U.S. patent application number 12/294037 was filed with the patent office on 2009-09-17 for microfluidic chamber assembly for mastitis assay.
Invention is credited to Charles F. Galanaugh, Rodolfo R. Rodriguez.
Application Number | 20090233329 12/294037 |
Document ID | / |
Family ID | 38541833 |
Filed Date | 2009-09-17 |
United States Patent
Application |
20090233329 |
Kind Code |
A1 |
Rodriguez; Rodolfo R. ; et
al. |
September 17, 2009 |
MICROFLUIDIC CHAMBER ASSEMBLY FOR MASTITIS ASSAY
Abstract
The present invention relates to a device and method for the
detection of mastitis or other disease from a body fluid of a
mammal for example from cow's milk. The device and method relates
to a wedge microfluidic chamber for using a minimal amount of fluid
and being able to use the device to observe leukocytes in a
mono-layer for the purpose of disease detection, cell counts or the
like.
Inventors: |
Rodriguez; Rodolfo R.;
(Durham, NC) ; Galanaugh; Charles F.; (West
Milford, NJ) |
Correspondence
Address: |
PASSE' INTELLECTUAL PROPERTY, LLC
1717 BRASSFIELD RD.
RALEIGH
NC
27614
US
|
Family ID: |
38541833 |
Appl. No.: |
12/294037 |
Filed: |
March 26, 2007 |
PCT Filed: |
March 26, 2007 |
PCT NO: |
PCT/US07/64893 |
371 Date: |
September 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60785877 |
Mar 24, 2006 |
|
|
|
Current U.S.
Class: |
435/39 ;
435/287.1 |
Current CPC
Class: |
G01N 15/1463 20130101;
G01N 15/1484 20130101; G01N 1/2813 20130101; G01N 33/04 20130101;
G01N 2015/008 20130101; B01L 3/5027 20130101 |
Class at
Publication: |
435/39 ;
435/287.1 |
International
Class: |
C12Q 1/06 20060101
C12Q001/06; C12M 1/34 20060101 C12M001/34 |
Claims
1-3. (canceled)
4. A microfluidic chamber assembly comprising: a) a wedge base; b)
a wedge top having a first top edge and an opposite second edge
positioned on the base such that the first top edge is in an
elevated configuration and the second edge is resting on the base
such that it forms a wedged shaped chamber sufficient for cell
differential determination; c) a cut out well positioned to add
liquid to a top portion of the wedge at the first top edge; wherein
the wedge top is secured to the base in the elevated position in a
manner that it does not move during normal use and wherein at least
one of the top or base is optically transparent.
5. A chamber assembly according to claim 4 wherein there are two or
more chamber assemblies on the wedge base.
6. A chamber assembly according to claim 4 wherein the chamber
further comprises a leucocyte observation colorant positioned in
the chamber.
7. A chamber assembly according to claim 4 wherein the first top
edge is elevated by means of a wedge forming device.
8. A system for detecting disease in milk comprising: a) a
microfluidic chamber assembly comprising: i. a wedge base; ii. a
wedge top having a first top edge and an opposite second edge
positioned on the base such that the first top edge is in an
elevated configuration and the second edge is resting on the base
such that it forms a wedged shaped chamber sufficient for cell
differential determination; and iii. a cut out well positioned to
add milk to a top portion of the wedge at the first top edge;
wherein the wedge top is secured to the base in the elevated
position in a manner that it does not move during normal use and
wherein at least one of the top or base is optically transparent;
b) a leucocyte observation colorant positioned within the chamber;
and c) a reader for automatically performing a leukocyte
differential on a milk sample placed in the well and reporting the
results of the differential.
9. A system according to claim 8 wherein the reader is programmed
to take the results of the differentiation and determine one or
more from the group consisting of: a) stage of a disease; b) an
indication for determining whether to do cultures; c) an indication
as to the likelihood that disease will get better; d) an early
warning of mastitis in peri-partum or early lactation; e) a
differentiation between a high SCC due to lactation and a high SCC
due to intra-mammary infection; and f) confirmation of the presence
of mastitis.
