U.S. patent application number 13/623958 was filed with the patent office on 2013-03-28 for sample carrier and method for microscopic examination of biological samples.
This patent application is currently assigned to Siemens Aktiengesellschaft. The applicant listed for this patent is Siemens Aktiengesellschaft. Invention is credited to Walter GUMBRECHT, Karsten HILTAWSKY, Daniel SICKERT.
Application Number | 20130075286 13/623958 |
Document ID | / |
Family ID | 47827640 |
Filed Date | 2013-03-28 |
United States Patent
Application |
20130075286 |
Kind Code |
A1 |
GUMBRECHT; Walter ; et
al. |
March 28, 2013 |
SAMPLE CARRIER AND METHOD FOR MICROSCOPIC EXAMINATION OF BIOLOGICAL
SAMPLES
Abstract
A sample carrier for microscopic examination of biological
samples includes a base body and a filter membrane. The base body
has at least one recess in which the filter membrane is disposed.
The filter membrane makes an essentially flush closure with the
surface of the sample carrier. The sample carrier has a circular
shape.
Inventors: |
GUMBRECHT; Walter;
(Herzogenaurach, DE) ; HILTAWSKY; Karsten;
(Schwerte, DE) ; SICKERT; Daniel; (Nurnberg,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Siemens Aktiengesellschaft; |
Munich |
|
DE |
|
|
Assignee: |
Siemens Aktiengesellschaft
Munich
DE
|
Family ID: |
47827640 |
Appl. No.: |
13/623958 |
Filed: |
September 21, 2012 |
Current U.S.
Class: |
206/216 |
Current CPC
Class: |
G01N 21/6458 20130101;
B01L 2200/026 20130101; B01L 2300/0806 20130101; B65D 15/00
20130101; B01L 2200/0647 20130101; B01L 2300/0681 20130101; G01N
21/6428 20130101; B01L 3/5085 20130101 |
Class at
Publication: |
206/216 |
International
Class: |
B65D 8/00 20060101
B65D008/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2011 |
DE |
102011083215.7 |
Claims
1. A sample carrier for microscopic examination of biological
samples, the sample carrier comprising: a base body with at least
one recess; and a filter membrane in the at least one recess, the
filter membrane essentially making a flush closure with a surface
of the sample carrier; wherein the sample carrier has a circular
shape.
2. The sample carrier as claimed in claim 1, wherein the sample
carrier has a central, circular through-opening.
3. The sample carrier as claimed in claim 2, wherein the
through-opening has a diameter of about 15 mm and an edge height of
about 1.2 mm.
4. The sample carrier as claimed in claim 1, wherein the at least
one recess and the filter membrane are circular in shape.
5. The sample carrier as claimed in claim 1, wherein the at least
one recess includes a plurality of recesses forming a ring
concentric to the sample carrier.
6. The sample carrier as claimed in claim 1, wherein the at least
one recess has an outlet channel for filtrate.
7. The sample carrier as claimed in claim 1, wherein a support
element in the at least one recess supports the filter
membrane.
8. The sample carrier as claimed in claim 1, wherein the filter
membrane has a pore size between about 5 .mu.m and about 20 .mu.m,
inclusive.
9. The sample carrier as claimed in claim 1, wherein the base body
is embodied from a transparent material.
10. A method for detecting fluorescence-marked objects in a
biological sample, the method comprising: applying the biological
sample to a circular sample carrier having at least one filter
membrane; filtering the biological sample through the filter
membrane; treating the retentate remaining on the filter membrane
with at least one fluorescence marker; rotating the sample carrier
and scanning the sample carrier with a laser while simultaneously
detecting fluorescence events with a photodetector, the frequency
of the laser corresponding to an excitation frequency of an
assigned fluorescence marker; and storing, upon detection of a
fluorescence event, coordinates of the fluorescence event on the
sample carrier.
11. The method as claimed in claim 10, wherein a plurality of
biological samples are applied to a plurality of filter membranes
of the sample carrier.
12. The method as claimed in claim 10, wherein the biological
sample is treated with a lysis agent at least one of before and
after the filtration.
13. The method as claimed in claim 10, wherein at least one of the
biological sample and a further fluid media is applied to the
sample carrier by a robotic pipetting system.
14. The method as claimed in claim 13, wherein the sample carrier
is disposed rotatably in the pipetting system and the sample
carrier is rotated to specific positions for application of the
further fluid media.
15. The sample carrier as claimed in claim 9, wherein the
transparent material is one of glass and polycarbonate.
16. The sample carrier as claimed in claim 2, wherein the at least
one recess and the filter membrane are circular in shape.
