U.S. patent application number 15/449859 was filed with the patent office on 2017-06-22 for method for detecting a plasmodium infection.
The applicant listed for this patent is Siemens Healthcare Diagnostics Products GmbH. Invention is credited to Oliver Hayden, Jan van den Boogaart.
Application Number | 20170175162 15/449859 |
Document ID | / |
Family ID | 45524485 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170175162 |
Kind Code |
A1 |
Hayden; Oliver ; et
al. |
June 22, 2017 |
METHOD FOR DETECTING A PLASMODIUM INFECTION
Abstract
The invention relates to a method for detecting a plasmodium
infection in a patient blood sample, wherein a differential
analysis of the polymorphonuclear neutrophil granulocytes in the
sample is performed, and the distribution of the cell volume and
the cell density, the number of thrombocytes in the sample, and the
distribution of the cell density of the thrombocytes in the sample
is determined.
Inventors: |
Hayden; Oliver;
(Herzogenaurach, DE) ; van den Boogaart; Jan;
(Someren, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Siemens Healthcare Diagnostics Products GmbH |
Marburg |
|
DE |
|
|
Family ID: |
45524485 |
Appl. No.: |
15/449859 |
Filed: |
March 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13996948 |
Aug 29, 2013 |
9605293 |
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PCT/EP11/73428 |
Dec 20, 2011 |
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15449859 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 15/1456 20130101;
Y02A 50/58 20180101; G01N 2015/008 20130101; C12Q 1/02 20130101;
C12Q 1/04 20130101; G01N 2333/445 20130101; Y02A 50/30 20180101;
G01N 2015/0084 20130101 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; G01N 15/14 20060101 G01N015/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2010 |
DE |
102010064131.6 |
Jan 25, 2011 |
DE |
102011003101.4 |
Claims
1. A method for detecting an indication of a possible plasmodium
infection in a patient blood sample, the method comprising the
steps of: a) obtaining a blood sample from a patient suspected of
having a possible plasmodium infection; b) performing a
differential analysis of polymorphonuclear neutrophil granulocytes
in the sample and determining a distribution of cell volume and
cell density by scattered light measurement using a hematology
system; c) coloring the neutrophil granulocytes and determining the
volume distribution of the colored neutrophil granulocytes; d)
determining a number of thrombocytes in the sample by scattered
light measurement using a hematology system; e) determining a
distribution of cell density of the thrombocytes in the sample by
scattered light measurement using a hematology system; f) obtaining
sample parameters from the values determined in steps b)-d),
wherein the sample parameters include the number of thrombocytes in
the blood sample (PLT), the distribution of cell density of the
thrombocytes in the blood sample (MPC), and the distribution of
cell density of polymorphonuclear neutrophil granulocytes in the
blood sample (PMNx); g) evaluating the parameters in relation to a
prespecified criterion, wherein, if the criterion is fulfilled,
presuming a possible plasmodium infection is present, and wherein
the criterion is expressed as (PLT.times.MPC)/100<47 and
PMNx+0.1635.times.(PLT.times.MPC)/100<33; and h) diagnosing a
patient as being possibly infected with plasmodium if the patient
blood sample indicates the presence of a possible plasmodium
infection.
2. The method as claimed in claim 1, wherein the parameters are
determined by scattered light measurements.
3. The method as claimed in claim 1, wherein the volume
distribution of the neutrophil granulocytes is additionally
determined after a coloring.
4. The method as claimed in claim 1, wherein values of the
parameters determined in b), c) d), and e) being below previously
determined criterion are predicative of the presence of a possible
plasmodium infection.
Description
PRIORITY STATEMENT
[0001] This application is a continuation of and claims priority
from U.S. patent application Ser. No. 13/996,948, filed Aug. 29,
2013 and titled "METHOD FOR DETECTING A PLASMODIUM INFECTION"
(Attorney Docket No. SAG-001), which is a 371 of International
Application No. PCT/EP2011/073428, filed Dec. 20, 2011, which
claims the benefit of European Patent Application No.