10. A method for performing a leukocyte differential assay on a
milk sample comprising: a) adding a milk sample to a microfluidic
chamber assembly comprising: i. a wedge base; ii. a wedge top
having a first top edge and an opposite second edge positioned on
the base such that the first top edge is in an elevated
configuration and the second edge is resting on the base such that
it forms a wedged shaped chamber sufficient for cell differential
determination; and iii. a cut out well positioned to add milk to a
top portion of the wedge at the first top edge; wherein the wedge
top is secured to the base in the elevated position in a manner
that it does not move during normal use, wherein at least one of
the top or base is optically transparent and wherein there is a
leucocyte observation colorant positioned within the chamber; b)
enumerating and calculating the leukocytes in the milk containing
chamber into sub-populations; c) deriving a differential count of
the leucocyte sub-populations.
11. An assay according to claim 10 wherein there are one or more
chambers on each base.
12. An assay according to claim 10 wherein the assay is used to
determine the presence of mastitis in the milk.
13. A method according to claim 10 wherein the assay is performed
in an automatic reader wherein a differential is performed by the
reader on a milk sampler placed in the microfluidic chamber and the
chamber placed in the reader.
Description
[0001] This application claims priority of U.S. provisional patent
application No. 60/785,877 filed on 24 Mar. 2006 incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to microfluidic chamber for
analyzing a subjects' body fluids, in particular milk, to determine
if the subject has a bacterial infection such as bovine mastitis.
Specifically, the invention relates to a chamber assembly which can
be used to detect mastitis or other bacterial disease in bovine or
other mammal species.
[0004] 2. Description of the Related Art
[0005] Mastitis is the inflammation of the mammary gland caused by
microorganisms that invade one or more quadrants of the bovine
udder, multiply, and produce toxins that are harmful to the mammary
gland. Economic loss to mastitis in the United States is estimated
to be approximately $185/cow annually. The total annual US cost of
mastitis is over $ 2 billion. This is approximately 10% of the
total value of farm milk sales, and about two-thirds of this loss
is due to reduced milk production in subclinically infected
cows.
[0006] The average production loss per lactation for one infected
udder quarter is about 1,600 pounds. Other losses are due to
discarded abnormal milk and milk withheld from cows treated with
antibiotic, costs of early replacement of affected cows, reduced
sale value of culled cows, costs of drugs and veterinary services,
and increased labor costs. Mastitis reduces milk yield and alters
milk composition. The magnitude of these changes in individual cows
varies with the severity and duration of the infection and the
causative microorganisms. Mastitis is almost always caused by
bacteria. These microorganisms produce toxins that can directly
damage milk-producing tissue of the mammary gland, and the presence
of bacteria initiates inflammation within the mammary tissue in an
attempt to eliminate the invading microorganisms. The inflammation
contributes to decreased milk production and is primarily
responsible for the compositional changes observed in milk from
infected quarters and cows. In general, compositional changes
involve an increase in blood components present in milk and a
decrease in normal milk constituents.
[0007] Clinical Mastitis includes visible signs of mastitis such as
the "mild" signs, for example, flakes or clots in the milk, or
slight swelling of an infected quarter. It also includes "severe"
signs such as abnormal secretion; hot, swollen quarter or full
udder; fever, rapid pulse, loss of appetite; dehydration and
depression; and in some cases death may occur.
[0008] In Subclinical Mastitis there are no visible signs of the
disease. Diagnosis of sublinical mastitis is characterized by the
Somatic Cell Count (SCC) of the milk being elevated and the
bacteriological culturing of milk will detect bacteria in the milk.
Subclinical mastitis causes the greatest financial loss to dairy
farmers through lowered milk production. For every clinical case of
clinical mastitis, there will be 15 to 40 sub-clinical cases.
[0009] The SCC is the number of leukocytes or white blood cells per
milliliter of milk. The SCC has become the standard procedure for
diagnosing sub-clinical mastitis and is also used worldwide as the
index of milk quality. The SCC enumerates the many cells that
populate the milk-producing gland as part of the immune defense
system, and then cross the blood/milk barrier, as well as the few
epithelial cells that line the udder, and also get into the milk.
In response to infection, the animals' immune systems produce an
"inflammatory response" in the gland and more of the
infection-fighting white cells (mostly neutrophils) find their way
into the milk. The SCC is reported as "the sum", the total of those
cells. Normal milk will have less than 200,000 cells per
milliliter. An elevated SCC (200,000 and over) is an indication of
inflammation in the udder. The SCC is the current measure for
commercial acceptability for milk in many countries, for example
milk with an SCC over 400,000 can not be sold in Europe. Other
countries limits include Canada, 500,000 and the United States
750,000 (600,000 in California). Currently there is no SCC
associated with Clinical Mastitis.
