JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Preparation of Human Erythrocytes for In-Air
Micro-PIXE Analysis
Y. Tokita a) , H. Kikuchi a) , T. Nagamine a) , T. Satoh b) , T. Kamiya b) and K. Arakawa b)
a) School of Health Sciences, Faculty of Medicine, Gunma University,
b) Department of Advanced Radiation Technology, TARRI, JAEA
1. Introduction
Essential elements play a pivotal role on homeo- stasis in
human body, and metal ions such as zinc, copper, and iron
are assayed in the clinical samples of serum, urine and
tissues. It is well known that erythrocytes (red blood cell)
contain various trace elements, which are altered along with
pathogenesis of disorders. Because erythrocytes can be
collected via peripheral vessels non-invasively, this blood
cell is a convenient sample material for in-air micro PIXE
analysis.
In order to establish an appropriate preparation with
erythrocytes for micro PIXE analysis, we investigated (i)
whether X-ray spectra can be affected by morphological
change of erythrocyte (color and shape) or ion contained in
washing solution, (ii) what element is most suitable to
demarcate the physical shape of erythrocyte.
2. Material and Method
Erythrocytes samples obtained from normal volunteers
and a patient of Wilson disease were analyzed by in-air
micro PIXE. The blood was collected via cubital vein
using EDTA 2Na as anticoagulant. Equal volume of
physiological saline or isotonic LiCl solution was added this
blood, centrifuged (1,400 rpm, 5 min), and discard
supernatant. Residual red blood cells were used for the
sample preparation.
PIXE samples were prepared by two methods. (i) After
washing with saline, blood sample was seep into 0.5 m
thick of mayler membrane, and floating on isopentane in a
stainless cup, then this cup was put on liquid nitrogen and
lyophilized by vacuum evaporation (Conventional method).
(ii) Blood sample was dropped on mayler membrane, and
this membrane was sunk into isopentane (-150 ºC), cooled
by liquid nitrogen previously, then lyophilized by vacuum
1)
evaporation (Ortega method ).
Three point zero MeV proton beams in 1-μm diameter
that was generated by the TIARA single-ended accelerator at
JAEA-Takasaki, was used to analyze elemental distribution
of erythrocytes.
3. Results and Discussion
To confirm the optimization as PIXE sample, we
compared the conventional method with the Ortega method.
Elemental distributions of erythrocytes were similar between
both methods. But, under optical microscope, the
erythrocytes made by the conventional method colored
black and changed its shape atrophic. The erythrocytes
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made by the Ortega method were clear and spherical. As a
next step, we analyzed elemental distribution in the
erythrocytes colored-black or clear; the black-colored
erythrocyte indicated higher K value than that of the clear
ones. This result suggests that spread of erythrocyte is
needed to prepare a proper PIXE sample.
It is unclear known whether erythrocyte’s X-ray spectra,
especially Na, is influenced by cations contained in washing
buffer, physiological saline (NaCl). Thus, to confirm this
problem, we compared the erythrocyte’s X-ray spectrum
between the sample washed with saline and that washed
with Li-contained solution. As a result, no difference was
identified in two X-ray spectrums. Therefore cation
contained in washing solution was not essential for sample
preparation.
We compared elemental maps of three elements (Cl, Fe,
and K) to confirm a suitable element to demarcate
erythrocyte’s shape, and distribution of Cl is well agreed
with erythrocyte’s shape (Fig. 1). Reproducibility is
confirmed by two normal erythrocyte’s data.
Reference
1) R. Ortega, Cell. Mol. Biol. 42 (1996) 77-88.
Wilson
Disease
Normal
Normal
Fig. 1 Comparison of microphotograph with
distribution of each element analyzed by PIXE.