Immunophenotypic Analysis of PNH Cells

Stephen J. Richards1, Peter Hillmen1

1 Haematological Malignancy Diagnostic Service, Leeds
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.11
DOI:  10.1002/0471142956.cy0611s20
Online Posting Date:  May, 2002
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Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem‐cell disorder in which a gene mutation results in cellular inability to synthesize the glycosylphosphatidylinositol (GPI) anchors needed to attach various proteins to the cell membrane. Traditional clinical laboratory PNH screening and diagnosis are based upon demonstration of increased erythrocyte susceptibility to lysis by activated complement. These methods are neither very specific nor very sensitive, and have largely been superseded by the rapid, sensitive, and specific flow cytometric analysis of GPI‐linked antigen expression. The protocols in this unit cover immunophenotypic analysis of red blood cells, peripheral blood granulocytes, and peripheral blood monocytes, as well as a four‐color modification for simultaneous analysis of peripheral blood granulocytes and monocytes.

Keywords: PNH; aplastic anemia; GPI anchor; CD55; CD59; hemolytic anemia; immunophenotyping

     
 
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Table of Contents

  • Basic Protocol 1: Immunophenotypic Analysis of GPI‐Linked Antigen Expression on Erythrocytes
  • Basic Protocol 2: Analysis of GPI‐Linked Antigen Expression on Peripheral Blood Neutrophils
  • Basic Protocol 3: Analysis of GPI‐Linked Antigen Expression on Peripheral Blood Monocytes
  • Basic Protocol 4: Simultaneous Analysis of GPI‐Linked Antigen Expression on Peripheral Blood Neutrophils and Monocytes
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunophenotypic Analysis of GPI‐Linked Antigen Expression on Erythrocytes

  Materials
  • <48‐hr‐old (room temperature) or <7‐day‐old (4°C) EDTA‐anticoagulated peripheral blood sample
  • Washing solution without protein support: phosphate buffered saline (PBS; appendix 2A), FACSFlow, or equivalent isotonic cell washing solution not containing protein support
  • FITC‐ and PE‐conjugated isotype‐matched negative controls, appropriately titered
  • PE‐conjugated monoclonal antibodies to CD235a (glycophorin A; Cymbus Biotechnology) and CD55 (DAF; Cymbus Biotechnology)
  • FITC‐conjugated monoclonal antibodies to CD59 (MIRL; Cymbus Biotechnology)
  • 96‐well round‐bottom microtiter plates
  • Microtiter plate mixer
  • 12 × 75–mm polystyrene tubes as required by flow cytometer
  • Flow cytometer with 488‐nm argon laser, at least two fluorescence detectors, and appropriate filter sets for detection of FITC and PE.

Basic Protocol 2: Analysis of GPI‐Linked Antigen Expression on Peripheral Blood Neutrophils

  Materials
  • <24‐hr‐old EDTA‐stabilized peripheral blood sample
  • Washing solution with protein support: FACSFlow containing 0.1% (w/v) BSA, or equivalent isotonic cell washing solution containing protein support
  • FITC‐, PE‐, and PE‐Cy5‐conjugated (directly) monoclonal antibodies to CD66abce (Dako), CD55 (Cymbus Biotechnology), and CD16 (Cymbus Biotechnology), appropriately titered
  • Lysing reagent—e.g., FACSLyse (Becton Dickinson), Uti‐Lyse (Dako), QuickLysis (Quest Biomedical)
  • CellFIX reagent (Becton Dickinson) or 1% (v/v) paraformaldehyde in PBS ( appendix 2A)
  • 12 × 75–mm polystyrene tubes suitable for the flow cytometer
  • Flow cytometer with 488‐nm argon laser, at least three fluorescence detectors, and appropriate filter sets for detection of FITC, PE, and PE‐Cy5
  • NOTE: PerCp or PerCP‐Cy5.5 conjugates can be used in place of PE‐Cy5.
  • Additional reagents and equipment for counting leukocytes ( appendix 3A)

Basic Protocol 3: Analysis of GPI‐Linked Antigen Expression on Peripheral Blood Monocytes

  Materials
  • <24‐hr‐old EDTA‐stabilized peripheral blood sample
  • Washing solution with protein support: FACSFlow containing 0.1% (w/v) BSA, or other equivalent isotonic cell‐washing solution containing protein support
  • FITC‐ or PE‐conjugated (directly) monoclonal antibodies to CD14 (Dako) and CD64 (Becton Dickinson)
  • Lysing solution—e.g., FACSLyse (Becton Dickinson), Uti‐Lyse (Dako), QuickLysis (Quest Biomedical)
  • CellFIX reagent (Becton Dickinson) or 1% paraformaldehyde in PBS
  • 12 × 75–mm polystyrene tubes suitable for the flow cytometer
  • Flow cytometer with 488‐nm argon laser, two fluorescence detectors, and appropriate filter sets for detection of FITC and PE.
  • Additional reagents and equipment for counting leukocytes ( appendix 3A)

Basic Protocol 4: Simultaneous Analysis of GPI‐Linked Antigen Expression on Peripheral Blood Neutrophils and Monocytes