[0010] The SCC is also used in bulk tank management, in determining
the suitability for shipment, in making culling decisions and in
the payment of quality bonuses. In summary, the SCC, a single
number, is everywhere in the dairy industry and it is used in
almost every area of milk production. The SCC led to increases in
productivity, losses due to clinical-stage mastitis being
stabilized, and the generally accepted notion that the milk supply
is safe.
[0011] The SCC is not a number representing a single type of
leucocyte, rather it is a mixture of multiple types of leukocytes,
each with its own significance. In milk from a healthy animal, the
predominant cell types are lymphocytes, followed by much lesser
numbers of neutrophils and macrophages. The percentages of each
kind of cell rise and fall as part of the immune response to
infection. Those percentages, "the differential milk leukocyte
count", represent the unique immune status of an individual quarter
udder, at a specific point in time. The literature has suggested
that a better understanding of the dynamics of mastitis could be
accomplished by specifically measuring the rise and fall in the
types of leukocytes involved in the disease process and recovery.
The literature has also specifically identified the normal
differential pattern of uninfected cows at various stages of
lactation, it has suggested algorithms to identify the various
stages of infection by looking at changes in those patterns and it
has suggested linkages of specific abnormal patterns to specific
pathogens. In 2005, Hamman used the differential pattern to show
quarters were inter-dependent. Essentially, the literature suggests
that the breakdown of the SCC into its component parts, the "milk
somatic cell differential", should be a better, more accurate and
specific indicator of udder health, and thus a better tool for the
management of mastitis. However, in spite of the evolving evidence,
the SCC continues to be reported as a total SCC, and the
differential information remains a research tool. This is primarily
been driven by the cost differential between the SCC and
computations for a differential.
[0012] At least two procedures are currently used in the
determination of the bovine SCC with a leucocyte differential. One
method is using flow-cytometry, an expensive, sophisticated tool
only found in top research laboratories. This method is not even
remotely practical for the farmer. Its only alternative, the
"manual milk differential smear" (MMDS), is a difficult and time
consuming procedure, subject to great variability, even when
performed by highly trained laboratory technologists. It is
impractical for field research or the barn environment as well.
[0013] In order to perform an MMDS, milk is centrifuged at 2500 g
for 10 min at 4 degrees Celsius, the fat layer and supernatant are
removed, leaving 500 ul or less of milk with a pellet. The pellet
is re-suspended via vortexing and pipetting. Microscope slides are
coated with Trypticase Soy Broth, air-dried and 10 ul of milk is
placed on the slide for a 30-degree angle smear to be made. The
slide is allowed to air dry and is then stained with Wright Giemsa
Stain. The slide is placed in the stain for 15 seconds, then water
for 30 seconds, then a 15 second dip in clean water and finally it
is allowed to air dry. Slides are examined on a transmitted light
microscope with a 100.times. oil immersion lens.
[0014] In U.S. Pat. No. 6,350,613 to Wardlaw, there is described a
device and method for determination of white blood cell
differential counts. The slide described in FIG. 1 is a wedge type
slide comprising a microscope slide base, a slip cover wedge top
and a rectilinear shim at the top part of the wedge. This design is
described primarily for blood but is taught to be useful for any
leucocyte containing liquid for example milk. The device suffers
from some difficulties in use. First since the cover slip end
opposite the shim is not fixed the end can leak, shift or get
caught on other objects during use. Second since the design
involves a shim the entire length of the cover slip there is no
easy way to place the liquid in the chamber.
[0015] FIG. 2 in the Wardlaw patent describes the appropriate angle
for creating a wedge sampling configuration for us in differential
testing. Accordingly, it would be very useful to have a different
wedge chamber design that overcomes the limitations of the prior
art teachings.
BRIEF SUMMARY OF THE INVENTION
[0016] It is an object of the invention to provide both an assay
and a novel microfluidic slide assembly using the wedge design that
can be used to test leucocyte containing body fluids such as bovine
milk for a leucocyte differential test. It is an object to make the
test and slide useful for the farmer in the field and easy enough
for a farmer to do or at least prepare. The test is relatively
quick, easy to do, accurate, wherein the slide is disposable. The
farmer can do the test in the field and use very small volumes of
milk. The above objectives and more are achieved by the present
invention and are distinct novel advantages as can be further seen
from the disclosure herein.
