Patent Application
20200096530
The present invention relates to the field of analysis of capillary blood samples using automated devices, in particular to determination of thrombocyte counts from such samples.
Methods and devices for determining blood cell counts from capillary blood samples in a point-of-care setting are well known. In a typical case, a small sample (microliters) of capillary blood (as opposed to venous blood) is drawn into a thin glass capillary coated with an anticoagulant, typically EDTA. The glass capillary with the blood sample is then placed in an automated device, which performs a dilution in an isotonic saline buffer followed by determination of cell counts, such as red blood cells, various classes of white blood cells and thrombocytes (also called platelets).
In the technical field, it is a recognized problem that when using capillary blood samples to determine the thrombocyte count from a patient using an automated integrated instrument, the results are often falsely low compared to counts obtained using venous samples from the same patient, sampled around the same time point. The problem stems from the tendency of thrombocytes to aggregate despite use of powerful anticoagulants such as EDTA coated on the capillary walls.
A proposed solution to the problem is described in U.S. Pat. No. 8,927,228, where the inventors have discovered that addition of chloroquine salts to the sample alleviates the problem of aggregates.
A distinct separate problem, which is not the aim of the present invention is EDTA-dependent pseudothrombocytopenia (PTCP), the rare phenomenon of a spurious low platelet counts due to EDTA-induced aggregation of platelets in some patients, which can be resolved e.g. with kanamycin. The unusual PTCP phenomenon is distinct from the general case of less accurate counts from capillary samples compared to venous samples.
However, there is still need in the field to provide alternative and/or improved methods and devices for thrombocyte determination from capillary blood using integrated automated devices, in particular obviating the need to use additional reagents and being cost-effective to implement.
The term EDTA refers to ethylenediaminetetraacetic acid, a chelating agent that binds calcium and other divalent metal cations.
The terms thrombocyte and platelet are used interchangeably.
The term capillary blood refers to a blood sample from a capillary of a subject, typically a puncture/stick of a finger. Capillary blood is distinct from venous blood, which is defined by being sampled from a vein, as well as from arterial blood sampled from an artery. In addition to the source, the sampling methods differ between capillary blood and venous blood, leading to distinct properties for samples of each category.
The term MPA refers to an adapter device designed for and used in the Medonic M-series M32 instruments and the Swelab Alfa Plus instruments. It is intended to receive and then dilute a small defined volume of a blood sample contained in a capillary tube open in both ends. The technology is described in U.S. Pat. No. 6,284,548B1.
The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.
Normally, the determination of thrombocyte counts from capillary blood involves a dilution step prior to determination. Furthermore, in case of automated analysis using integrated instruments, the determination after the dilution is performed in the shortest possible time, no more than a few seconds, to optimise instrument throughput. However, the known protocols used in such instruments lead to falsely low thrombocyte counts due to thrombocyte aggregation, unless additional reagents such as chloroquine salts are added to the diluted sample.
The inventors have unexpectedly found that the problematic aggregation of thrombocytes in capillary blood samples can be reversed by incubating a diluted capillary blood sample for a short time period (23 s or more), under certain conditions, before the thrombocyte counting is performed (see Examples 1-5).
The discovery provides a method for thrombocyte counting from capillary blood samples, which gives more accurate results without need for any additional reagents, such as chloroquine salts. Thus, existing reagents and kits can be used in the improved method with more accurate results, which is of great practical importance in many settings. Furthermore, in many instances, existing automated integrated devices for thrombocyte counting can be adapted to the more accurate method by a simple software update or other minor modification, whereby the inventive method is cost-effective to implement.
In a first aspect, the present invention provides a method for determining the thrombocyte count in a capillary blood sample, comprising the steps of:
That the capillary blood sample is subjected to anticoagulant treatment with EDTA at sample collection means that the blood sample is brought into contact with EDTA immediately after being sampled, preferably within less than 1 s, more preferably the sample is collected into an EDTA-containing container. The anticoagulant treatment necessarily implies that the blood sample is subjected to an amount of EDTA sufficient for inhibiting coagulation.
The capillary blood sample provided may be a blood sample collected in an EDTA-coated sampling device containing an amount of EDTA sufficient to have a coagulation-inhibiting effect on the sample. The sampling device may be an EDTA-coated capillary.
The non-lytic buffer may be a buffered physiological saline solution, such as phosphate buffered saline or HEPES-buffered saline. Many different known types of buffers may be used, as long as they do not damage or change the thrombocytes to be counted to such an extend as to impede the counting.
