DURAClone RE ALB Protocol

This protocol is for reference purposes only.  It may be necessary to adapt the method for specific research needs.

Materials

KIT BOX CONTENTS

25 tests of the DURAClone RE ALB Antibody Panel

3 Compensation Kits, each kit containing eight single color tubes:

  • CD4-FITC
  • CD34-ECD
  • CD10-PC5.5
  • CD19-PC7
  • CD38-APC-Alexa Fluor 700
  • CD20-APC-Alexa Fluor 750
  • CD8-Krome Orange

NOTE: Do not store the reagent tubes in the refrigerator; do not freeze/thaw the tubes. Minimize the exposure of the tubes to light, especially during processing and incubation of sample(s) prior to acquisition.

MATERIAL REQUIRED BUT NOT SUPPLIED

Blood collection tube containing anticoagulant, K2 EDTA

Calibrated pipettes

Vortex mixer

VersaLyse Solution (Part Number A09777)

IOTest 3 Fixative Solution (Part Number A07800)

VersaLyse Fix-and-Lyse Mixture:

  • Prepared fresh each day by adding 25 μL of 10X IOTest 3 Fixative Solution to 1 mL of VersaLyse Solution. Each sample and compensation control requires 2 mL.

Phosphate Buffered Saline, PBS (Part Number 6603369, or equivalent)

  •  Prepared following the IFU.

1X PBS/Fixative:

  • Prepared fresh each day by adding 1 mL of 1X PBS to 12.5 μL of IOTest®3 10X Concentrate. Each sample and compensation control requires 500 μL.

VersaComp Antibody Capture Beads (Part Number B22804)

Flow cytometer equipped with the following lasers and detectors:

  • FSC/SSC
  • 405 nm: 430 – 470 nm and 530 – 570 nm
  • 488 nm: 504 – 545 nm, 560 – 600 nm, 605 – 635 nm, 680 – 710 nm and >755 nm
  • 633 nm: 650 – 670 nm, 715 – 735 nm and >755 nm

Sheath fluid

Flow cytometer calibration beads

Staining Procedure

SAMPLE PREPARATION 

  1. Determine the total number of leukocytes in the bone marrow samples.

  2. Transfer the volume of sample equivalent to 1.5 to 2.0 million leukocytes to an empty tube.

  3. Add 2 mL of VersaLyse Fix-and-Lyse Mixture per 100 μL of bone marrow cells. Vortex the tube at high speed for 1-3 seconds and incubate the tube for 15 minutes between 18 and 25 °C. Protect from light.

  4. Centrifuge the tube at 300 x g for 5 minutes; aspirate the supernatant. Gently tap to dissociate the pellet.

  5. Add 100 μL of PBS and transfer to the DURAClone RE ALB dried reagent tube. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 18 and 25 °C. Protect from light.

  6. Add 3 mL 1X PBS;  centrifuge at 200 x g for 5  minutes; aspirate the supernatant.

  7. Resuspend cells in 500 μL of 1X  PBS/Fixative solution.

  8. The sample is now ready for acquisition.

COMPENSATION SETUP

  1. Add 800 μL of whole blood into an empty polypropylene tube and process by following steps 3-4 in the Sample Preparation procedure.

  2. Add 3 mL 1X PBS; centrifuge at 300 x g for 5 minutes; aspirate the supernatant. Resuspend the cell pellet in 800 μL of 1X  PBS.

  3. Add 100 μL into each of the single color tubes in the Compensation Kit.  All tubes should be from the same pouch.

  4. Add one drop of the positive VersaComp Antibody Capture Beads to the following compensation tubes:

    • CD34-ECD
    • CD10-PC5.5
    • CD19-PC7
    • CD38-APC-Alexa Fluor 700
    • CD20-APC-Alexa Fluor 750

  5. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 20 and 30 ºC. Protect from light.

  6. Follow steps 6-8 in the Sample Preparation procedure.

  7. Follow standard procedures and instrument manufacturer instructions for compensation setup.

Analysis

  1. Create a FS INT vs FS TOF dot plot and a singlet gate to eliminate any doublets (identified by higher values on FS TOF)

  2. Create a FSC vs. SSC dot plot and apply the singlets gate. Set the discriminator on the FS parameter to a value low enough to assure lymphocytes are not excluded from acquisition.

  3. Create a CD45-Krome Orange vs. SSC dot plot and apply the scatter gate.

  4. Create a CD19-PC7 vs. SSC dot plot. Draw a region to surround the CD19+ low SSC cells.

  5. Create a CD34-ECD vs CD38-APC-Alexa Fluor 700 dot plot and apply the gate from step 5. Create a region to encompass the CD38-APC-Alexa Fluor 700 dim/+ CD34+ cells.

  6. Create a CD10-PC5.5 vs CD20-APC-Alexa Fluor 750 dot plot and apply the gate from step 5. Create a region to encompass the CD20-APC-Alexa Fluor 750 dim/-ve+ CD10+ cells

  7. Create a CD10-PC5.5 vs CD58-FITC dot plot and apply the gate from step 5. Create a region to encompass the CD58+ CD10+ cells.

  8. Create a Boolean AND gate which encompass all the gates from step 6, 7 and 8. Label the Boolean gate as “Rare B cells”.

  9. Create a CD45-Krome Orange vs CD19-PC7 dot plot and apply the “Rare B cells” gate. Draw a gate on CD19+ CD45 dim population and label the gate as “Anomalous B progenitors”.

  10. Create a CD10-PC5.5 vs CD34-ECD dot plot. Apply the gate “Anomalous B progenitors” on to this plot.

  11. Use the above gates to analyze the immunophenotype expression of the markers on the gated CD34+ CD38+ CD10+ CD58+ B cells.