4th International Workshop on Monoclonal Antibodies
against Human Red Blood Cells and Related Antigens

Paris 2001

Section 1 B - Rh Flow Cytometry - Version 1.1

1.	Introduction
You have elected to evaluate some or all of the monoclonal antibodies (MoAbs) in the Section 1 Rh by flow cytometry. There are 80 IgG anti-Rh in all. Anti-D comprise 65 MoAbs; there are also 4 MoAbs of anti-C or anti-c specificity, and 8 MoAbs of anti-E. The specificity of one MoAbs each was designated anti-CD, anti-D/G and anti-G, respectively. No anti-e of IgG was submitted to this workshop. For a very few of these MoAbs their Ig type has not been provided. Please consider that they may actually be IgM, if you have problems with agglutination or with labeling by the secondary antibody.
You are asked to evaluate all IgG anti-Rh MoAbs that you receive. Please try to coordinate your laboratory that the Workshop testing can best be done between 15 September and 30 October 2000.
1.1	Link to the previous 3rd Workshop's Rh flow cytometry section
It is well known that there is variation between laboratories when performing flow cytometric analyses. Some of this variation may be attributed to differences in staining protocols and reagents. In the previous 3rd Workshop the participants endeavored to overcome a number of these problems by adhering to the same basic protocol. In order to provide comparable data of the highest quality, it was recommend that the standard protocol was used in combination with fluorescent beads and a second antibody supplied. The second antibody was a Fluorescein (FITC)-conjugated Fab Fragment Goat Anti-Human IgG (H+L) and was anticipated to reduce the amount of agglutination produced when staining sensitized red cells. Fluorescent calibration beads should have been run at the commencement and at the end of each day's work. They should also be run again if the instrument settings are adjusted during the day. Both reagents had been provided to the previous workshop by companies. 
A number of these features will be utilized in the current 4th Workshop: A fluorescence-labeled Fab fragment anti-human IgG (H+L) antibody should be used as secondary antibody throughout (there is an exception for scFv clones). You may choose to use the 3rd Workshop's staining protocol, which is reproduced below. If you are going to use fluorescent calibration beads, which is optional, please provide the pertinent data. However, less emphasis will be put on protocol and reagent standardization, and the use of fluorescent beads will be optional.
1.2	New features of the current Workshop
To evaluate the utility of another layer of standardization, "control RBC" (red blood cells; see paragraph 6.2.1) will be provided and must be used in all tests. The calculation of epitope densities will be based on the relative fluorescence detected with "test RBC" in comparison to the "control RBC". Hence, the "controls" differ quite a bit from the 3rd Workshop's procedure. Please apply this new feature of the current Workshop carefully, as it is a major difference and will be most critical for the data evaluation. 
There are two scFv clones (single chain fragments; Workshop no. 1-6 and 1-71). Neither MoAb may be detected by an anti-human IgG (H+L) antibody. Both MoAbs carry two "tags": 6-His and c-myc. To label these clones, use the anti-c-myc-antibody (no. 9E10) that was supplied. Please note, that an anti-mouse antibody, FITC-labeled, is required. Don't hesitate to contact me, if you have questions how to handle these two clones.

2.	Aim
To evaluate the following technical features and objectives for their utility in analyzing Rh variant phenotypes: a) "control RBC" as internal standards, b) determination of antigen D density (epitope density profile) of RBC by using control RBC, c) assessment of assay variability among the participating laboratories, and d) assessment of the robustness of an "Rhesus D similarity index".

3.	Reagents supplied to you by the Workshop
3.1	Monoclonal antibodies
The MoAbs are shown on the enclosed spreadsheet ("Rh1Bantibody.xls") and will be sent to you directly from the submitters. Information about the code number and subclass of the MoAb is also found in this file. At the time of writing (August 2000) it is not known whether all the offered MoAbs will actually be distributed. The number of MoAbs sent to you to evaluate may therefore be less than the enclosed list. The antibodies are supplied as culture supernatant containing 0.1% azide as preservative. Many of the MoAbs have been dispatched to you by the submitters, the remaining MoAbs will hopefully be sent soon. 

3.2	Control RBC
I am currently collecting a set of up to ten different RBC samples, including "control RBC" samples and RBC of various molecularly defined weak D and partial D phenotypes, and will be sending this set in September 2000. You will be notified by email one week before the samples are expected to arrive in your lab. 