17. The sample carrier as claimed in claim 2, wherein the at least
one recess includes a plurality of recesses forming a ring
concentric to the sample carrier.
18. The method of claim 10, wherein after scanning the sample
carrier with the laser, the method further includes, moving a
high-resolution microscope according to the stored coordinates; and
recording microphotographs of the biological sample at the stored
coordinates.
19. The method as claimed in claim 18, further comprising: treating
the biological sample with a lysis agent at least one of before and
after the filtration.
20. The method as claimed in claim 18, wherein the sample carrier
is disposed rotatably in the pipetting system and the sample
carrier is rotated to specific positions for application of fluid
media.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] The present application hereby claims priority under 35
U.S.C. .sctn.119 to German patent application number DE 10 2011 083
215.7 filed Sep. 22, 2011, the entire contents of which are hereby
incorporated herein by reference.
BACKGROUND
[0002] 1. Field
[0003] Example embodiments relate to sample carriers for
microscopic examination of biological samples, and methods for
detecting fluorescence-marked objects in a biological sample.
[0004] 2. Description of Related Art
[0005] The separation of particles, such as cells, bacteria or the
like from biological fluids, such as blood or urine, is
increasingly undertaken nowadays using microfiltration. This makes
use of the fact that the particles sought (e.g., tumor cells
circulating in blood) are significantly larger than most of the
other particles or cells contained in the biological fluid. As a
result, the particles separated in this way by filtration can be
colored with the usual cytological or immunochemical methods and
can subsequently be viewed, identified and documented under a
microscope.
[0006] Thin polymer films having a porosity of the desired order of
magnitude are mostly used for microfiltration. After filtration
these microfilters are transferred onto an object carrier so that
they are able to be viewed in the microscope.
[0007] This step is time-consuming and also prone to the risk of
incorrect transfer or destruction of the membrane.
[0008] A further problem in analysis of particles obtained by
microfiltration lies in the large filter surface on which the
particles must be searched for in the microscope. In many
applications the number of particles sought is so small that the
entire filter surface must be evaluated in order to obtain usable
and reliable results. Since the particles sought are small relative
to the filter surface, a large number of greatly enlarged
microscope images of the filter surface must be made in order to
cover the entire filter surface, but not overlook any of the
particles sought. This too is very time-consuming and
labor-intensive.
[0009] A microfilter is known from DE 10 2010 001 322 A1, which is
integrated directly into an object carrier for microscopy. This
enables the complex step of transferring the filter to the object
carrier to be dispensed with. Even with these types of filter
devices, however, the coverage of the entire filter surface with
microphotographs is still extremely time-consuming and complex.
SUMMARY
[0010] Example embodiments provide sample carriers and methods with
which more rapid detection of marked particles in biological
samples is possible.
[0011] At least one example embodiment provides a sample carrier
for microscopic examination of biological samples. The sample
carrier comprises a base body with at least one recess in which a
filter membrane is disposed. The filter membrane essentially makes
a flush closure with a surface of the sample carrier. In this case,
the sample carrier may be embodied as a circular carrier. The round
shape of the sample carrier makes it possible to carry out an
especially rapid microscopic sampling of the filter surface. In
contrast with sample carriers having integrated microfilters known
from the prior art, such a sample carrier can be accommodated
rotating under a fluorescence microscope or the like, so that the
sampling of the entire filter surface can be carried out more
rapidly than with an object carrier accommodated on a conventional
cross table. This merely requires the sample carrier to be set into
rotation, wherein a relative movement in a radial direction between
sample carrier and microscope is sufficient to capture the entire
surface of the sample carrier microscopically. This is also
relatively simple mechanically.
[0012] According to at least some example embodiments, the sample
carrier has a central, circular through-opening. A spigot of a
corresponding drive spindle can engage in this opening in order to
make the sample carrier rotate for analysis.
[0013] This through-opening may have a diameter of about 15 mm and
an edge height of about 1.2 mm. This corresponds to the compact
disk (CD), digital video disc (DVD) or Blu-ray standard, so that
apparatuses for handling such a sample carrier can be manufactured
simply and at lower cost from components which are produced in
large volumes and are thus cheaper. Other dimensions or adaptation
to other standards are of course also possible.
[0014] In at least one example embodiment, the at least one recess,
as well as the corresponding filter membrane, is circular. This
makes possible an especially simple homogeneous application of the
fluid to be filtered to the filter membrane, which can for example
be done easily by pipetting the fluid into the center of the
circular membrane.