In accordance with the invention, the dilution, incubation and determination steps (b, c and e), as well as the optional second dilution step (d) if present, are performed using an automated integrated device for thrombocyte counting (100) (optionally including readout on other blood parameters as well), such as a device of the second aspect described below. Adding an extra delay for incubation according to the present invention in integrated devices runs counter to conventional design principles, since such devices are normally designed to perform the analysis in the shortest possible time to optimise performance.
Incubations of the EDTA-coated capillary in which the blood sample was taken prior to dilution steps was also tested (see Table II in Example 3) for 2 minutes, 6 minutes and 10 minutes as compared to 0 minutes, i.e. with all these 4 incubations before dilution then following the normal analytical cycle. The outcome was that thrombocyte count showed an increase up to the 6 minutes and then stabilized. An incubation before dilution thus seems to be able to give similar results as the method of the present invention, but at a much slower rate. The improvements seen after a 6-minute incubation of the undiluted sample are equivalent to only a few seconds incubation after dilution. Given that rapid results and high throughput are highly desirable, the method of the present invention provides a significant improvement compared to the option of simply incubating the samples in the capillaries.
Preferably, the sample is not from a patient afflicted with EDTA-dependent pseudothrombocytopenia, a rare phenomenon than can be resolved e.g. using kanamycin.
As discussed above and shown under Examples 3 and 5, the platelet counts also can be improved by an incubation before dilution occurs. However, this is much less effective from a time performance perspective, and incubation at a dilution 1:45000 was not effective at all. Therefore, the incubated sample needs to be diluted to within a certain range (1:10 to 1:2000) during incubation for optimal results. Preferably, the dilution factor is 1:30 to 1:1000, more preferably 1:50 to 1:450, yet more preferably 1:150 to 1:300, still more preferably 1:175 to 1:225 and most preferably 1:225.
As shown in Example 4, the incubation must take place in the presence of an amount of EDTA efficient for reducing platelet aggregation to achieve improved results. What constitutes an efficient EDTA concentration in each specific case depends on other components in the non-lytic buffer, since EDTA has the effect of binding divalent metal cations in solution. The binding of Ca2+, thus reducing the concentration of free Ca2+ is generally considered the main mechanism for anticoagulant action of EDTA. While not wishing to be bound by theory, it is theorized that the divalent cation (in particular Ca2+) binding activity is behind the observed effect of incubation on platelet aggregation. Several classes of cell adhesion molecules relevant for platelet aggregation, such as integrins and cadherins are Ca2+ dependent so aggregation mediated by such cell adhesion molecules would be inhibited by an EDTA concentration sufficient for lowering the level of free Ca2+ below a certain threshold.
It is apparent from the above reasoning, that a higher concentration of Ca2+ ions in the diluent means that a higher concentration of EDTA is required, in order to achieve the sufficiently low concentration of free Ca2+ ions. Thus, the efficient amount of EDTA will depend on the concentration of divalent metal cations in the buffer. If other chelating agents that also bind Ca2+ are present, the required concentration of EDTA is lowered. Therefore, the effective concentration of EDTA needs to be determined in view of the other compounds present in the buffer. It is a matter of routine design and experimentation for a person having ordinary skill in the art to determine the effective EDTA concentration for the circumstances at hand, given the teachings herein.
Preferably, the incubation takes place in the presence of at least 0.1 mM EDTA, more preferably at least 0.2 mM EDTA, yet more preferably 0.3 mM EDTA, still more preferably at least 0.4 mM EDTA, most preferably at least 0.5 mM EDTA.
Also preferably, the incubation takes place in the presence of 0.2-4 mM EDTA, more preferably 0.3-4 mM EDTA, yet more preferably 0.3-0.7 mM EDTA.
The incubation may take place in the presence of 0.1-5 mM EDTA, preferably 0.2-4 mM EDTA, more preferably 0.3-3 mM EDTA, yet more preferably 0.3-2 mM EDTA, still more preferably 0.3-1 mM EDTA, even more preferably 0.4-0.7 mM EDTA, yet even more preferably 0.4-0.6 mM EDTA, most preferably 0.50-0.56 mM EDTA.
In the present context, by incubation it is meant that the sample is within the appropriate effective limits of dilution and EDTA concentration irrespective of whether other activities (such as mixing) are simultaneously ongoing or not.
