APPLICATION NOTE

FlowDSA-XM™
Catalog # FLDSA

For Research Use Only. Not for use in diagnostic procedures.

REAGENTS

A. Identification
 The FlowDSA-XM uses a pooled panel of microparticles coated with anti-HLA Class I
and II antibodies.
B. Warning or Caution
1. For Research Use Only
2. Warning: The FlowDSA-XM test reagents contain 0.02% sodium azide (NaN3) as a
preservative. Under acidic conditions, sodium azide yields hydrazoic acid, an extremely toxic
compound. Reagents containing sodium azide should be diluted in running water prior to being
discarded. These conditions are recommended to avoid deposits in plumbing where explosive
conditions may develop. (Refer to Safety Data Sheet for detail).
3. Warning: All blood products should be treated as potentially infectious. Source material from
which this product was derived was found negative when tested in accordance with current FDA
required tests. No known test methods can offer assurance that products derived from human
blood will not transmit infectious agents.
4. Caution: For manual “flicking” of trays, use a quick downward arm motion without wrist
movement to prevent repetitive motion effects.
5. Refer to the Safety Data Sheet for detailed information.
C. Preparing Reagents for Use
1. See Directions for Use below.
D. Storage Instructions
1. The entire package may be stored in a freezer at -65°C or below until first use, up to the labeled
expiration date.
2. Once reagents are thawed, DO NOT REFREEZE. Store at 2 – 8°C for up to four weeks or until
the expiration date (if earlier).
3. The FlowDSA-XM Capture Beads and Lysis/Stain Buffer are sensitive to light and must be
stored in the dark.
E. Purification or Treatment Required for Use
See Directions for Use below.
F. Instability Indications
None

INSTRUMENT REQUIREMENTS
A. Required Equipment
 Flow Cytometer
 Centrifuge
 Swinging bucket rotor for 96-well microplate (2000 g)

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 Vortex mixer
 Plate shaker or rotating platform
 Cell counter
 37°C water bath
B. Equipment Calibration
Follow manufacturer’s instructions for calibration of the Flow Cytometer.
C. Recommended Software
Not applicable.

SPECIMEN COLLECTION AND PREPARATION

A. Unopened blood specimens may be kept at room temperature up to 4 days. Separated serum (from clotted
samples) may be refrigerated up to 7 days, or aliquots may be frozen at -20 to -80°C or below for up to 3
years, and thawed just before the assay. However, aggregates should be removed from the test serum by
centrifugation (8,000 – 10,000 g for 10 minutes) or filtration (0.2 um) prior to testing. Any aggregates or
contamination of the sample may generate invalid results.
B. Test serum should not be heat inactivated.
C. Undiluted serum is used for the test.

PROCEDURE
A. Materials Provided
Catalog ID Product Name Product Components
FLDSA FlowDSA-XM  FlowDSA-XM Capture Beads
 FlowDSA-XM Lysis/Stain Buffer
 FlowDSA-XM Wash Buffer 1
 FlowDSA-XM Wash Buffer 2
 FlowDSA-XM Wash Buffer 3

B. Materials Required, But Not Provided

1. Pipette tips (Rainin GPS)
2. 96-well microplate (Fisher Scientific cat. No. 0720095; Corning 96 Well Clear Round Bottom TC-Treated
Microplate Individually Wrapped, with Lid, Sterile Product #3799)
3. Tray seal (OLI Cat. # SSPSEA300 or equivalent)
C. Directions for Use

Notes:
 For lymphocyte isolation methods, refer to the ASHI Laboratory Manual1.
 In order to reach required lymphocyte purity (>90%) and maximally eliminate monocytes or/and
granulocytes contamination, a depletion procedure should be performed.
 Take special care in the aliquoting process. Failure to follow the steps described below may result in
reagent loss.
 Unless specified otherwise, all steps are performed at room temperature.
 Keep all sera, Capture Bead vials, Lysis/Stain Buffer vials, Wash Buffer 1, and Wash Buffer 3 on ice.
Keep Wash Buffer 2 at room temperature.
 For Data Acquisition (Results Section A), run a negative control serum and a Class I positive control
serum in replicates for Voltage Adjustment and Fluorescence Compensation. This only needs to be
performed when setting up the assay for the first time or when modifying an existing setup.