DE102010064131.6, filed Dec. 23, 2010 and European Patent
Application No. DE102011003101.4, filed Jan. 25, 2011, each of
which is hereby incorporated by reference herein in its entirety
for all purposes.
FIELD OF THE INVENTION
[0002] The invention relates to a method for detecting a plasmodium
infection in a patient blood sample.
BACKGROUND OF INVENTION
[0003] Plasmodium infections, such as malarial sicknesses caused
for example by the pathogens Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovale, Plasmodium malariae and also Plasmodium
knowlesi, are the cause of hundreds of millions of new infections
worldwide per year. According to figures from the World Health
Organization (WHO) the estimate of new infections per year ranges
from 300 to 500 million people.
[0004] In respect of the increasing development of resistance to
the existing medicines for treating plasmodium infections there is
therefore an increasing demand for reliable, low-cost and
quick-to-perform diagnostic methods, which can additionally largely
exclude false-positive and false-negative results. For example
highly-sensitive diagnostic methods would be necessary in regions
with a low prevalence of malarial infections in order to recognize
the few people with the illness as actually having it.
[0005] On the other hand, in regions of the world with a higher
prevalence/incidence, which are usually among the poorer regions of
the world, there is a high demand for test methods with a high
specificity, in order to exclude false-positive results (i.e. also
recognize healthy people as being healthy).
[0006] As a rule the problem also exists of not only an extremely
high number of patient samples having to be examined, but of
this--as discussed above--also having to be done quickly. This is
because the results of the diagnosis should be available within
less than 2 hours. If this is not possible the patient may possibly
have to be treated on the basis of a superficial clinical
analysis.
[0007] The results of an incorrect or false-negative/false-positive
malaria diagnosis are wide-ranging: with a false-positive diagnosis
the (pointless) use of medicines can be accompanied by avoidable
side-effects, quite apart from the financial load on the healthcare
system, as well as the possibility of the plasmodia building up
resistance. In a study from the year 2006 it was concluded that a
diagnostic test with the sensitivity and specificity of 95% in each
case which only needs minimal infrastructure could save more than
100,000 deaths and more than 400 million unnecessary treatments
(Rafael M E, Taylor T, Magill A et al. Reducing the burden of
childhood malaria in Africa: The role of improved diagnosis.
Nature. 2006; 444 (suppl 1): 39-48)
[0008] In practice the main problem arising in the diagnosis of
parasitic illnesses, for example malaria, is that the laboratory
diagnosis is only undertaken when a clinical suspicion arises that
the patient is actually suffering from this type of infection. This
can lead, especially in regions with a low prevalence/incidence, to
persons with the illness not being treated or not being treated in
good time. For example in a Canadian study (Kain et al, 1998,
Clinics in Infectious Diseases 27, 142-149) it was reported that
the correct diagnosis was not initially made for 59% of all
travelers returning infected with malaria. On average 7.6 days
elapsed before the correct diagnosis was made and before the
beginning of therapy for Plasmodium falciparum and 5.1 days for
Plasmodium vivax. These types of delays can lead to significant
complications and to an increased mortality rate (Humare et al,
1997, Canadian Medical Association Journal 156, 1165-1167).
[0009] There are a number of methods available for the diagnosis of
plasmodium infections: the safest method consists of a microscopic
blood examination, however this method is very labor-, time- and
equipment-intensive. With the conventional microscopic method
trained expert personnel can reliably determine the type and the
stage of the infection.
[0010] So-called Rapid Diagnostic Tests (RDT) also exist. For
example monoclonal antibodies are used here for the verification of
parasitic antigens. This test is usually used to detect Plasmodium
falciparum infections.
[0011] A far more sensitive method for malaria diagnosis consists
of the polymerase chain reaction which, because of the high outlay
in materials and time however, is little suited to acute cases.