The principle is best understood when explained in the framework of a concrete example, but this is not to be understood as limiting the scope of the invention. As illustrated in a non-limiting fashion in Table A, a typical automated thrombocyte determination method comprises a number of handling steps, with a defined duration. In the case illustrated in Table A, the sample is placed in the instrument (such as an instrument of
The aliquot is then flushed with diluent to the counting chamber thus creating a second dilution. As the flushing duration is 4 s and the dilution factor is 1:225, it can be deduced that it takes 1 second at most to dilute the sample beyond the dilution factor specified in the claims. The second dilution is allowed to mix and cell counting performed in the detector.
Thus, the standard procedure can be regarded as incubating the sample within the appropriate limits of dilution and EDTA concentration for the invention for a total of 18 s, whereas the inventive method shown in comparison results in a total of 33 s incubation.
Example 3 was performed in an instrument that follows the standard cycle illustrated in Table A. As shown in Table I of Example 3, a significant improvement in platelet count can be discerned when an added time delay of 5 s was added to the incubation step. From an added delay of 15 s onwards the effect is better than at 5 s and remains the same for longer incubations. There does not appear to be any clear upper limit, but longer incubation periods than necessary are undesirable as they increase the analysis time and reduce throughput. An optimal incubation period seems to be about 33 s, since this incubation time results in stable, full effect on platelet counts in the shortest time.
Thus, the incubation step duration may be 23-300 s, preferably 25-90 s, more preferably 27-60 s, yet more preferably 28-40 s, still more preferably 30-36 s and most preferably 33 s.
It may be desirable for practical reasons to perform a second dilution step subsequent to the incubation but prior to platelet count determination. Depending on the counting device (detector), additional dilution may be necessary for the determination to be feasible. In the case illustrated in Table A, there is a second dilution step with 1:200 dilution, resulting in a total dilution of 1:45000 before the platelet count is determined.
The second dilution step may involve diluting the sample by a second dilution factor of 1:50-1:1000, preferably 1:100-1:300, more preferably 1:175-1:225 and most preferably 1:200.
Existing devices that are possible to configure to perform the method according to the first aspect of the present invention are known in the prior art and commercially available.
The absolute majority of haematology devices used for making a blood count use a system with two dilution steps. Such a system can readily be modified by addition of a receiver for a sampling device for a capillary blood sample and configured to perform the method of the first aspect of the present invention e.g. by a software update.
Detection of cells is usually achieved either via the “Coulter principle” or the “Flow cytometry principle”. Both are well known methods, and both the methods are impaired by aggregation of the thrombocytes when determining the thrombocyte count.
The table below lists four representative example systems including the M-series M32 system used in the examples, using different means to achieve a two-step dilution and thrombocyte count, in an integrated and automated fashion. All the systems discussed here could readily be modified to perform the method as exemplified by the M-series M32 system, with guidance from the teachings herein.
The dilution elements and detectors for determining the cell count of red blood cells and thrombocytes employed in each instrument are shown in distilled schematic overviews (
Receiver shown as blood inlet 1 is in position to receive a blood sample. Syringe 2 pulls a specific volume of the blood sample into blood inlet 1. Receiver blood inlet 1 is moved over the dilution element 8. Syringe 3 ejects the sample with diluent 6 into the dilution element 8 to create the first dilution. Receiver blood inlet 1 is dipped into the dilution element 8 and syringe 2 pulls a specific volume of the first dilution into blood inlet 1. Blood inlet 1 is moved over the second dilution element 9. Syringe 3 ejects the sample with diluent 6 into the second dilution element 9 to create the second dilution. Valve 10 is opened and syringe 4 pulls the second and final dilution into third dilution element 16. Valve 10 is closed and syringe 4 pushes the dilution through the detector 17 using the coulter principle to determine the thrombocyte cell count before finally being ejected as waste 15.
Pipette System with a Bath and Flow Injection Principle Schematic and Operation for the Count of Thrombocytes (
Receiver shown as blood inlet 1 is in position to receive a sample. Syringe 2 pulls a specific volume of the blood sample into receiver blood inlet 1. The receiver blood inlet 1 is moved over the dilution element 8. Syringe 3 ejects the sample together with diluent 6 into the dilution element 8 to create the first dilution. Valve 10 is opened and syringe 4 pulls the first dilution into holding element 11. Valve 10 is closed and syringe 4 moves the first dilution via the injector needle 12 into the hydrodynamic focusing chamber 13, at the same time syringe 5 moves diluent 7 into the hydrodynamic focusing chamber 13. The ratio of dispensed diluent from syringe 5 and first dilution from syringe 4 defines the ratio of the second dilution of the sample as it is pushed through the detector 14 using the flow cytometry principle for the thrombocyte cell count and then finally to be ejected as waste 15.