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1. Vortex lymphocytes and dispense 0.2X106 (less than 250 µl in volume) into all pre-selected wells of a 96-
well plate.
2. Seal and centrifuge the plate at 1360 g for 3 minutes.
3. Remove supernatant from wells of plate by flicking. Blot until dry on paper towels before turning plate
over. Vortex plate for 30 seconds.
4. Add 50 µl of serum to each pre-selected well of the plate. Do not pipette mix.
5. Seal plate and vortex for 30 seconds.
6. Incubate for 20 minutes at 20-25°C with shaking.
7. Add 150 µl of cold Wash Buffer 1 (WB-1) to each pre-selected well of the plate. Pipette up and down 4-5
times while adding to mix thoroughly.
8. Seal and centrifuge the plate at 1360 g for 3 minutes.
9. Remove supernatant from wells of plate by flicking. Blot until dry on paper towels before turning plate
over. Vortex plate for 30 seconds.
10. Repeat steps 7 – 9 for a total of two washes.
11. Mix the Capture Beads well by vortexing for 10 seconds prior to use.
12. Add 5 µl of Capture Beads to each pre-selected well of the plate. Do not pipette mix. Seal plate and
vortex for 30 seconds.
13. Add 150 µl of cold WB-1 to each pre-selected well of the plate. Pipette up and down 4-5 times while
adding to mix thoroughly.
14. Seal and centrifuge the plate at 2000 g for 3 minutes.
15. Remove supernatant from wells of plate by flicking. Blot until dry on paper towels before turning plate
over. Vortex plate for 30 seconds.
16. Mix Lysis/Stain Buffer (LSB) vial gently by pipetting up and down 4-5 times prior to use. Do not vortex.
17. Add 25 µl of LSB to each pre-selected well of the plate. Do not pipette mix. Seal plate and vortex for 30
seconds.
18. Incubate in the dark for 30 minutes at 20-25°C with shaking.
19. Add 150 µl of cold WB-1 to each pre-selected well of the plate. Pipette up and down 4-5 times while
adding to mix thoroughly.
20. Seal and centrifuge the plate at 2000 g for 3 minutes.
21. Remove supernatant from wells of plate by flicking. Blot until dry on paper towels before turning plate
over. Vortex plate for 30 seconds.
22. Add 200 µl of Wash Buffer 2 (WB-2) to each pre-selected well of the plate. Pipette up and down 4-5
times while adding to mix thoroughly.
23. Seal and centrifuge the plate at 2000 g for 3 minutes.
24. Remove supernatant from wells of plate by flicking. Blot until dry on paper towels before turning plate
over. Vortex plate for 30 seconds.
25. Add 200 µl of Wash Buffer 3 (WB-3) to each pre-selected well of the plate. Pipette up and down 4-5
times while adding to mix thoroughly.
26. The sample is ready for flow analysis. Store at 2 - 5°C before reading.

RESULTS
A. Data Acquisition
Notes:
 All compensation settings should be set to 0. Compensation settings should not be linked to any
other values as compensation must be performed independently for this assay. This only needs to be
performed when setting up the assay for the first time or when modifying an existing setup.

1. Align and quality control the flow cytometer daily according to the manufacturer’s recommended start-up
procedure.

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2. Instrument Setup
a. Run a control test using 5 µl of FlowDSA-XM Capture Beads in 200 µl of Wash Buffer 3 (WB-3) to set
up the FSC and SSC gains in order to locate the bead population on your flow cytometer (Figure 1).
Note: Because the beads are smaller than regular lymphocytes, higher FSC gains may be expected
in order to visualize the beads.

Figure 1. An example of FSC vs SSC bead location

b. Voltage Adjustment
(1) Run a FlowDSA-XM test with a negative control serum and a Class I positive control serum.
(2) Adjust the FL2 and FL3 Voltages to place the HLA-I negative bead population in the lower-left
quadrant of the FL2 vs FL3 plot (Figure 2).