[0012] The group of Rapid Diagnostic Tests (RDT) has recently also
come to include automated methods which, because of their high
throughput, are outstandingly suitable for wide-area-coverage
verification methods. Cf. Hanscheid T, Pinto B G, Pereira I,
Christino J M, Valadas E (1999) Avoiding misdiagnosis of malaria: a
novel automated method allows specific diagnosis, even in the
absence of clinical suspicion. Emerging Infectious diseases [1999,
5(6): 836-838].
[0013] For automatic use so-called automated cell counters are
being employed with increasing success. Examples of such counters
are the Advia 2120, Sysmex XE-2100 and also CellaVision DM96. These
automated devices, apart from their high throughput rate, provide a
number of benefits, such as for example higher objectivity (no
observer-dependent variability), elimination of statistical
variations which are usually associated with manual counting
(counting high cell numbers), as well as the determination of
numerous parameters which would not be available during manual
counting and, as mentioned, a more efficient and cost-effective
treatment. A few of these devices can process between 120 and 150
patient samples per hour.
[0014] The technical principles of the automatic single cell
counting are based either on an impedance measurement or on an
optical system (scattered light or absorption measurement).
[0015] With the impedance method the counting of the cells and also
the determination of their size is done on the basis of the
detection and the measurement of changes in the electrical
conductivity (impedance) which are caused by a particle which is
moving through a small opening. Particles, such as blood cells for
example, are not themselves conductive, but are suspended in an
electrically-conductive thinning medium. If such a suspension of
cells is passed through an opening, during the passage of a single
individual cell the impedance of the electrical path between the
two electrodes which are located on each side of the opening
temporarily increases.
[0016] For example a method is described in WO 2005/088301 for
verifying malaria and other parasitic infections by means of such
an impedance measurement (see experimental part) and also in the
article entitled "Development of an Automated Malaria Discriminant
Factor Using VCS Technology", Briggs C et al. Am J Clin Pathol
2006.
[0017] By contrast with the impedance methods, the optical method
comprises the passing of a laser light beam through a thinned blood
sample which is detected in a continuous stream by the laser beam.
Each cell which passes through the detection zone of the
throughflow cells scatters the focused light. The scattered light
is then detected by a photo detector and converted into an
electrical impulse. The number of impulses generated here is
directly proportional to the number of cells which pass through the
detection zone within a specific period of time.
[0018] In the optical methods the light scattering of the
individual cells which pass through the detection zone is measured
at different angles. Information about cell structure, shape and
reflection ability is detected through this. These properties can
be used to differentiate between different types of blood cells and
use the derived parameters for diagnosis of deviations of the blood
cells from the norm.
[0019] The values obtained by the two measurement methods are
logically linked by means of differential diagnostics into a
meaningful diagnosis result.
[0020] The sensitivity and specificity of diagnostic methods plays
a major role within the framework of differential diagnostics,
accordingly work on improving these properties is constantly being
undertaken.
[0021] For example in WO 2005/088301 the measured values relating
to the cell volume of lymphocytes and monocytes are detected and
included as parameters for a malarial disease. In more precise
terms the standard deviation of the volumes of the monocyte and
lymphocyte populations is assessed, i.e. their heterogeneity. It
has transpired however that this parameter is not specific enough
for the diagnosis of a parasitic infection, since other infectious
diseases (for example colds) can also result in a change in volume
of the lymphocytes and monocytes. In addition impedance
measurements of blood samples have also proved to be susceptible to
errors, e.g. measurement results can be falsified (for example by
varying viscosity of the suspension to be tested).
[0022] In a similar manner a description is given in "Development
of Automated Malaria Discriminant Factor Using VCS Technology" (see
above) of how the standard deviation of the volume of lymphocytes
and monocytes deviates significantly from the standard value when a
malaria infection is present.
[0023] An example presentation of different sensitivities and
specificities using automated blood test devices can be found in
Table 3, in accordance with which the sensitivity in some cases
only amounts to 48.6 or 52%. Success is thus not achieved to a
sufficient extent with the conventional test methods in providing
methods with high sensitivity and specificity, i.e. methods for
detecting a plasmodium infection, which also recognize ill patients
as such, as well as on the other hand being able to recognize a
healthy patient as healthy.