[0015] As an alternative, the at least one recess as well as the
assigned filter membrane can also form a ring concentric to the
sample carrier. On the other hand this simplifies scanning of the
membrane surface on the rotating sample carrier since the membrane
surface can be captured continuously and without interruption
during a single rotation of the sample carrier.
[0016] The at least one recess may have an outlet channel for the
filtrate. This makes possible a residue-free observation of the
cells or other particles remaining on the filter surface as
retentate.
[0017] In at least one other example embodiment, a support element
for supporting the filter membrane may be disposed in the at least
one recess. This makes it possible to use especially thin and
fragile filter membranes which, because of the support element, can
easily withstand the mechanical stress when the sample is applied,
during the filtration and during the rotational sampling.
[0018] The filter membrane may have a maximum pore size of about 20
.mu.m, but may also have a pore size between about 5 .mu.m and 20
.mu.m, inclusive. Such a filter is especially suitable for
separation of suspended tumor cells in blood, since a large part of
the leukocytes, erythrocytes and other cellular blood components
can easily pass through this type of filter while the considerably
larger tumor cells will be held back on the filter surface.
[0019] In order to simplify microscopy, the base body may be
embodied from transparent material, such as glass, polycarbonate or
the like.
[0020] At least one other example embodiment provides a method for
detecting fluorescence-marked objects, such as cells, in a
biological sample. For this purpose the sample is initially applied
to a circular sample carrier with at least one filter membrane and
is filtered through this filter membrane. The pore size of the
filter membrane in this case is selected such that the objects to
be detected will be held back, while other particles contained in
the sample can pass through the filter. To simplify the detection
and to be able to distinguish the objects actually sought from
other objects also present in the retentate, the retentate
subsequently remaining on the filter membrane is treated with at
least one fluorescence marker. Immunologically-coupled fluorescence
markers are especially useful for this purpose, which for example
bind themselves specifically to surface structures of the sought
cells.
[0021] After the marking of the sought objects the sample carrier
is accommodated rotatably in a holder. The holder and thereby the
sample carrier are made to rotate and the sample carrier is sampled
with a laser, the frequency of which corresponds to the excitation
frequency of an assigned fluorescence marker. At the same time,
fluorescence events are detected with a photodetector.
High-resolution microscopy is not yet undertaken at this stage.
Instead, the coordinates of the recognized fluorescence events are
first stored. This may be done by using a polar coordinate system
because of the circular shape of the sample carrier. Only after the
complete sampling of the sample carrier with the laser is the
actual microscopy undertaken. To do this a high resolution
microscope is moved to the stored coordinates and microphotographs
are taken in each case.
[0022] Thus an especially rapid method is produced overall for
detecting fluorescence-marked objects or particles or cells in
biological samples. Through the filtration of the sample, which
removes a majority of the disruptive cellular or particulate
components of the sample, especially low-noise viewing is made
possible. Sensitivity and selectivity are further increased by the
subsequent fluorescence marking. The method is also compatible to
using sampling facilities known per se for circular sample
carriers, which are currently used to analyze samples brushed onto
their surface.
[0023] A plurality of biological samples may be applied to a
plurality of filter membranes of the sample carrier. This makes
possible the simultaneous or contemporaneous analysis of a
plurality of samples, so that the throughput of the method can be
further increased.
[0024] The biological sample may be treated before and/or after the
filtration with a lyze agent for lysis of the given, desired or
predetermined cell type. For example, an ammonium chloride lysis
can be carried out during the examination of blood samples in order
to fragment the erythrocytes present in large numbers and
facilitate filtering them away.
[0025] The biological sample and/or further fluid media may be
applied to the sample carrier by a robotic pipetting system. Such
automation allows the method to be carried out in an especially
rapid and reliable manner.
[0026] It is expedient in such cases to dispose the sample carrier
rotatably in the pipetting system and to rotate the sample carrier
to specific positions for applying fluid media. This allows
mechanically simple pipetting systems to be used and exploits the
advantages of the circular sample carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Example embodiments will be described in more detail
hereinbelow by referring to the accompanying drawing, in which:
[0028] The single FIGURE in this application shows a schematic view
of an example embodiment of a sample carrier.
[0029] It should be noted that these Figures are intended to
illustrate the general characteristics of methods, structure and/or
materials utilized in certain example embodiments and to supplement
the written description provided below. These drawings are not,
however, to scale and may not precisely reflect the precise
structural or performance characteristics of any given embodiment,
and should not be interpreted as defining or limiting the range of
values or properties encompassed by example embodiments. For
example, the relative thicknesses and positioning of molecules,
layers, regions and/or structural elements may be reduced or
exaggerated for clarity.