[0017] One embodiment of the device is microfluidic chamber
assembly for use in performing leucocyte differential assay on a
leucocyte containing fluid sample from a subject in a wedge
sampling configuration chamber comprising: [0018] a) an wedge base;
[0019] b) a wedge top forming the angle part of the wedge; [0020]
c) a wedge forming device which secures a first edge of the top to
the base forms the bottom edge of the wedge chamber, elevates and
secures the opposite second edge of the top from the base to form a
wedge chamber and provides for a liquid entry well for liquid
addition to the chamber; [0021] wherein at least one of the base or
top is optically transparent.
[0022] The assay itself comprises in one embodiment, method for
performing a leukocyte differential assay comprising: [0023] a)
adding a leukocyte containing liquid to the chamber of a slide
assembly of claim 1; [0024] b) enumerating and calculating the
leucocytes in the liquid containing chamber into sub-populations;
and [0025] c) deriving a differential count of the leucocyte
sub-populations.
[0026] Other embodiments and variations will be obvious from the
disclosure, teachings and examples herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows an embodiment of the invention with a dual
chamber mounted on a base.
[0028] FIG. 2 displays enhanced images produced by an embodiment of
the invention.
[0029] FIG. 3 displays graphs of the resultant cell count produced
by an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention relates to a novel microfluidic wedge
chamber and a leucocyte differential assay (LDA) for determination
if a subject has mastitis in a given sample of milk. The general
description of both the device and method are stated in the Brief
Summary above. This detailed description defines the meaning of the
terms used herein and specifically describes embodiments in order
for those skilled in the art to practice the invention. The assay
is done exposing leucocyte containing body fluid such as bovine
milk, whole blood or other leucocyte containing body fluid such as
lymph fluid, spinal fluid or the like, to a predetermined cell
count in the device of the invention. The test is compared for
difference in leucocyte types and those differences determine if
the subject has mastitis i.e. if the leucocyte SCC is 200,000 or
greater and what the breakdown of different leucocytes within that
population is. A distinct advantage of the assay of the invention
is that it is not only qualitative in nature, it is quantitative in
nature and therefore leucocyte such measurements are more telling
of the exact condition of the subject bovine patient.
[0031] By "subject" is meant herein to be any animal especially
bovine and especially bovine milk containing leucocytes. I addition
other birds, mammals and especially humans, that have body fluids
containing leucocytes. The primary assay of the invention is use of
the chamber to test for bovine mastitis but the chamber could
simply be used to make a leucocyte determination for other bodily
fluids for example as taught in the Wardlaw patent described
above.
[0032] By "leucocyte" is meant any of the sub categories of
leucocytes that are known to exist in milk or blood or other bodily
fluid. These sub-categories can be identified and quantified with
the device and assay of the invention especially for the detection
of mastitis or other leucocyte based disease states.
[0033] By "wedge base" is meant a substrate sufficient for
performing a leucocyte differential determination. In one
embodiment the base would be a microscope slide made of glass or
plastic and optionally optically transparent. It is clear however
that where the determination is made with observation and lighting
from the top of the assembly, that the base could be a
non-transparent material such as paper or opaque plastic.
[0034] By "Wedge top" is meant a substrate which forms the angle
top wall of a chamber such as described in Wardlaw. In one
embodiment that top portion is a slide cover slip of glass or
plastic which is positioned to form a wedge chamber for making cell
count differential determinations. It is an embodiment that the
wedge top be optically transparent but where a reading is taken
from the bottom the top may be opaque and be of a material other
than plastic or glass including paper cardboard, metal or the
like.
[0035] By "wedge forming device" is meant a device which by its
design accomplishes 4 important functions. First, it secures an
edge of the wedge top to the base. Second, it secures the opposite
edge of the top in an elevated configuration such that the wedge
shaped chamber is formed sufficient for cell differential
determination. One skilled in the art as taught above would easily
be able to determine the proper angles for such chamber. Third, it
secures the opposite side in such a manner that it may not move
during normal use and fourthly, it provides a cut-out well so that
liquid may be added to the top portion of the wedge instead of from
the side as in previous prior art wedge chambers. This is
accomplished by use of an adhesive backed film in one embodiment.