[0024] In accordance with more recent investigations the
expressiveness of the existing automated test systems (and thus
also of the specified sensitivities/specificities) is also
questionable and allows scope for improvements. In this connection
see the recently published paper "Automated hematology analysis to
diagnose malaria", Malaria Journal 2010, 9:346.
SUMMARY OF INVENTION
[0025] It is therefore an object of the present invention to
provide a method for detecting a plasmodium infection in a blood
sample which produces test results with higher sensitivity and
specificity. It is a further object of the invention to provide a
detection method for malaria infections which by the use of a
number of independent parameters, allows a high specificity and
sensitivity for malaria independently of other infections or
different states of health of the patients. It is yet another
object of the present invention to provide such a method which can
be performed with the aid of automated blood analysis devices.
[0026] This object is inventively achieved by a method with the
features claimed in claim 1.
[0027] The invention creates a method for detecting a plasmodium
infection in a patient blood sample, comprising [0028] a)
Performing a differential analysis of the polymorphonuclear
neutrophil granulocytes in the sample and determining the
distribution of the cell volume and the cell density; [0029] b)
Determining the number of thrombocytes in the sample; [0030] c)
Determining the distribution of the cell density of the
thrombocytes in the sample; [0031] d) Obtaining sample parameters
from the values determined in steps a) to c); and [0032] e)
Assessment of the parameters against a prespecified criterion,
wherein a plasmodium infection is present if the criterion is
fulfilled.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] A more complete appreciation of the present invention and
many of the attendant advantages thereof will be readily understood
by reference to the following detailed description when taken in
conjunction with the accompanying drawings, in which:
[0034] FIG. 1 shows a schematic diagram of a flow cytometry
facility 1 for performing the inventive method.
[0035] FIG. 2 shows a schematic diagram of the large angle
scattered light on the basis of a red blood cell.
[0036] FIG. 3 shows the determination of the cell volume.
[0037] FIG. 4 shows scattered light diagrams executed with the
device ADVIA 2120i.
[0038] FIG. 5 shows the results of an analysis after peroxidasic
coloring with the device ADVIA 2120i.
DETAILED DESCRIPTION OF INVENTION
[0039] The patient blood sample examined in accordance with the
invention as a rule involves a human blood sample. It is however
also possible to examine blood samples of mammals.
[0040] The term "differential analysis", as used herein, means the
recording of a number of measurable individual values of the
components of the patient blood sample which will ultimately be
evaluated combined for the diagnosis to be performed.
[0041] In step a) of the inventive method, to perform this method,
first of all a differential analysis of the polymorphonuclear
neutrophil granulocytes in the sample is carried out in relation to
determining the distribution of the cell volume and the cell
density. However this does not exclude further leukocyte types, for
example eosinophils or basophils also being included in an
examination.
[0042] A deviation of the distribution of the cell volume as well
as of the cell density from the norm normally point to a
pathological state. In the present case, compared to the usually
measured normal values, lower values point to a plasmodium
infection. The reduction of the cell volume and of the cell density
associated with the plasmodium infection is to be explained by the
defense mechanisms of the leukocytes occurring during the course of
the infection. For example neutrophil granulocytes migrate from the
blood vessel into the tissue, secrete proteolytic enzymes there in
order to release intercellular compounds and phagocytize bacteria
there. This results in a change to the cell volume and the cell
density.
[0043] It has surprisingly been shown that the sensitivity and
specificity of the detection method for a plasmodium infection can
be greatly increased if, as well as the values determined above
(i.e. sequentially or simultaneously) both the thrombocyte number
in the patient blood sample and also the distribution of the cell
density of the thrombocytes in the sample is determined. Here too a
value deviating downwards from the norm is predictive of the
presence of a plasmodium infection. In accordance with the
invention this leads to a greatly increased sensitivity and
specificity of the detection method compared to existing
methods.
