Method and a system for determination of particles in a liquid sample

• US 6,710,879 B1
• Filed: 11/01/1999
• Issued: 03/23/2004
• Est. Priority Date: 05/05/1997
• Status: Expired due to Term

• Current Assignee: ChemoMetec A/S
• Original Assignee: ChemoMetec A/S
• Inventors: Jeppesen, Jesper Myron, Glensbjerg, Martin, Hansen, Frans Ejner Rvan, Arnvidarson, Borkur
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): MERLINO, AMANDA H
• Application Number:
  US09/403,958
• Time in Patent Office: 1,604 Days
• Field of Search: 356/436-440, 356/39-43
• US Class Current: 356/436
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/025 Methods for single or grouped particles
• CPC Class Codes: G01N 2015/1452 Adjustment of focus; Alignment
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2021/5957 using an image detector type detector, e.g. CCD
• CPC Class Codes: G01N 2021/6491 Measuring fluorescence and transmission; Correcting inner filter effect
• CPC Class Codes: G01N 21/31 Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry G01N21/72 takes precedence
• CPC Class Codes: G01N 21/59 Transmissivity G01N21/25 takes precedence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 33/04 Dairy products

Abstract

The present invention relates to a method for the assessment of quantity and quality parameters of biological particles in a liquid analyte material. The method comprises applying a volume of a liquid sample to an exposing domain from which exposing domain electromagnetic signals from the sample in the domain can pass to the exterior, and exposing, onto an array of active detection elements such as CCD-elements, a spatial representation of electromagnetic signals having passed from the domain, the representation being detectable as an intensity by individual active detection elements, under conditions permitting processing of the intensities detected by the array of detection elements during the exposure in such a manner that representations of electromagnetic signals from the biological particles are identified as distinct from representations of electromagnetic signals from background signals. The size of the volume of the liquid sample is sufficiently large to permit the assessment of the quantity and quality parameters to fullfil a predetermined requirement to the statistical quality of the assessment based on substantially one exposure.

Claims

1. A method for the assessment of at least one parameter of a species of biological particles in a liquid analyte material, comprising applying a volume of a liquid sample representing the analyte material and comprising a plurality of particles, or a plurality of particles isolated from a volume of liquid sample representing the analyte material, to sample compartment from which sample compartment electromagnetic signals from the sample in the compartment can pass to the exterior, the size of the volume allowing identification of at least 10 of the biological particles,

performing one exposure of electromagnetic signals from the sample onto an array of active detection elements forming an image of the plurality of particles, the ratio of a linear dimension of the image on the array of detection elements to the original linear dimension in the sample compartment being from 40:

1 to 1;

10 when the size of the particles is between ⅓

Tm and 3 Tm, and from 3;

1 to 1;

100, when the size of the particles is between 3 T, and 100 Tm, detecting the image as intensities by individual active detection elements,

processing the intensities in order to identify the image of electromagnetic signals from the species of biological particles as distinct from representations of electromagnetic signals from background signals

correlating the results of the processing to the at least one parameter of the liquid analyte material, and

assessing the at least one parameter with a repeatability error of at most 33%.

2. A method according to claim 1, wherein the sample compartment has a wall part defining an exposing area, the wall part allowing electromagnetic signals from the sample into he compartment to pass through the wall and to be exposed to the exterior.

3. A method according to claim 2, wherein the image of the electromagnetic signals is a one-dimensional image.

4. A method according to claim 1, wherein the image of the electromagnetic signals is a two-dimensional image.

5. A method according to claim 1, wherein the array of detection elements is arranged in such a way that a series of detection elements form a substantially straight line.

6. A method according to claim 5, wherein the array of detection elements is arranged in arranged in two directions in such a way that the detection elements form a series of substantially parallel straight lines, the series forming a rectangle.

7. A method according to claim 1, wherein the exposure of the image of electromagnetic signals onto the array of detection elements is performed by focusing an image of electromagnetic signals from at least a part of the exposing domain onto the array of detection elements by means of a focusing means.

8. A method according to claim 7, wherein the focusing means is a lens consisting of one or several elements.

9. A method according to claim 1, wherein the particles the parameter or parameters of which is/are to be assessed are of a size of between ⅓

Tm to 3 Tm, and the ratio is in the range between 10;

1 and 1;

10.

10. A method according to claim 1, wherein the particles the parameter or parameters of which is/are to be assessed are of a size between 3 Tm and 100 Tm, and the ratio is in the range between 2:

1 and 1;

2.

11. A method according to claim 1, wherein the individual particles the parameter or parameters of which is/are to be assessed are imaged on at the most 5 detections elements.

12. A method according to claim 1, wherein the interior of the sample compartment has an average thickness of between 20 Tm and 200 Tm.

13. A method according to claim 1, wherein the sample compartment has dimensions, in a direction substantially parallel to the array of detection elements, in the range between 1 mm by 1 mm and 10 mm.

14. A method according to claim 1, wherein the volume of the liquid sample from which electromagnetic radiation is detected on the array is in the range between 0.01 Tl and Tl.

15. A method according to claim 14, wherein the particles the parameter or parameters of which is/are to be assessed are of a size of between 1/3 Tm to ⅓

Tm, and the volume of the liquid sample from which electromagnetic radiation is detected on the array is in the range between 09.01 Tl and 1 Tl.

16. A method according to claim 14, wherein the particles the parameter or parameters of which is/are to be assessed are of a size of between 3 Tm to 100 Tm, and the volume of the liquid sample from which electromagnetic radiation is detected on the array is in the range between 0.04 Tl and 4 Tl.