  Materials
  • <24‐hr‐old EDTA‐stabilized peripheral blood sample
  • Washing solution with protein support: FACSFlow containing 0.1% (w/v) BSA, or other equivalent isotonic cell‐washing solution containing protein support
  • FITC‐, PE‐, and PE‐Cy5‐conjugated (directly) monoclonal antibodies to CD66abce, CD55 and CD16
  • APC‐conjugated monoclonal antibody to CD33
  • Lysing reagent (e.g., FACSLyse (Becton Dickinson), Uti‐Lyse (Dako), QuickLysis (Quest Biomedical)
  • CellFIX reagent (Becton Dickinson) or 1% paraformaldehyde in PBS
  • 12 × 75–mm polystyrene tubes suitable for flow cytometer
  • Flow cytometer with 488‐nm argon laser and 635‐nm red diode laser, at least four fluorescence detectors, and appropriate filter sets for detection of FITC, PE, PE‐Cy5, and APC.
  • Additional reagents and equipment for counting leukocytes ( appendix 3A)
NOTE: PerCP or PerCP‐Cy‐5.5 conjugates can be used in place of PE‐Cy5.
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Figures

Videos

Literature Cited

Literature Cited
   Alfinito, F., Del Vecchio, L., Rocco, S., Boccuni, P., Musto, P., and Rotoli, B. 1996. Blood cell flow cytometry in paroxysmal nocturnal hemoglobinuria: A tool for measuring the extent of the PNH clone. Leukemia 10:1326‐1330.
   Bessler, M., Mason, P.J., Hillmen, P., Miyata, T., Yamada, N., Takeda, N., Luzzatto, L., and Kinoshita, T. 1994. Paroxysmal nocturnal hemoglobinuria (PNH) is caused by somatic mutations in the PIG‐A gene. E.M.B.O. J. 13:110‐117.
   Hall, S.E. and Rosse, W.F., The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 87:5332‐5340.
   Hillmen, P., Lewis, S.M., Bessler, M., Luzzatto, L., and Dacie, J.V. 1995. Natural history of paroxysmal nocturnal hemoglobinuria. N. Engl. J. Med. 333:1253‐1258.
   Hillmen, P. and Richards, S.J. 1999. Flow cytometry in PNH: Serial analysis and the prediction of outcome. Blood 94:412a.
   Nakakuma, H., Nagakura, S., Kawaguchi, T., Iwamoto, N., Hidaka, M., Horikawa, K., Kagimoto, T., Tsuruzaki, R., and Takatsuki, K. 1994. Persistence of affected T lymphocytes in long‐term clinical remission in paroxysmal nocturnal hemoglobinuria. Blood 84:3925‐3928.
   Navenot, J.M., Bernard, D., Harousseau, J.L., Muller, J.Y., and Blanchard, D. 1996. Expression of glycosyl‐phosphotidylinositol‐linked glycoproteins in blood cells from paroxysmal nocturnal hemoglobinuria patients. A flow cytometry study using CD55, CD58 and CD59 monoclonal antibodies. Leuk. Lymphoma 21:143‐151.
   Regan, F., Newlands, M., and Bain, B. 2001. Acquired haemolytic anaemias. In Dacie and Lewis Practical Haematology (S.M. Lewis, B.J. Bain, and I. Bates, eds.) pp. 199‐229. Harcourt Health Sciences, St. Louis.
   Reid, M.E., Mallinson, G., Sim, R.B., Poole, J., Pausch, V., Merry, A.H., Liew, Y.W., and Tanner, M.J. 1991. Biochemical studies on red blood cells from a patient with the Inab phenotype (decay‐accelerating factor deficiency). Blood 78:3291‐3297.
   Richards, S.J. and Hillmen, P. 2001. Advances in the laboratory diagnosis of paroxysmal nocturnal hemoglobinuria. Clin. Applied Immunol. Rev. 1:315‐330.
   Richards, S.J., Norfolk, D.R., Swirsky, D.M., and Hillmen, P. 1998. Lymphocyte subset analysis and glycosylphosphatidylinositol phenotype in patients with paroxysmal nocturnal hemoglobinuria. Blood 92:1799‐1806.
   Richards, S.J., Rawstron, A.C., and Hillmen, P. 2000a. The application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry (CCC) 42:223‐233.
   Richards, S.J., Morgan, G.J., and Hillmen, P. 2000b. Immunophenotypic analysis of B cells in PNH: Insights into the generation of circulating naive and memory B cells. Blood 96:3522‐3528.
   Socie, G., Mary, J.Y., de Gramont, A., Rio, B., Leporrier, M., Rose, C., Heudier, P., Rochant, H., Cahn, J.Y., and Gluckman, E. 1996. Paroxysmal nocturnal haemoglobinuria: Long term follow‐up and prognostic factors. French society for haematology. Lancet 348:573‐577.
   Takeda, J., Miyata, T., Kawagoe, K., Iida, Y., Endo, Y., Fujita, T., Takahashi, M., Kitani, T., and Kinoshita, T. 1993. Deficiency of the GPI anchor caused by a somatic mutation of the PIG‐A gene in paroxysmal nocturnal hemoglobinuria. Cell 73:703‐711.
   van der Schoot, C.E., Huizinga, T.W., van't Veer‐Korthof, E.T., Wijmans, R., Pinkster, J., and van dem Borne, A.E. 1990. Deficiency of glycosyl‐phosphatidylinositol‐linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay. Blood 76:1853‐1859.
   Yamashina, M., Ueda, E., Kinoshita, T., Takami, T. Ojima, A., Ono, H., Tanaka, H., Kondo, N., Orii, T., Okada, N., Okada, H., Inoue, K., and Kitani, T. 1990. Inherited complete deficiency of 20‐kilodalton homologous restriction factor (CD59) as a cause of paroxysmal nocturnal hemoglobinuria. N. Engl. J. Med. 323:1184‐1189.
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