different set of monoclonal antibodies and different samples for the partial D and weak D. With the exception of the weak D type 2, the maximum deviation was less than 30% for the median antigen densities. The weak D type 2 sample may have deviated by a wider margin, because it represented inadvertently a sample with an unusually high antigen density (see Methods).
Table 7. Antigen densities by laboratory
		Antigen density of aberrant RhD (antigen D per cell)	
	No. of	Partial D		Weak D types	
Lab	MoAb	DVI type III	DVII		Type 3	Type 1 B	Type 1 A	Type 2	
A	50	15,242	9,441		3,087	1,542	1,457	1,277	
B	48	14,106	8,213		2,440	1,152	903	947	
C	35	13,945	6,370		2,004	922	814	842	
D	22	13,940	8,951		2,401	894	977	1,044	
E	54	13,539	7,101		1,987	1,026	907	864	
F	38	12,450	7,496		2,361	921	856	1,023	
G	50	11,805	8,672		2,866	1,465	1,211	1,226	
H	50	11,534	7,041		1,908	898	754	688	
I	40	11,462	5,585		1,885	726	592	797	
K	55	10,191	6,671		1,887	1,057	830	781	
L	30	9,581	7,787		2,444	1,036	867	944	
M	45	8,299	5,541		1,800	963	964	971	
Table 8. Aggregate statistics of antigen densities for all monoclonal antibodies tested
Sample	Mean	SD	Range	CV	
DVI type III	12,175	2,090	8,299 - 15,242	17%	
DVII	7,406	1,264	5,541 - 9,441	17%	
Weak D type 3	2,256	416	1,800 - 3,087	18%	
Weak D type 1 B	1,050	237	726 - 1,542	23%	
Weak D type 1 A	928	221	592 - 1,457	24%	
Weak D type 2	950	175	688 - 1,277	18%	

Assessment of assay variability among the laboratories
The aggregate statistics (Table 8) showed a standard deviation (SD) of about 20% of the mean (i. e., CV of about 20% or 0.20). Further analysis of the assay variability among the laboratories was done to delineate possible causes of this variability. Three aspects were evaluated: Comparison for twelve antibodies used by all laboratories; systematic variation among the different laboratories; and order of antigen densities within the same laboratory.
(i) Twelve antibodies investigated by all laboratories. Restricting the analysis to twelve antibodies (see Methods) did hardly affect mean antigen densities of DVII and weak D samples (Table 9). These results indicated that the antigen densities were not much influenced by the number of monoclonal antibodies used. The CV became smaller, because the influence of varying sets of monoclonal anti-D was eliminated. In contrast, the CV for DVI type III increased considerably, because the number of monoclonal antibodies in the set of 12 anti-D that were actually binding to this partial D became too low for a reliable antigen density determination.

Table 9. Aggregate statistics of antigen densities for twelve monoclonal antibodies
Sample	Mean	SD	Range	CV	
DVI type III	11,366	3,279	4,332 - 16,135	29%	
DVII	7,489	1,427	5,620 - 10,220	19%	
Weak D type 3	2,192	364	1,828 - 2,878	17%	
Weak D type 1 B	1,087	202	809 - 1,510	19%	
Weak D type 1 A	937	176	732 - 1,303	19%	
Weak D type 2	936	166	708 - 1,221	18%	