Figure 2. An example of FL2 vs. FL3 dot plot

c. Fluorescence Compensation
(1) Using the negative control serum, adjust the FL2-%FL3 so that the HLA-IIb beads are aligned
along the FL2 axis with the HLA-I beads (Figure 2).
(2) Using the Class I positive control serum, adjust the FL3-%FL2 so that the HLA-I beads are
aligned along the FL3 axis with the Class I negative control population (Figure 3 Right Dot Plot).

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Figure 3. An example of FL2 vs. FL3 compensation. Left and center dot plots are examples of
poor compensation. Right dot plot is an example of correct compensation.
3. Acquire Samples
a. Using settings from Voltage Adjustment and Fluorescence Compensation, acquire samples.
b. Acquire 1000 – 5000 events for each sample.

B. Data Analysis
1. Gate the major population of FlowDSA-XM Capture Beads on the FSC vs. SSC dot plot (Figure 4).

Figure 4. An example of FSC vs. SSC gating

2. Obtain the FSC vs. FL3 dot plot of the R1 (BEADS) gated region. If available, FL4 can also be used in
place of FL3. R2 (CI) is the gate for HLA-I beads; R3 (CIIa) is the gate for HLA-IIa beads; and R4 (CIIb)
is the gate for HLA-IIb beads (Figure 5). Obtain FL2 histograms of each sample on the gated R1 and R2
regions for Class I analysis, on the gated R1 and R3 regions for Class IIa analysis, and R1 and R4
regions for Class IIb analysis.

Figure 5. An example of FSC vs. FL3 Capture Bead gating

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3. The abbreviations used in this section are defined below.

MCS Median Channel Shift

MCV Median Channel Value

4. Determine the MCS for sera and Positive Controls.

Calculation of MCS:
MCS = MCV of the sera (or Positive controls) - MCV of the Negative Controls

5. Record the results.

C. Determination of Positive/Negative Cut-Off

1. The suggested method for determining the cutoff values are outlined here. The cutoffs for FlowDSA-XM
are determined by the results from 20 pre-tested AB male sera against 5 different target cells. MCS for
each tested AB serum are calculated. The means and standard deviations (SD) of FlowDSA-XM MCSs
are calculated from all MCSs obtained. The suggested FlowDSA-XM cutoffs are the mean plus 3SD.

LIMITATIONS OF THE PROCEDURE

 Sera samples that contain contaminants or aggregates may clog the flow cytometer and generate inaccurate
data. Aggregates in the test specimen should be removed by centrifugation prior to testing.
 Accurate data acquisition depends on proper performance of the flow cytometer. The machine must be
properly calibrated and maintained.
 Assignment of antibody is limited to the anti-HLA Class I and Class II antibodies presented on each Capture
Bead (see lot specific worksheet).
 The antibodies used for each anti-HLA Class I and Class II Capture Bead may change from lot-to-lot of
product (see lot specific worksheet).
For Research Use Only. This product is not intended to provide information for the diagnosis, prevention or
treatment of disease or to aid in the clinical decision making process. This product is not cleared or approved for
clinical use by the FDA or approved in the EU as an in vitro diagnostic assay, nor is it CE marked.
BIBLIOGRAPHY
1. ASHI Laboratory Manual, 3rd Edition, ASHI, Lenexa, KS.

TRADEMARKS AND DISCLAIMERS

FlowDSA-XM™

EXPLANATION OF SYMBOLS (reference EN ISO 15223-1: Medical devices – Symbols to be used with medical device labels,
labeling and information to be supplied)

Symbol Description

Catalog number

Consult instructions for use*

Caution, consult accompanying documents

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Biological risks

Temperature limitation

Manufacturer

*Please consult Application Note for Research Use Only product

REVISION HISTORY
Revision Date Revision Description
0 06/12/2017 Initial Release
1 Current Revision to Open Vial Stability and Directions for Use, updated: 90% purity
required, plate sealing for vortexing, washing steps combined, and vortexing
speed clarified

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