[0044] From the measurement results obtained in steps a) to c)
sample parameters are obtained and assessed in relation to a
previously defined criterion, wherein a plasmodium infection is
present if the criterion is fulfilled.
[0045] The parameters involve derived variables, for example the
term cell volume parameter stands for a parameter related to the
cell volume distribution such as for example the average cell
volume or the standard deviation of the cell volume distribution of
a given cell subpopulation.
[0046] The term "previously determined criterion" as used herein
relates to a criterion which was established on the basis of one or
more sample parameters, in the case of the present invention
especially based on cell volume parameters, cell number parameters
and cell density parameters. The criterion is determined on the
basis of a comparison between infected blood samples and
corresponding values of normal blood samples, for example, for the
experimental investigations on which the present invention is
based, a comparison of 204 P. falciparum infected blood samples
with corresponding values of 3240 normal blood samples was carried
out.
[0047] By combination and assessment of sample parameters of
polymorphonuclear neutrophil granulocytes (cell volume and cell
density) and additionally, by including the thrombocyte number and
also the distribution of the cell density of the thrombocytes in
the sample, in accordance with the invention unexpectedly high
values for the sensitivity and specificity of the detection method
were able to be obtained. As is presented in the examples, success
rates for specificity values of 99% and sensitivity values of 98%
have been achieved here. This means that with full-coverage
automated blood examinations the number of false-positive or
false-negative diagnosis results could be reduced to a previously
unknown extent. This means great progress in respect of a secure
plasmodium infection diagnosis, especially malaria diagnosis and in
countries which are affected by a high prevalence/incidence of
malaria infections will lead to a marked improvement of the overall
state of health and timely and sensible medical action.
[0048] In an embodiment of the inventive method the parameters are
determined by scattered light measurement. By contrast with
impedance measurement, scattered light measurement involves, as
stated above ("optical methods") a method in which laser light is
used, wherein the blood samples (cell by cell) are passed through
the laser light and the deflection of the laser beams can be
detected by a suitable facility. The method performed using the
laser beams is explained in the enclosed FIG. 1. In this method the
light beams scattered by the individual cells are detected in
various angular ranges (low angle and high angle), which
respectively allow information to be obtained about volume (low
angle) and density (high angle). In accordance with the invention
an angle of around 2.degree. to 3.degree. deviation from the laser
light axis is referred to as a "low angle", an angle of around
5.degree. to 15.degree. as a "high angle". It has transpired that
scattered light measurement is superior to impedance measurement in
respect of lower susceptibility to errors of the measurement
results.
[0049] In an embodiment the plasmodium infection involves an
infection with P. ovale (Malaria tertiana), P. vivax (Malaria
tertiana), P. malariae (Malaria quartana) or P. falciparum (Malaria
tropica). The inventive detection method can be used equally for
these plasmodium infections.
[0050] In an embodiment, in addition to the above method steps, a
spherization and differential analysis of the reticulocytes and
erythrocytes in the patient blood sample is additionally carried
out by scattered light measurement. Here too a differential
analysis is involved, wherein the parameters ultimately to be
evaluated involve the cell volume, the cell density and also the
hemoglobin content of the cells and of the reticulocyte portion.
Reticulocytes involve young red blood corpuscles which, by contrast
with the erythrocytes, still contain RNA themselves and can be
differentiated by this method.
[0051] The spherization of the erythrocytes or reticulocytes is
necessary in order to convert the blood cells independently of
their original form into a form able to be evaluated with the
scattered light measurement. For this purpose the blood sample has
a reagent added to it which leads to a spherization of the
reticulocytes and erythrocytes. Typical reagents are disclosed in
U.S. Pat. No. 5,045,472, U.S. Pat. No. 5,284,771, U.S. Pat. No.
5,633,167 and also U.S. Pat. No. 6,114,173. For example U.S. Pat.
No. 5,045,472 discloses a reagent mixture comprising an isotonic
aqueous solution, a spherization means (for example a detergent
such as alkali metal salts of an alkyl sulfate) and also a protein,
which reversibly binds the spherization means.