(ii) Systematic variation among the different laboratories. There was some systematic influence based on the laboratory (Table 7). For example, laboratory A detected the highest antigen densities for all aberrant RhD. This effect did not seem to be caused by instruments or flow cytometry setting. However, factors like different saturation rates for monoclonal antibodies [14] or handling of the standard RBC may well cause the observed systematic variation among some laboratories and will need further attention to improve standardization among laboratories.
(iii) Order of antigen densities within the same laboratory. The influence of the laboratory as mentioned in the previous paragraph could have explained part of the observed antigen density overlap. The ranking of the antigen densities is shown for each laboratory (Table 10). The ranking of antigen densities was DVI type III > DVII > weak D type 3 > weak D type 1/type 2 without exception in all laboratories. The variable ranking of the weak D type 1 and type 2 samples indicated the lack of a reproducible difference among these molecularly defined weak D samples.
Table 10. Ranking of antigen density
	Rank of antigen density in each laboratory	
	Partial D		Weak D types	
Lab	DVI type III	DVII		Type 3	Type 1 B	Type 1 A	Type 2	
A	1	2		3	4	5	6	
B	1	2		3	4	6	5	
C	1	2		3	4	6	5	
D	1	2		3	6	5	4	
E	1	2		3	4	5	6	
F	1	2		3	5	6	4	
G	1	2		3	4	6	5	
H	1	2		3	4	5	6	
I	1	2		3	5	6	4	
K	1	2		3	4	5	6	
L	1	2		3	4	6	5	
M	1	2		3	6	5	4	
Robustness of an Rhesus D similarity index (RI)
The Rhesus indices were determined for each laboratory and aberrant RhD sample (Table 11). Median and range of these results were calculated (Table 12). The absolute values for the Rhesus index varied considerably among the laboratories. However, nine of the twelve participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices: D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. As with the antigen densities, the results for RI obtained in this Workshop section correlated with previous experiments [3] that utilized a different set of monoclonal antibodies and different samples for the partial D and weak D types. Weak D type 2 made no exception in regard to RI (in difference to antigen density, see above).
Basing the RI determination on the twelve monoclonal antibodies (see Methods) decreased the reproducibility of the RI among the laboratories (Table 11, data in parentheses), indicating that a sufficiently high number of monoclonal antibodies, preferably directed to many different epD, is advantageous for RI determination.
The results from laboratory D deviated considerably from the main trend. Inspections of its raw data (not shown, but retrievable online from the website) revealed that at least two anti-D binding to weak D type 3 and one anti-D binding to weak D type 2 the reported fluorescence results were  below background, raising the possibility of technical issues. In difference to RI, these issues won't affect the antigen density determination, though, because of the robustness of the median to fluorescence data outliers.
Table 11. Rhesus indices by laboratory
		Rhesus index of aberrant RhD 1	
	No. of	Partial D		Weak D types	
Lab	MoAb	DVI type III	DVII		Type 3	Type 1 B	Type 1 A	Type 2	
A	50	0 (0)	0.42 (0.63)		0.59 (0.58)	0.41 (0.58)	0.45 (0.52)	0.60 (0.57)	
B	48	0 (0)	0.48 (0.38)		0.70 (0.74)	0.45 (0.65)	0.44 (0.54)	0.77 (0.80)	
C	35	0 (0)	0.32 (0.48)		0.57 (0.59)	0.45 (0.50)	0.39 (0.61)	0.61 (0.74)	
D	22	0 (0.01)	0.43 (0.35)		0.03 (0)	0.25 (0.41)	0.04 (0.33)	0.25 (0.36)	
E	54	0 (0)	0.61 (0.67)		0.75 (0.92)	0.54 (0.78)	0.60 (0.74)	0.71 (0.85)	
F	38	0 (0)	0.37 (0.49)		0.48 (0.71)	0.31 (0.45)	0.30 (0.49)	0.56 (0.65)	
G	50	0 (0.01)	0.48 (0.67)		0.60 (0.70)	0.42 (0.63)	0.45 (0.68)	0.62 (0.73)	
H	50	0 (0)	0.38 (0.53)		0.76 (0.84)	0.32 (0.62)	0.29 (0.57)	0.73 (0.78)	
I	40	0 (0)	0.36 (0.64)		0.75 (0.84)	0.35 (0.52)	0.38 (0.55)	0.74 (0.81)	
K	55	0.01 (0.01)	0.47 (0.65)		0.84 (0.81)	0.74 (0.74)	0.73 (0.76)	0.81 (0.86)	
L	30	0 (0)	0.53 (0.71)		0.64 (0.66)	0.63 (0.71)	0.45 (0.61)	0.56 (0.57)	
M	45	0.01 (0.01)	0.48 (0.65)		0.55 (0.71)	0.39 (0.44)	0.49 (0.61)	0.44 (0.63)	
1	The Rhesus index as determined by all monoclonal antibodies (MoAb) is shown. Determination by the twelve monoclonal antibodies is given in parentheses.

Table 12. Aggregate statistics of Rhesus indices for all monoclonal antibodies tested
Sample	Mean	SD	Range	CV	
DVI type III	0.003	0.004	0 - 0.01	137%	
DVII	0.44	0.08	0.32 - 0.61	18%	
Weak D type 3	0.60	0.21	0.03 - 0.84	35%	
Weak D type 1 B	0.44	0.14	0.25 - 0.74	32%	
Weak D type 1 A	0.42	0.17	0.04 - 0.73	41%	
Weak D type 2	0.61	0.16	0.25 - 0.81	25%	

Lack of correlation between Rhesus index and antigen density
We pondered the possibility that the variable antigen densities determined in the laboratories might have affected the RI determination. The correlation coefficients among the laboratories between antigen density and RI were computed for each sample tested. For samples with non-zero RI (weak D types and DVII) , r2 ranged from 0.002 (weak D type 1, sample A) to 0.20 (weak D type 2) excluding a relevant confounding among both variables. In DVI  type III, RI was 0 in ten of twelve laboratories; r2 was formally calculated as 0.45 (r = -0.67).
Conclusion
The testers in the twelve participating laboratories of this Workshop section evaluated between 22 to 55 different anti-D in flow cytometry with a RBC panel comprising 2 partial D and 4 weak D types. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities represented as antigen density (antigen D per RBC).
The panels of anti-D used had little effect on the determination of antigen density. However, for grossly distorted antigens like in D category VI a sufficient number of anti-D that can bind the partial‑D need to be included in a panel. Inter-laboratory comparison of antigen density data seemed possible, when a variability typical for biologic assays is acceptable.
Technical issues had more influence on the determination of the Rhesus index. Hence, a sufficient number of anti-D is mandatory and a large panel of anti-D should be used. Comparison of aberrant RhD within a laboratory seemed possible, but at the current state of standardization inter-laboratory comparison of Rhesus indices should be restricted to an ordinal scale. A representative set of aberrant RhD with known Rhesus indices should be used as reference.
The results of the twelve laboratories showed that the previously published [3] antigen densities as well as the Rhesus index difference between weak D type 1 and type 2/type 3 were reproducible, whilst using different samples and a different set of anti-D. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

Acknowledgement
Several parts of this Workshop section were based on the work of the previous Rh flow cytometry section of the 3rd Workshop in Nantes 1996, which was coordinated by Dr. Anne Fletcher. The coordinator acknowledges the expert technical assistance of Marianne Lotsch, Anita Hacker, Sabine Kaiser and Sabine Zahn in Ulm. The head of the laboratories were in Reims Jacques H. M. Cohen and in Rennes Anne Martin. The principal investigators of the participating laboratories thank the following persons for their contributions to the work: Ruth R. Rupreht (VCS); Toru Miyazaki (HI); Lene Christiansen (JJ); David J Jackson (BK); Claude Patereau (PYLP); Yuri Marinov (MP); J. Takahashi (YT); and Rita Fontão Wendel (SW).

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