4.	Reagents required but not provided by the Workshop
As less emphasis is put on standardizing the reagents and protocols, you will have to procure your own secondary antibodies and, if required, your fluorescent calibration beads. 
1.	Secondary antibody must be an Fab fragment antibody, preferably an Fab fragment anti-human IgG (H+L) antibody, fluorescence-labeled (for 78 IgG MoAbs). Please provide exact details for this reagent (for example, see Figure 1, page 5).
2.	For the two scFv clones, use the anti-c-myc-antibodies (no. 9E10) that was supplied. Secondary antibody should be an anti-mouse antibody, fluorescence-labeled (not supplied). Please provide exact details for the latter reagent.
3.	As control RBC, one RBC sample of ccddee phenotype (negative control for anti-D, anti-C and anti-E antibodies) and one RBC sample of ccDEe phenotype and (positive control for anti-D, anti-c and anti-E antibodies).
4.	Feel free to include any additional RBC samples in your testing and add the data to the spreadsheet. For each MoAb, your RBC samples should be analyzed along with the RBC samples provided by the Workshop during the same experiment (during the same flow cytometer session).
5.	Fluorescent calibration beads (optional).

5.	Reagent storage and handling
5.1	Monoclonal antibodies (MoAbs) 
It is recommended that the MoAbs are stored at +4°C, and precautions are taken to avoid microbial contamination when sampling. 

5.2	RBC provided by the Workshop
It is strongly recommended to aliquot all RBC samples that will be provided through Rh Flow Cytometry Section and freeze them immediately upon receiving the samples. Any analyses with reconstituted RBC must be performed within 5 days after thawing and reconstitution. Freezing and storing may be done on liquid nitrogen or - for the limited storage time involved - at ‑80°C.
If you are not familiar with freezing RBC samples, they should be kept at +4°C and all analyses must be done as soon as possible but no later than 3 weeks after receiving the RBC samples. It is known that antigen densities may be diminished or increased after prolonged storage, which may potentially introduce a major bias in the analyses.
Please document carefully, how long RBC samples were kept at +4°C before being frozen or, if you don't freeze them, before being tested.

6.	Instructions for flow cytometry methods
Some of the workshop antibodies may be able to be diluted (particularly when evaluating strongly antigen positive RBC). However, you are asked to supply your results for the undiluted antibody to ensure that near saturating conditions are used for all antibodies. 
Please feel essentially free to choose the protocols that you are used to and most familiar with. You may use one of the following protocols. Please provide details, which of the following protocols you were using or, if you prefer, provide a copy of your own protocol.

6.1	Staining Procedure
6.1.1	Method 1 (adopted from the 3rd Workshop's protocol): 
1.	Stain 2 x 106 cells (as a pellet or in a volume of not more than 50 L of PBS/0.1% BSA/0.1% NaN3 with 50 L of antibody. Incubate cells with antibody for 30 min at room temperature.
2.	Wash the cells at least twice using PBS/0.1% BSA/0.1% NaN3 or your own preferred washing buffer.
3.	Stain with 20 L of the second antibody. The second antibody should be used at a dilution of 1:100 in PBS/0.1% NaN3. Incubate cells with antibody for 30 min at room temperature.
4.	Wash and resuspend in fixative (1% paraformaldehyde). Fixation is optional.

6.1.2	Method 2 (recommended protocol, adopted from Wagner & Flegel 1997): 
1.	Prepare 2% suspensions of sample RBC in, for example, PBS.
2.	Prepare 2% suspensions of control RBC in, for example, 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.

6.2	Mandatory: Control RBC
6.2.1	Positive control RBC (will be provided by the Workshop)
You will receive a RBC sample of CcDee phenotype, which must be used as positive control and must prepared twice (independently) and must be tested twice during each flow cytometer session. Please test this RBC sample at the beginning and at the end of each flow cytometer session. The results of either testing should not deviate from each other by more than 15%. It is recommended to repeat the whole flow cytometer session if both test result differ by more than 15%; however, it remains at your discretion to still submit the results of such a session without repetition. Please note the exact test result of this control RBC in the appropriate fields at the beginning and at the end of the spreadsheets.
Those control RBC may be very useful for your future testing: You may want to keep many surplus aliquots as frozen stock.

6.2.2	Negative control RBC (mandatory, but not provided by the Workshop) 
RBC negative for the appropriate antigen (e.g. ccddee for anti-D) should be run as a negative control. Do not use a pool of ccddee. Rather use a RBC sample of an individual ccddee donor. Keep two or three samples of this control frozen until the end of the Workshop, in case that DNA or epitope analysis of your ccddee control should be required. 