[0052] By including the reticulocytes and a erythrocytes in the
inventive detection method specificity and sensitivity can be
further increased. The ratio of reticulocytes to erythrocytes is an
important indicator of the presence of a plasmodium infection in
blood. The higher the ratio of reticulocytes, i.e. younger, more
immature erythrocytes in relation to mature erythrocytes, the
higher is the probability of the presence of a plasmodium
infection.
[0053] In a further embodiment, after a peroxidasic coloring of all
leukocytes in the blood sample, a differential analysis is carried
out by scattered light measurement and absorption.
[0054] The peroxidasic coloring is usually undertaken by cellular
peroxidasic activity after conversion with 4-Chloro-1-naphthol.
4-Chloro-1-naphthol serves here as a substrate which makes it
possible for hydrogen peroxide to form a dark precipitate at
endogen locations of the peroxidasic activity in the granula of the
leukocytes. In a scattered light measurement the cells with a small
or moderate peroxidasic activity will then absorb less light, while
cells with a high peroxidasic activity will absorb more light.
[0055] In a further embodiment, after the peroxidasic coloring, as
previously explained, the volume distribution of the neutrophil
granulocytes is additionally measured. It is also true here that
the greater the volume distribution of the neutrophil granulocytes
is, the greater is the probability of the presence of a plasmodium
infection in the patient blood sample.
[0056] In a further embodiment the standard deviation of the volume
distribution of all leukocytes after peroxidasic coloring is used
as an additional parameter. The greater the standard deviation of
the volume distribution of all leukocytes is, the greater is the
probability of the presence of a plasmodium infection.
[0057] In an embodiment the determination in step a), i.e. the
performing of a differential analysis of the polymorphonuclear
neutrophil granulocytes in the sample after lysis of at least the
eosinophils and neutrophils, but not of the basophil leukocytes
(BASO lysis reagent), is undertaken by a suitable lysis reagent.
Here too the presence of a wide volume or density distribution is a
symptom of the presence of a plasmodium infection. Mixtures based
on phatalic acids and detergents have proved to be advantageous
here as lysis reagents, for example such a reagent can contain
hydrochloric acid, phthalic acid, a surfactant and also optionally
a conservation means. The ratio of phthalic acid and hydrochloric
acid is around 2.5:1 here (based on their concentration in mmol/l.
Through this reagent the red blood corpuscles, the thrombocytes,
and all leukocytes (except for the basophil granulocytes) are
lysed. The plasmodium-specific prediction value can also be
increased by this method.
[0058] In a further embodiment, after a specific lysis of all cells
except for the basophil leukocytes, the non-specific portion from
the scattered light diagram (low angle and high angle distribution)
is determined. The greater this portion is the greater is the
probability that a plasmodium infection is present.
[0059] The determination steps a), b) and c) performed in the
inventive detection method are, as explained above, predictive for
the presence of a plasmodium infection. The term "low" values in
the context of the present invention means a lower value of the
determination performed in the individual case compared to the
standard values obtained for normal patient samples (i.e.
non-infected patient samples).
[0060] By contrast, the additional inclusion of the parameters
which are obtained by peroxidasic coloring as well as in
determination of the volume distribution of the neutrophil
granulocytes as well as the standard distribution of the volume
distribution of all leukocytes, as well as the determination of the
unspecific portion from the scattered light diagram after specific
lysis of all cells, means that the higher the values are, the
higher is the probability that a plasmodium infection is
present.
[0061] Embodiments of the inventive method are also described in
greater detail below, which refer to the enclosed figures, in
which:
[0062] FIG. 1 shows a schematic diagram of a flow cytometry
facility 1 for performing the inventive method. The flow cytometry
facility 1 includes a laser 2, a sensor module 3, optical lenses 4,
a semi-transparent mirror 5, an aperture 6, a reflector 7, and
sensors 8, 9, 10.
[0063] FIG. 2 shows a schematic diagram of the large angle
scattered light on the basis of a red blood cell 11, which
correlates with the granularity and density of the cell and is
measured in a typical range of 5.degree. to 15.degree.. This
corresponds to the determination of the cell density.
