High‐Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells

D. Robert Sutherland1, Andrea Illingworth2, Michael Keeney3, Stephen J. Richards4

1 Contact author, 2 Dahl‐Chase Diagnostic Services, Bangor, Maine, 3 Pathology and Laboratory Medicine, London Health Sciences Centre, London, Ontario, 4 Haematological Malignancy Diagnostic Service, Department of Clinical Haematology, St. James University Hospital, Leeds
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.37
DOI:  10.1002/0471142956.cy0637s72
Online Posting Date:  April, 2015
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Abstract

Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub‐clinical PNH samples. A CD235a‐FITC/CD59‐PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four‐color FLAER‐based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single‐tube 5‐color FLAER/CD157‐based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra‐ and inter‐laboratory performance characteristics even in laboratories with little prior experience performing PNH testing. © 2015 by John Wiley & Sons, Inc.

Keywords: PNH; high‐sensitivity flow; RBCs; WBCs

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Sensitivity Detection of PNH Red Blood Cells
  • Alternate Protocol 1: High Sensitivity Detection of Immature PNH Red Cells Combined with Detection of Membrane Opsonized Complement C3d
  • Basic Protocol 2: High‐Sensitivity Detection of PNH Neutrophils Using a FLAER‐Based 4‐Color Assay
  • Basic Protocol 3: High‐Sensitivity Detection of PNH Monocytes Using FLAER‐Based 4‐Color Assay
  • Alternate Protocol 2: Simultaneous High‐Sensitivity Detection of PNH Neutrophils and Monocytes Using FLAER and CD157‐Based 5‐Color Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: High‐Sensitivity Detection of PNH Red Blood Cells

  Materials
  • EDTA (preferred) or heparin anti‐coagulated peripheral blood sample <48 hr old if stored at room temperature or at 4°C (samples kept at 4°C remain suitable for red cell analysis up to 7 days); bone marrow samples are not usually used due to the presence of immature blood cells that may mimic PNH phenotypes
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Antibodies (the volumes of antibodies described in the method below are for guidance only, and each laboratory should establish optimal antibody titers for each reagent in the combination; once individual antibodies have been optimally titrated, it is recommended that they be combined or cocktailed and diluted as shown in the example below for the KC16:MEM43 combination):
    • CD235a‐FITC [clone KC16 (Beckman Coulter, cat. no. IM2212U) or 10F7MN (eBioscience, cat. no. 11‐9886)]
    • CD59‐PE [clone MEM43 (Beckman Coulter, cat. no. MHCD5904) or OV9A2 (eBioscience, cat. no. 8012‐0596)]
  • Sheath fluid
  • 12 × 75–mm polypropylene tubes,
  • Centrifuge (or cell washer)
  • Metal or hard plastic test tube rack for racking samples (see step 10) before acquisition
  • Flow cytometer able to detect emissions from FITC and PE, appropriately compensated (see unit 1.14; Roederer, )

Alternate Protocol 1: High Sensitivity Detection of Immature PNH Red Cells Combined with Detection of Membrane Opsonized Complement C3d

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Antibodies:
    • CD71‐FITC (clone LO1.1; BD Biosciences, cat. no. 333151)
    • CD59‐PE (clone MEM43; Abnova, cat. no. MAB5011)
    • CD235a‐PECy5 (clone GA‐R2; BD Biosciences, cat. no. 559944)
    • Anti‐C3d‐biotin (clone A702; Quidel, cat. no. A702)
  • EDTA anti‐coagulated peripheral blood sample
  • Sheath fluid
  • Streptavidin‐APC (BD Biosciences, cat. no. 349024)
  • 96‐well U‐bottom microtiter plate
  • Plate mixer
  • Plate centrifuge
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer able to detect emissions from FITC, PE, PECy5, and APC, appropriately compensated (see unit 1.14; Roederer, )

Basic Protocol 2: High‐Sensitivity Detection of PNH Neutrophils Using a FLAER‐Based 4‐Color Assay