The adhesive film is cut out in such a manner that a flap for
adhesion of one side of the top is formed. The film also has a cut
out portion allowing for an unobstructed view of the top and base.
Further it provides a means for securing the top edge of the top
and it also provides a cut out portion for a liquid addition well.
The wedge configuration of the chamber is then formed by one edge
of the top resting against the base and the other opposite edge of
the top resting on the film, the thickness of the film determines
the shape and volume of the chamber. In general, where the base is
a microscope slide and the top a slide slip cover the film would be
chosen to be about 0.04 to 0.06 mm more or less in thickness. In
one embodiment, the film is 0.05 mm in thickness. As can be seen in
the figures, the adhesive allows the film to be attached to the
base and the top simultaneously because of the adhesive backing and
the unique cut-out design. The film is in an embodiment a polymer
which gives it both flexibility and ease of putting an adhesive
backing thereon. The adhesive should be such that it adheres to
both the top and base and one skilled in the art would be able to
make optimum choices depending on the materials chosen for the top
and base.
[0036] In the assay, by "enumerating and calculating" is meant that
the liquid in the chamber is observed by methods taught for
differentiation (see Example) and thus the chamber allows for
differentiation of the leucocyte sub-population. By deriving a
differential count" is meant that once the sub populations of the
ample are determined that a count of each of the sub populations is
made such that the sub population of the total SCC count can be
determined.
[0037] "Leucocyte observation colorants" are compounds known to
differentially color morphological factors, in a leucocyte and
cause various colorations (spectral factors) at various wavelengths
based on the leucocytes reaction or lack of reaction to the
antigen. Examples of such colorants include but are not limited to:
Astrozone Orange, Also known as Basic Orange 21 which is
3-trimethyl-2(2-(2-methyl-1H-indol-3-yl)-vinyl)-3H-indolium
chloride. Other possible colorants include Acridine Orange,
Ethidium Bromide, Griefswalder's Blue, Blue Borrel, Rhodanile Blue,
Toluidine Blue, Night Blue, Prune Pure, Hofmann's Violet, Basic Red
13, Basic Violet 16, Carbocyanine K-5, and mixtures of above. Many
of the colorants are cytotoxic. When selecting a cytotoxic colorant
it is preferable to allow it to be in contact with cells the
minimum time. In the embodiment of the invention where the
observation and reaction chambers are separate the minimum time in
contact is achieved. Where the predetermined time is short enough
or the colorant is not cytotoxic the embodiment where the reaction
and observation microchambers of each test are the same
microchamber can be used.
[0038] The "chamber" is a chamber for which leucocytes measurement
factors can easily be observed by optical scan. The is designed to
spread out the fluid sample in such a manner to make a field by
field, YYZ scan possible. See, for example, U.S. Pat. No. 6,350,613
which describes such chamber and optical scan thereby.
[0039] In one embodiment of the invention the device is made in a
disposable format. This device would be made of plastic, glass or
other inexpensive disposable material. The device of the invention
containing the subject sample can be discarded in an appropriate
manner and the tester need never come in contact with the contents.
The disposable microfluidic device can be constructed credit card
size more or less similar to other microfluidic assays such that it
fits in a reader portion of an image analyzer that can read the
colorimetric data from the tests by either moving the test device
around or moving a reader in the analyzer or both to take readings
of the type in the above referenced patents and also described
herein. A microscopic slide size will also be useful.
[0040] Turning now to the figures FIG. 1 is an embodiment of the
invention wherein two wedge chambers are shown suitable for two
assays. The microfluidic assembly 1 consists of the wedge base 2,
which in this case is a microscopic glass slide. Other materials
including plastics can be used for the wedge base 2 Wedge base 2 is
shown as a rectangle but one skilled in the art could chose what
ever shape necessary or desired to accommodate the assay or assay
machine. In this view the Wedge base 2 is a microscope slide. A
slide is a good embodiment since slides are readily available in
both glass and plastic and normally come optically transparent. In
the embodiment in FIG. 1 there are actually two Microfluidic
chambers since based on the size of the chambers it is easy to
place multiple chambers on the base 2. However, it is clear that
one skilled in the art could place one or depending on their size
more than two on the base 2. The wedge top 6 in this embodiment is
a microscope slide slip cover and has a first edge 15 and an
opposite second edge 16. Thin glass is used in this embodiment but
plastic and other thicknesses as desired could be substituted as
well.