[0064] FIG. 3 shows the determination of the cell volume (or of the
cell size) of the cell 11 in the low angle scattered light range
(2.degree. to 3.degree.).
[0065] FIG. 4 shows scattered light diagrams executed with the
device ADVIA 2120i, which shows the typical displacement of the
volume 16 and the density 17 for thrombocytes and polymorphonuclear
leukocytes (PMNx) for malaria-infected blood samples. FIG. 4
includes a scattered light diagram showing Baso volume 12, Baso
configuration 13, large-angle scatter PLT 14, small-angle scatter
PLT 15, and a nonspecific component from the scattergram 18.
[0066] FIG. 5 shows the results of an analysis after peroxidasic
coloring with the device ADVIA 2120i, which shows examples of the
volume and density distribution (PEROX Y 26 or PEROX X 27) between
a malaria-infected sample 24 and a normal sample 25. "PEROX Y" 26
refers to the width of the peroxidasic-negative populations along
the Y axis. With a malaria sample this appears longer by comparison
with a normal sample. The results for the malaria sample 24 have a
more diffuse effect than the normal sample 25, which is
attributable to the greater volume distribution 26 and density
distribution 27. In FIG. 5, lymphocytes 19, large unstained cells
20, monocytes 21, neutrophils 22, and eosinophilic leukocytes 23
are represented.
[0067] The performance of the inventive method is shown by way of
example below as well as two algorithms which, on the basis of the
inventive method, make possible the evaluation of the determined
parameters and a prediction of a plasmodium infection with high
specificity and sensitivity. The measurements are performed with
the ADVIA 2120i system from SIEMENS AG.
TABLE-US-00001 TABLE 1 The following abbreviations are used:
Parameter Description PMNx Density distribution of the
polymorphonuclear leukocytes MPC Distribution of the cell density
of the thrombocytes PLT Total quantity of thrombocytes in the blood
sample PLT mode Device setting of ADVIA 2120i % Baso Noise
Unspecific portion from the scattered light diagram (small and
large angle distribution) after specific lysis by BASO lysis
reagent % Abnorm Standard deviation of the cluster distribution of
all leukocytes PEROX Y Sigma Volume distribution of the neutrophil
granulocytes after peroxidasic coloring
[0068] In accordance with a first inventive evaluation the
following algorithm 1 was produced for a detection method for a
malaria infection with high specificity:
PLT.times.MPC/100<47 and
PMNx+0.1635.times.(PLT.times.MPC/100)<33
[0069] On the basis of this algorithm it can be clearly seen (see
below) that the setting of the results of a differential analysis
of the polymorphonuclear neutrophil granulocytes in relation to one
another in relation to the distribution of the cell density (PMNx)
with the number of thrombocytes and the distribution of the cell
density of the thrombocytes in the sample, possibly with the
inclusion of further sub algorithms (1b and 1c) leads to a very
high specificity.
[0070] With the inclusion of the further sub algorithms
PLT mode-30.times.% Baso Noise<27 and also
Perox Y Sigma+12/70.times.% Abnorm>12
a test specificity for malaria of 99% and a sensitivity of 76.5%
was produced.
[0071] Algorithm 1 can thus advantageously be used in countries
with a high prevalence of malaria.
[0072] For countries with a lower malaria prevalence the following
algorithm 2 would make sense:
PMN peak+14/140.times.(PLT.times.MPC)/100<34 2a
Baso Noise>0.074 2b
PLT Mode+25.times.% Baso Noise<45 2c
Perox Y Sigma>6.6 2d.
[0073] The observed specificity amounted to 90.2%, the sensitivity
to 98.9%. In other words an extremely high sensitivity can be
achieved by the algorithm 2.
[0074] Thus almost all malaria positive samples can be detected by
the algorithm 1 in a large sample population, while algorithm two
is especially suitable for screening examinations. A combination of
the two algorithms can additionally be of advantage.
* * * * *