17. A method according to claim 1, wherein the sample in the sample compartment is at stand still during the exposure.

18. A method according to claim 1, wherein the sample in the sample compartment is moved through the sample compartment during the exposure, and wherein the exposure is performed over a sufficiently short period of time to substantially obtain stand still condition during the exposure.

19. A method according to claim 1, wherein at least a major part of the electromagnetic radiation emitted from the sample during exposure originates from or is caused by electromagnetic radiation supplied to the sample from a light source, at least a major part of the radiation from the light source having a direction which is transverse to the wall of the sample compartment or a plane defined by the compartment.

20. A method according to claim 1, wherein the parameter to be assessed is the number of the biological particles per volume of the liquid analyte material.

21. A method according to claim 1, wherein the parameter(s) to be assessed is the size and/or shape of the biological particles in the liquid analyte material.

22. A method according to claim 20, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least 100 of the biological particles.

23. A method according to claim 1, comprising applying a volume of between 0.01 Tl and 20 Tl of a liquid sample representing the liquid analyte material, or particles isolated from a volume of a liquid sample representing the liquid analyte material, to the sample compartment the sample in the sample compartment being at stand still during the exposure, and in the case where at least a major part of the electromagnetic radiation emitted from the sample during exposure originates from or is caused by electromagnetic radiation supplied to the sample from a light source, then at least a major part of the radiation from the light source having a direction which is transverse to the wall of the sample compartment or a plane defined by the compartment, and the individual particles the parameter or parameters of which is/are to be assessed are imaged on at the most 25 detection elements of the array of detection elements.

24. A method according to claim 1, wherein the parameter to be assessed is the presence or non-presence of a particular type of particles in the liquid analyte material.

25. A method according to claim 1, wherein particles isolated from a liquid sample representing the analyte are applied to the sample compartment or arranged in the sample compartment, the particles being retained on a particle retaining means selected from means chemically binding the particles, means capable of electronically or magnetically retaining the particles, and filtering means.

26. A method according to claim 1, wherein the signal which is detected by the detecting elements originates from one or several types of molecules of types which bind to, are retained within, or interact with, the biological particles, such molecules being added to the sample or the isolated particles before or during exposure, the molecules being molecules giving rise to one or several of the following phenomena:

attenuation of electromagnetic radiation, photoluminescence when illuminated with electromagnetic radiation, scatter of electromagnetic radiation, raman scatter.

27. A method according to claim 26, wherein an effective amount of one or more nucleic acid dyes and/or one or more potentiometric membrane dyes is added.

28. A method according to claim 1, wherein the duration of the exposure is in the range from 100 milliseconds to 5 seconds.

29. A method according to claim 28, wherein the duration of the exposure is in th range of 0.5 to 3 seconds.

30. A method according to claim 28, wherein the exposure is performed as a single exposure.

31. A method according to claim 21, wherein compression of information of the intensities representing distinct objects scattered over an area, an object being represented by a variation in the intensity information

said information existing in the form of varying degrees of measurable intensity of a physical property distributed over a confined area divided into sub-areas, each of which sub-areas having assigned thereto an index uniquely identifying the sub-area, the method comprising

determining the intensity of the physical property,

a) defining a sub-area of interest situated in a group of sub-areas comprising of at least 2× 2 sub-areas situated adjacent to each other,

b) evaluating in said sub-area of interest at least one directional derivative(s) of the measurable intensity in the sub-area of interest with respect to predetermined geometrical direction(s) in the plane of the confined area, the directional derivative(s) is (are) based on measurable intensities in sub-areas situated adjacent to or in proximity of the group of sub-area,

c) based on th evaluation of the at least one directional derivative an attribute is assigned to the value assigned to said sub-area of interest;

the attribute represent an adjusted measurable intensity and/or information(s) related to a predetermined strategy for adjustment of the measurable intensity in the sub-area of interest or sub-areas situated adjacent to or in proximity to the sub-area of interest,

d) repeating the step a)-c) for substantially all sub-areas of the confined area.

32. A method according to claim 1, wherein the correlation comprises:

identifying and counting substantially all detection elements having intensities which are distinct from background signals,

adjusting the result of the counting by a predefined scaling value,

the scaling value being directly related to the number of detection elements representing a signal from a biological particle,

the result of the scaling being correlated to the number of particles represented exposure.

33. A method according to claim 32, where the measured intensities of the detection elements have been adjusted prior to counting, the adjustment comprising the steps of:

a) defining a range of predetermined size in a co-ordinate system representing the intensity values of the detection elements, the size of the range being determined such that it is bigger than the representation of a biological particle having an average extension,

b) choosing a first detection elements, the first detection element being one of which the intensity is subject to an adjustment,

c) positioning the range such that the detection element of which the intensity is to be adjusted is substantially in the centre of the range,

d) adjusting the intensity of the detection element in the centre of the range based on the result of an investigation of at least one gradient describing the variation of the signal intensities inside the range and around the centre of the range by considering intensities of detection elements describing the gradient,

and repeating the step b) through c) until a predetermined number of detection elements has been adjusted a predetermined number of times.

34. A method according to claim 33, wherein the sample compartment form which electromagnetic signals from the sample in the sample compartment can pass to the exterior is adapted to allow the assessment of substantially only one sample of liquid analyte material.

35. A method according to claim 34, wherein the sample compartment is connected with a reagent container, the reagent container containing one or several reagent component(s).

36. A method according to claim 35, wherein the reagent container contains one or several reagent component(s) in an amount substantially adequate for substantially only one assessment.