[0041] The wedge forming device 8 is a piece of cut out plastic
sheeting with an adhesive backing facing the wedge base 2. The
wedge forming device 8 is placed against the base 2 so that it
adheres. There are extra portions 12 that extend beyond the base 2.
These extra portions can be folded underneath the base 2 to form a
stronger bond to the base2. The functioning of the wedge forming
device 8 will be clear now upon looking at this embodiment. The
scored hold down flap 20, adheres to the top 6 and holds the first
edge 15 securely against the base 2 forming the bottom point of a
chamber 30. The second opposite edge 16 rests on the upper surface
of the wedge forming device 8 such that it forms the high point
under the top 6 of chamber 30. The top 6 is held down in place on
the upper surface of wedge forming device 8 by two arms 35 which
fold over the top and adhere to the tops upper surface. A last
feature in the wedge forming device 8 is a cut out for a liquid
addition well 38. to perform the assay a liquid for example bovine
milk is placed in well 38 and spreads out evenly in chamber 30. The
design of the chamber is such that at the first end of the top the
area under the top is such that a single layer of cells is created
within chamber 30.
EXAMPLE
Assay for Milk Somatic Cell Differential Counts
[0042] In one embodiment of the assay, 80 .mu.l of milk is mixed
with 20 .mu.l of a meta-chromatic stain, gently mixed, and a small
drop of the mixture is placed in the deposition well of a slide of
the invention. The wedge of the slide chamber fills automatically
by capillary action, the cells in the milk are distributed evenly
at optimum locations, and are ready for observation in seconds. A
pre-concentration step may be required for very low SCC samples.
The wedge can be aptly described as a "self preparing wet
smear."
[0043] Once the wedge slide has self-prepared, it is ready for
immediate analysis by one of three methods:
(a) Visual identification by direct observation of the various
live, intact, fluorescing cells, using a simple fluorescence
microscope (for use by the experienced milk researcher); (b) Visual
identification of the various cells using computer-enhanced digital
camera images in a computer screen (for the use of a laboratory
cytology technician) or (c) Automatic counts of the cells by a
simple imaging instrument requiring minimum operator training (for
use by non-laboratory personnel in the milking barn).
Principle of the Image Computer Enhancement
[0044] Multiple fluorescence images at different wavelengths are
captured and the resulting "enhanced image" is displayed for easy
identification by the lab technician as shown in FIG. 2.
Principle of the Simple Automatic Counter
[0045] In the on-site simple reader version, the enhanced imaged is
analyzed using mathematical features captured by software derived
from face-recognition/machine-vision research, and a report of the
percent of each of the three inflammatory cells is presented, as
well as total SCC.
Variety of Differential Patterns
[0046] The variance of result is illustrated by a few examples of
the variety of patterns present in cows. The graphs below suggest
that not all cows with the same SCC may be assumed to have the same
health status, and that the ratio of differential cells may indeed
have clinical relevance.
[0047] The dark areas are % PMN, the light areas are % lymphocytes
and the cross hatched areas are % macrophages. SCC is .times.1000
cells/ml for all examples. See FIG. 3.
Visual Identification of Computer-Enhanced Images Vs. Manual
Differential Smear
[0048] Composite samples (n=85) from Holstein dairy cows were
collected from North Carolina farms. For each milk sample, a
smear.sup.4 was prepared from an aliquot, using the Wright-Giemsa
stain method, and a "one hundred cell differential" was performed
using light microscopy. The test method was prepared from a second
aliquot and enhanced images were collected. A certified
technologist identified one hundred cells from those images.
Comparison of results is shown.
TABLE-US-00001 Correlation of Visual ID vs. One Hundred Cell Smear
Neutrophils (PMN) R.sup.2 = 0.763 Lymphocytes R.sup.2 = 0.786
Macrophages/Epithelial R.sup.2 = 0.713
Automatic Counts of the Inflammatory Cells by the Simple Imaging
Instrument
[0049] Quarter samples (n=122) from Holstein dairy cows were
collected from North Carolina farms. For each milk sample, a smear
was prepared from an aliquot, using the Wright-Giemsa stain method,
and a "two hundred cell differential" was performed using light
microscopy. The test method was prepared from a second aliquot and
enhanced images were collected. The instrument software identified
two hundred cells from those images. Comparison of results is
shown.