DETAILED DESCRIPTION
[0030] Various example embodiments will now be described more fully
with reference to the accompanying drawings in which only some
example embodiments are shown. Specific structural and functional
details disclosed herein are merely representative for purposes of
describing example embodiments. The present invention, however, may
be embodied in many alternate forms and should not be construed as
limited to only the example embodiments set forth herein.
[0031] Accordingly, while example embodiments of the invention are
capable of various modifications and alternative forms, embodiments
thereof are shown by way of example in the drawings and will herein
be described in detail. It should be understood, however, that
there is no intent to limit example embodiments of the present
invention to the particular forms disclosed. On the contrary,
example embodiments are to cover all modifications, equivalents,
and alternatives falling within the scope of the invention. Like
numbers refer to like elements throughout the description of the
figures.
[0032] It will be understood that, although the terms first,
second, etc. may be used herein to describe various elements, these
elements should not be limited by these terms. These terms are only
used to distinguish one element from another. For example, a first
element could be termed a second element, and, similarly, a second
element could be termed a first element, without departing from the
scope of example embodiments of the present invention. As used
herein, the term "and/or," includes any and all combinations of one
or more of the associated listed items.
[0033] It will be understood that when an element is referred to as
being "connected," or "coupled," to another element, it can be
directly connected or coupled to the other element or intervening
elements may be present. In contrast, when an element is referred
to as being "directly connected," or "directly coupled," to another
element, there are no intervening elements present. Other words
used to describe the relationship between elements should be
interpreted in a like fashion (e.g., "between," versus "directly
between," "adjacent," versus "directly adjacent," etc.).
[0034] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
example embodiments of the invention. As used herein, the singular
forms "a," "an," and "the," are intended to include the plural
forms as well, unless the context clearly indicates otherwise. As
used herein, the terms "and/or" and "at least one of" include any
and all combinations of one or more of the associated listed items.
It will be further understood that the terms "comprises,"
"comprising," "includes," and/or "including," when used herein,
specify the presence of stated features, integers, steps,
operations, elements, and/or components, but do not preclude the
presence or addition of one or more other features, integers,
steps, operations, elements, components, and/or groups thereof.
[0035] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which example
embodiments belong. It will be further understood that terms, e.g.,
those defined in commonly used dictionaries, should be interpreted
as having a meaning that is consistent with their meaning in the
context of the relevant art and will not be interpreted in an
idealized or overly formal sense unless expressly so defined
herein.
[0036] Spatially relative terms, such as "beneath", "below",
"lower", "above", "upper", and the like, may be used herein for
ease of description to describe one element or feature's
relationship to another element(s) or feature(s) as illustrated in
the figures. It will be understood that the spatially relative
terms are intended to encompass different orientations of the
device in use or operation in addition to the orientation depicted
in the figures. For example, if the device in the figures is turned
over, elements described as "below" or "beneath" other elements or
features would then be oriented "above" the other elements or
features. Thus, term such as "below" can encompass both an
orientation of above and below. The device may be otherwise
oriented (rotated 90 degrees or at other orientations) and the
spatially relative descriptors used herein are interpreted
accordingly.
[0037] Referring to the FIGURE, according to at least one example
embodiment, a sample carrier for microscopic examination of
biological samples identified overall by the number 10 comprises a
circular base body 12 made of a transparent material, such as
glass, polycarbonate or the like. The base body 12 has a central
through-opening on which it can be rotatably supported by, for
example, suitable spindles, spigots or the like. In one example, it
is possible to design the central through-opening 18 in accordance
with the CD, DVD or Blu-ray standard, so that widely-used
components can be employed to drive the sample carrier 10.
[0038] The sample carrier 10 also has a plurality of recesses 14
let into the base body 12. Each recess is spanned by a
microfiltration membrane 16. Support elements are also provided in
the recesses 14. The support elements support and stabilize the
microfiltration membrane 16, so that it withstands the mechanical
stresses of the application of the sample, filtration and
rotational movement.
[0039] The properties of the microfiltration membrane 16 are
governed by the actual analysis task for which the sample carrier
10 is to be employed.
[0040] In this example embodiment, the detection of tumor cells in
the blood is to be illustrated by the sample carrier 10. The use of
polymer microfiltration membranes 16 with a pore diameter of
between about 5 and about 20 .mu.m, inclusive, is suited for this
purpose.