  Materials
  • Fresh EDTA (preferred) or heparin anti‐coagulated peripheral blood sample <48 hr old if stored at 4°C; bone marrow samples are not generally used due to the presence of immature precursors that may mimic PNH phenotypes
  • Antibodies:
    • For Beckman Coulter instruments: FLAER‐Alexa488 (liquid formulation; Cedarlane, cat. no. FL2S‐C), CD24‐PE [clone SN3 (eBioscience, cat. no. 8012‐0247) or ALB9 (Beckman Coulter, cat. no. IM1428U)], CD15‐PECy5 (clone 80H5; Beckman Coulter, cat. no. IM2641U), and CD45‐PECy7 (clone J33; Beckman Coulter, cat. no. IM3548U)
    • For BD Biosciences instruments: FLAER‐Alexa488 (liquid formulation; Cedarlane, cat. no. FL2S‐C), CD24‐PE [clone SN3 (eBioscience, cat. no. 8012‐0247) or ML5 (BD Biosciences, cat. no. 555428)], CD45‐PerCP (clone 2D1; BD Biosciences, cat. no. 347464), and CD15‐APC (clone HI98; BD Biosciences, cat. no. 561716)
  • Lysing agent: ImmunoPrep (Beckman Coulter), VersaLyse (Beckman Coulter), or FACSlyse (BD Biosciences)
  • PBA: phosphate‐buffered saline (PBS; appendix 2A) supplemented with 1% (w/v) bovine serum albumin (BSA)
  • Sheath fluid
  • 12 × 75–mm polypropylene tubes
  • Centrifuge or cell washer
  • Flow cytometer able to detect emissions from FITC, PE, PECy5, and PECy7 (for Beckman Coulter instruments), or FITC, PE, PerCP, and APC (for BD Biosciences instruments)

Basic Protocol 3: High‐Sensitivity Detection of PNH Monocytes Using FLAER‐Based 4‐Color Assay

  Materials
  • Fresh EDTA (preferred) or heparin anti‐coagulated peripheral blood sample <48 hr old if stored at 4°C; bone marrow samples are not generally used due to the presence of immature precursors that may mimic PNH phenotypes
  • Antibodies:
    • For Beckman Coulter instruments: FLAER‐Alexa488 (liquid formulation; Cedarlane cat no FL2S‐C), CD14‐PE [clone 61D3 (eBioscience, cat. no. 8012‐0149) or RMO52 (Beckman Coulter, cat. no. A07764)], CD64‐PECy5 (clone 22; Beckman Coulter, cat. no. IM3606U), and CD45‐PECy7 (clone J33; Beckman Coulter, cat. no. IM3548U)
    • For BD Biosciences instruments: FLAER‐Alexa488 (liquid formulation; Cedarlane, cat. no. FL2S‐C), CD14‐PE [clone 61D3 (eBioscience, cat. no. 8012‐0149) or M0P9 (BD Biosciences, cat. no. 347497)], CD45‐PerCP (clone 2D1; BD Biosciences, cat. no. 347464), and CD64‐APC (clone 10.1; BD Biosciences, cat. no. 561189)
  • Lysing agent: ImmunoPrep (Beckman Coulter), VersaLyse (Beckman Coulter), or FACSlyse (BD Biosciences)
  • PBA: phosphate‐buffered saline (PBS; appendix 2A) supplemented with 1% (w/v) bovine serum albumin (BSA)
  • Sheath fluid
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer able to detect emissions from FITC, PE, PECy5, and PECy7 (for Beckman Coulter instruments), or FITC, PE, PerCP, and APC (for BD Biosciences instruments)

Alternate Protocol 2: Simultaneous High‐Sensitivity Detection of PNH Neutrophils and Monocytes Using FLAER and CD157‐Based 5‐Color Assay

  Materials
  • Fresh EDTA (preferred) or heparin anti‐coagulated peripheral blood sample <48 hr old if stored at 4°C; bone marrow samples are not generally used due to the presence of immature precursors that may mimic PNH phenotypes
  • Antibodies:
    • Beckman Coulter instruments: FLAER‐Alexa488 (liquid formulation; Cedarlane, cat. no. FL2S‐C), CD157‐PE (clone SY11B5; eBioscience, cat. no. 9012‐1579‐025), CD64‐ECD (clone 22; Beckman Coulter, cat. no. A98434), CD15‐PECy5 (clone 80H5; Beckman Coulter, cat. no. IM2641U), and CD45‐PECy7 (clone J33; Beckman Coulter, cat. no. IM3548U)
    • BD Biosciences instruments: FLAER‐Alexa488 (liquid formulation; Cedarlane, cat. no. FL2S‐C), CD157‐PE (clone SY11B5; eBioscience, cat. no. 9012‐1579‐025), CD45‐PerCP (clone 2D1; BD Biosciences, cat. no. 347464), CD64‐APC (clone 10.1; BD Biosciences, cat. no. 561189), and CD15‐eFluor450 (or CD15‐v450) (clone MMA; eBioscience, cat. no. 8048‐0158 )
  • Lysing agent: ImmunoPrep (Beckman Coulter), VersaLyse (Beckman Coulter), or FACSlyse (BD Biosciences)
  • PBA: phosphate‐buffered saline (PBS; appendix 2A) supplemented with 1% (w/v) bovine serum albumin (BSA)
  • Sheath fluid
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with at least five PMTs able to detect emissions from FITC, PE, ECD, PECy5, and PECy7 (for Beckman Coulter instruments) or FTIC, PE, PerCP and APC and v450 (or eFluor450) (for BD Biosciences Canto II instruments), appropriately compensated.
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Figures

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Literature Cited

Literature Cited
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