TABLE-US-00002 Correlation of Instrumented ID vs. Two Hundred Cell
Smear Neutrophils (PMN) R.sup.2 = 0.794 Lymphocytes R.sup.2 = 0.863
Macrophages/Epithelial R.sup.2 = 0.724
[0050] The results obtained with this assay match the statistically
expected performance when evaluating manual/visual differential
leukocyte counting methods, and we therefore conclude there is
reasonably good agreement between it and the more difficult milk
differential smear. This new method is a tool to help in the
routine management of mastitis. Possible applications of the newly
available information include: (a) Stage the disease, (b) an
indicator for determining whether to culture, (c) an indicator as
far as which fully symptomatic clinical cows are likely to get
better, (d) an early warning of mastitis in peri-partum or early
lactation, (e) differentiate between a high SCC due to lactation
and a high SCC due to intra-mammary infection, (f) confirm quarters
with mastitis after positive "in-line" conductivity
measurement.
REFERENCES
[0051] 1. Anderson K L, et al. PMN leukocyte function in clinical
bovine patients and in cows with or without Staphylococcus aureus
mastitis. Vet Res Com1992; 16(2):107-115 [0052] 2. Doboo I R, et
al. Use of total and differential somatic cell counts from
composite milk samples to detect mastitis in individual cows. Can J
Comp Med. 1981; 45 (1): 8-14 [0053] 3. Dosogne H, et al.
Differential leukocyte method for bovine low somatic cell count
milk. J Dairy Sci. 2003; (3): 828-34 [0054] 4. Dulin A M, et al.
Cytospin centrifuge in differential counts of milk somatic cells. J
Dairy Sci. 1982; 65: 1247-1251 [0055] 5. Emanuelson U, et al.
Potential of differential somatic cell counts as indicators of
mastitis in quarter milk samples from dairy cows. Acta Vet Scand
1989; (4): 475-81 [0056] 6. Hamman J, et al. Differential cell
count and interdependence of udder quarters, Proceedings IDF
Congress on Mastitis and Milk Quality, June 2005 [0057] 7. Kelly M
et al. Correlation between bovine somatic cell counts and PMN
Leukocyte levels for samples of bulk milk and milk from individual
cows, J Dairy Sci 2000; 83:77:619-627 [0058] 8. Kitchen B J. Review
of the progress of dairy science bovine mastitis, milk
compositional changes and related diagnostic tests. J Dairy Sci
1981; 48:167-188 [0059] 9. Koepke J A, et al. A critical evaluation
of the manual/visual differential leukocyte counting method. Blood
Cells 1985: 11:173-186 [0060] 10. Leitner G, et al. Milk leucocyte
population patterns in bovine udder infection of different
aetiology. Journal Vet Med B 2000; 47, 581-589 [0061] 11. Miller R
H, et al. Flow cytometric analysis of neutrophils in cow's milk. Am
Vet Res 1993; 54:1975-1979 [0062] 12. Paape, M J, et al. Historical
perspective on the evolution of the milk somatic cell count. Flem.
Vet. J. Suppl., 66:93 [0063] 13. Pillai, S R et al. Application of
differential inflammatory cell count as a tool to monitor udder
health. J Dairy Sci 2001; 84:1413-1420 [0064] 14. Redelman D. A
mastitis monitoring program using the differential inflammatory
cell count (DICC) Pages 219-220, Proc 34.sup.th Annual Meeting,
NMC, 1997 [0065] 15. Rivas, A L, et al. Longitudinal evaluation of
bovine mammary gland health status by somatic cell counting, flow
cytometry and cytology. 2001; J Vet Diagn Invest 13:399-407 [0066]
16. Rumke C L. Expected Variability in Differential Leukocyte
Counting. In John A. Koepke (Ed.), Differential Leukocyte Counting
(pp. 39-45). Aspen: CAP 1977 [0067] 17. Schroder A C, et al. The
influence of technical factors on differential cell count in milk.
J Dairy Research 2005; 72: 153-158
[0068] The previous examples are not intended to be limiting. One
skilled in the art would be able to form other cut-outs, make
various material choices and be able to apply the novel design to
other assays involving cell differential and the like. The
disclosure and the claims are therefore not intended to be
limiting.
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