[0041] Since tumor cells circulating in the blood are only present
in an extremely small number, it is expedient to first separate
these from other blood components or to concentrate them for
analysis. For this purpose the blood to be investigated is first
applied to the microfiltration membranes 16. If necessary, the
separation of the tumor cells from other blood components can also
be supported by an erythrocyte lysis, such as and ammonium chloride
lysis. After application of the sample prepared in this way to the
microfiltration membranes 16, the pore size of which allows the
passage of lysated erythrocyte fragments, leukocytes and other
small particulate blood components, while the microfiltration
membrane 16 holds back the significantly larger tumor cells as
retentate on its surface, the actual filtration follows. The
filtrate can in this case run out of the recesses 14 through
channels not shown in any greater detail.
[0042] The sought tumor cells now remain on the membrane surface,
as well as if necessary other blood components which may not have
been filtered away. To facilitate the detection of the tumor cells
present in extremely small numbers, a fluorescence coloring is
undertaken in the next step. This can likewise be carried out on
the surface of the microfiltration membranes 16. To this end, an
immunofluorescence marker, which is specific for surface proteins
of the sought tumor cells, is applied to the membrane surfaces by a
pipette, where it binds itself specifically to the corresponding
targets. Likewise, depending on the cytological or
immunohistochemical coloring method used, further treatment steps
are necessary. Surplus markers can finally be washed away.
[0043] Like the application of the sample, these coloring steps can
also be undertaken by an automatic pipetting system. In such cases
it is expedient to support the sample carrier 10 by the recess 18
rotatably in the pipetting system, so that each point of the
surface of the sample carrier 10 can be reached by a radial
translation movement of the pipetting robot as well as by rotation
of the sample carrier 10, so that the pipetting system is simple to
design.
[0044] After successful coloration or marking of the sought tumor
cells on the membrane surface, the sample carrier 10 is brought
into a corresponding detection device. In this device, the sample
carrier 10 is once again supported rotatably on the through-opening
15. Using, for example, at least one laser, the entire surface of
the sample carrier 10 is sampled and simultaneously observed with a
photo detector. The wavelength of the at least one laser
corresponds to the excitation wavelength of the fluorescence
colorant used. If fluorescence events are recognized, the
respective coordinates are stored. Naturally in such cases it is
possible to also carry out a multiple fluorescence coloration
(e.g., for different surface proteins of different tumor types) to
carry out specific antibody fluorescence label complexes. Ideally
the fluorescence colorants of these complexes have different
excitation and emission wavelengths. The sampling is then
undertaken in accordance with a plurality of lasers, wherein for
each detected fluorescence event, not only the coordinates but also
the detected emission wavelength--and thus the type of fluorescence
label used--is determined.
[0045] If the entire surface of the sample carrier 10 or the entire
surface of the microfiltration membranes 16 has been sampled in
this way, then the detected fluorescence events are observed
microscopically in greater detail on the basis of the stored
coordinates. A high-resolution microscope moves in such cases to
the stored coordinates and creates corresponding microphotographs.
Because of the transparent nature of the base body 12, this can
initially be done in simple available light. A fluorescence
excitation is also possible here in order to recognize the presence
of the sought tumor cells in the sample on the basis of the
specific and selective fluorescence marking. All known techniques
of fluorescence microscopy, such as confocal fluorescence
microscopy, can be employed here.
[0046] During the application of the sample and the analysis the
sample carrier 10 can be rotated in such cases at considerable
speeds of several hundred to several thousand rpm, so that
especially rapid scanning is possible. The separation of the
detection of fluorescence events from the actual microscopic
recording further speeds up the scanning in such cases since the
entire membrane surfaces do not have to be recorded
microphotographically.
[0047] A further accelerated variant of the method is well suited
to the routine laboratory analysis of the large number of samples.
In this case blood samples of a number of patients are applied to
the individual microfilter surfaces and simultaneously analyzed in
the way described.
[0048] An alternative design of the recesses 14 and the assigned
microfiltration membrane 16 is also possible. For example, the
recesses 14 and membranes 16 can run around the circumference of
the sample carrier 10 in the form of concentric rings, so that an
interruption-free observation of the membrane surface is made
possible during a complete rotation of the sample carrier 10.
Naturally, such ring structures are then to be attached coaxially
to the central through opening 18.
[0049] Overall the method illustrated is to be carried out
especially quickly in this way, but also at lower cost and/or in a
highly selective and sensitive manner.
[0050] Example embodiments being thus described, it will be obvious
that the same may be varied in many ways. Such variations are not
to be regarded as a departure from the spirit and scope of the
present invention, and all such modifications as would be obvious
to one skilled in the art are intended to be included within the
scope of the following claims.
* * * * *