6.3	Optional: Calibration Standards (reagents not provided by the Workshop) 
1.	Shake the bottle of calibration standards vigorously and place one drop in a sample tube.
2.	Add 0.5 mL of diluent used in the final sample suspension to the standards,
3.	Vortex the suspension prior to acquiring the data in the flow cytometer.
4.	Collect ungated data on 20,000 events for the calibration standards.
5.	Make fresh preparations each time calibration data is acquired (i.e. beginning and end of work day).
6.	Gate the singlet population and determine the mean peak channels of each of the five populations of mircobeads.
7.	Enter the instrument, PNT setting and peak channels of standards into the spreadsheet to be provided.

6.4	Flow cytometric analysis
1.	Collect the data on 20,000 events for each sample. 
2.	The "Analysis Region" or "Marker" should be to count all events observed in the analysis (see Figure 2, page 6). Please note: This Analysis Region may include Channel 0 or the lowest channel on your flow cytometer. For most flow cytometers it is appropriate to select "All" for evaluation.
3.	Please note: Do not try to exclude events representative for the negative control. (This is a major difference to the approach taken during the 3rd Workshop.)
4.	Please supply the median result (not mean or geometric mean). There is no need to supply mean channel number, geometric mean or coefficient of variation (CV); however, you may add these data to the spreadsheet, if you prefer, and these data may be used to resolve possible discrepancies.
5.	A disk containing an EXCEL spreadsheet for data entry will be sent to you no later than the set of Workshop RBC samples.

6.5	Determination of antigen density on RBC
The relative antigen densities will be automatically computed from the median fluorescence ratios in the spreadsheet. Using this relative antigen densities, the absolute antigen densities will be calculated in the spreadsheet using the known antigen density of the control RBC. 

7.	Reporting of results
Record results on the Microsoft EXCEL spreadsheet that will be provided in due time. The file is named RHFLOW.XLS. The sheet can be printed out, photocopied and used for recording results at the bench as you work.
As you complete one spreadsheet, save it as an EXCEL file under a new file name, e.g. COHEN.XLS. In this way you will create one copy of the spreadsheet for your results. The spreadsheet will automatically calculate the antigen densities in "antigen D per cell" from the numerical scores entered. All data should be entered in one spreadsheet, if possible.
Please supply full details of the red cells you have tested in addition to the RBC samples provided by the workshop. These details should usually include the molecular structure of the RHD gene.
Please retain a copy of your own results.

7.1	Deadline for data submission
The results of your experiments should be sent by 30 Oct 2000. Return copies of your results by email to: willy.flegel@medizin.uni-ulm.de

8.	References
Avent ND. The Rhesus blood group system: insights from recent advances in molecular biology. Transfus Med Rev 13(1999)245-266
Fletcher A, Bryant JA, Stavliotis M. Coordinator's report on the flow cytometric analysis of the Rh antibodies. Transfus Clin Biol 6(1996)407-413
Fletcher A et al. Rh Flow Cytometry. Transfus Clin Biol. 6(1996)405-435
Jones JW, Lloyd-Evans P, Kumpel BM. Quantitation of Rh D antigen sites on weak D and D variant red cells by flow cytometry. Vox Sang 71(1996)176-183
Scott ML, Voak D, Liu W, Jones JW, Avent ND. Epitopes on Rh proteins. Vox Sang 78, suppl 2 (2000)117-120
Wagner FF, Flegel WA: Analysis by flow cytometry of chimerism after bone marrow transplantation and of erythrocyte antigen density. In: Gutensohn K, Sonneborn HH, Kühnl P (Eds.) Aspects of the Flow-Cytometric Analysis of Red Blood Cells. Clin Lab Publications 1997, ISBN 3-9805785-1-8
Wagner FF, Flegel WA: Principles and applications of red cell flow cytometry. Infusionsther Transfusionsmed 25(1998)342–346
Wagner FF, Frohmajer A, Ladewig B, Eicher NI, Lonicer CB, Muller TH, Siegel MH, Flegel WA. Weak D alleles express distinct phenotypes. Blood 95(2000)2699-2708

9.	Acknowledgment
Several parts of this manuscript were written by closely following the protocol of the previous 3rd Workshop in Nantes 1996 by Dr Anne Fletcher, Sydney, and the Rh serology protocol by Dr Marion Scott, Bristol, both of whom I am hereby thanking. 

Willy A Flegel, Priv.-Doz. Dr. med.
Abteilung Transfusionsmedizin, Universität Ulm 
DRK Blutspendedienst Baden-Württemberg, Institut Ulm 
Helmholtzstrasse 10 
D-89081 Ulm 
Germany 
Telefon +49-731-150-601
FAX +49-731-150-602 
Email: willy.flegel@medizin.uni-ulm.de or waf@ucsd.edu 
Homepage: http://www.uni-ulm.de/~wflegel/

Figure 1

Figure 2