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NMR q-space analysis of canonical shapes of human erythrocytes: stomatocytes, discocytes, spherocytes and echinocytes

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Abstract

q-Space plots obtained experimentally using pulsed field-gradient stimulated echo (PGSTE) nuclear magnetic resonance (NMR) spectroscopy from water diffusing in red blood cells (RBCs) of different canonical (distinct variant) morphologies have “signature” features. The experimental q-space plots from suspensions of stomatocytes, echinocytes and spherocytes generated chemically had no diffraction features; in contrast a sample of blood from a patient with hereditary spherocytosis showed diffraction minima. To understand the forms of q-space plots, mathematical/geometrical models of discocytes, stomatocytes, echinocytes and spherocytes were used as restricting boundaries in simulations of water diffusion with Monte Carlo random walks. These simulations indicated that diffusion-diffraction minima are expected for each of the cell shapes considered. The absence of diffusion-diffraction minima in stomatocytes generated by dithiothreitol treatment was surmised to be due to non-alignment of the cells with the magnetic field of the NMR spectrometer. Differential interference contrast microscopy images of the chemically generated spherocyte and echinocyte suspensions showed them to be heterogeneous in cell shape. Therefore, we concluded that the shape heterogeneity caused the loss of the diffusion-diffraction features, which were observed in the more homogeneous sample from a patient with hereditary spherocytosis, and in the simulations of homogeneous cell suspensions. This understanding of factors that affect q-space plots from RBC suspensions will assist morphological studies of other cell and tissue types.

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Abbreviations

BSA:

Bovine serum albumin

CDTA:

Trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid

DIC:

Differential interference contrast

DTT:

Dithiothreitol

EGTA:

Ethylene glycol-bis(2-aminoethylether)-N,N′-tetraacetic acid

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulphonic acid

Ht :

Haematocrit, or packed cell volume

MI:

Morphological index

MRT:

Mean residence time

NMR:

Nuclear magnetic resonance

PBS:

Phosphate-buffered saline

PGSE:

Pulsed field-gradient spin echo

PGSTE:

Pulsed field-gradient stimulated echo

RBC:

Red blood cell

RMSD:

Root-mean-square displacement

WBC:

White blood cell

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Acknowledgments

The authors thank Professor Joy Ho for facilitating access to the patient with hereditary spherocytosis. The work was funded by a Discovery grant from the Australian Research Council to P.W.K., from Cure the Future (Cell and Gene Trust) to J.E.J.R., and a University of Sydney postgraduate award to T.J.L.

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Author notes

  1. Timothy J. Larkin

    Present address: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, CB2 0RE, UK

Authors and Affiliations

  1. School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia

    Timothy J. Larkin, Bogdan E. Chapman & Philip W. Kuchel

  2. Singapore Bioimaging Consortium (SBIC), A*Star, Agency for Science Technology and Research, #02-02 Helios Building, Singapore, 138667, Singapore

    Guilhem Pages & Philip W. Kuchel

  3. Gene and Stem Cell Therapy Program, Centenary Institute, University of Sydney, Sydney, NSW, 2050, Australia

    John E. J. Rasko

  4. Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, NSW, 2050, Australia

    John E. J. Rasko

Authors
  1. Timothy J. Larkin
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  2. Guilhem Pages
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  3. Bogdan E. Chapman
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  5. Philip W. Kuchel
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Correspondence to Timothy J. Larkin.

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Special Issue: From kinetics to imaging: An NMR odyssey.

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Larkin, T.J., Pages, G., Chapman, B.E. et al. NMR q-space analysis of canonical shapes of human erythrocytes: stomatocytes, discocytes, spherocytes and echinocytes. Eur Biophys J 42, 3–16 (2013). https://doi.org/10.1007/s00249-012-0822-8

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  • DOI: https://doi.org/10.1007/s00249-012-0822-8

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