Shear Valve System with Two Baths Principle Schematic and Operation for the Count of Thrombocytes (
Shear valve 18 connects the receiver shown as blood inlet 1 and syringe 2. Syringe 2 pulls the blood sample via receiver blood inlet 1 into the shear valve 18. The shear valve 18 connects diluent 6 and dilution element 8. Syringe 2 pushes diluent 6 and the blood in the shear valve 18 into dilution element 8 creating the first dilution. Shear valve 18 connects syringe 2 and dilution element 8. Syringe 2 pulls the first dilution into the shear valve 18. Shear valve 18 connects diluent 6 and dilution element 16. Syringe 2 pushes diluent 6 and the first dilution in shear valve 18 into dilution element 16 creating the second and final dilution. Shear valve 18 connects receiver blood inlet 1 and syringe 2, closing the path to/from the dilution element. Syringe 3 pushes the second and final dilution through the detector 17 using the coulter principle to determine the thrombocyte cell count before finally being ejected as waste 15.
M-series M32M with a Micro Pipette Adapter and a Shear Valve System with Two Baths Principle Schematic and Operation for the Count of Thrombocytes (
Two different dilution operations can be performed.
If valve 10 is closed throughout the operation, then the procedure as described for
If valve 10 is used to divert the diluent through the micro pipette adapter, then the following procedure can be performed. Refer to Table A above for durations of the various stages.
Shear valve 18 is closed to all connections. A capillary tube with a blood sample is inserted into the receiver, a micro pipette adapter 19. Valve 10 is open and syringe 2 pushes diluent 6 through valve 10 and the blood in the receiver micro pipette adapter 19 into the dilution element 8 creating the first dilution. The device may be configured to perform the inventive incubation at this stage (see Table A). Valve 10 is closed.
From this Position the Procedure Continues as in
Shear valve 18 connects syringe 2 and dilution element 8. Syringe 2 pulls the first dilution into the shear valve 18. Shear valve 18 connects diluent 6 and (second) dilution element 16. Syringe 2 pushes diluent 6 and the first dilution in shear valve 18 into dilution element 16 creating the second and final dilution. Shear valve 18 connects blood inlet 1 and syringe 2, closing the path to/from the dilution element. Syringe 3 pushes the second and final dilution through the detector 17 using the coulter principle to determine the thrombocyte cell count before finally being ejected as waste 15.
All these haematology devices could either replace the first blood aspiration step with an adapter or use a parallel adapter, as exemplified by the M-series M32M, to serve as a receiver for a sampling device containing a capillary blood sample subjected to anticoagulant treatment with EDTA at sample collection. This adapter would be intended to receive and dilute a small defined volume of a blood sample contained in a capillary tube open in both ends (see U.S. Pat. No. 6,284,548B1 for an exemple of a suitable adapter).
Thus, in a second aspect, the present invention provides a device for determining the thrombocyte count in a capillary blood sample, comprising:
During operation, the device may be configured such that the incubation during operation takes place in the presence of an amount of EDTA efficient for reducing platelet aggregation. The non-lytic buffer used for dilution in the dilution element (8) during operation may have an EDTA concentration resulting in an effective EDTA concentration as specified under the disclosure of the first aspect.
The device may have a physical configuration as shown in
The device may be configured such that incubation duration is 23-300 s, preferably 25-90s, more preferably 27-60 s, yet more preferably 28-40 s, still more preferably 30-36 s and most preferably 33 s.
The device may be configured such that the dilution factor is 1:30 to 1:1000, preferably 1:50 to 1:400, more preferably 1:150 to 1:300, yet more preferably 1:200 to 1:250, most preferably 1:225.
The device may comprise a second and/or further dilution element(s) (9, 13, 16) arranged, during operation, for subjecting the sample to a second and/or further dilution step(s) prior to determining the thrombocyte count from the incubated sample, for example as shown in
The second dilution step may involve diluting the sample by a second dilution factor of 1:50-1:1000, preferably 1:100-1:300, more preferably 1:175-1:225 and most preferably 1:200.
The term “comprising” is to be interpreted as including, but not being limited to. All documents cited herein are hereby incorporated by reference in their entirety. The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other.
The following examples are not limiting to the scope of the invention. For further experimental details, the skilled reader is directed to the section Material and Methods.
Prolonged Post-Dilution Incubation Time Improves Thrombocyte Counting from Capillary Blood
It was serendipitously discovered during product development that increasing the incubation time after the first dilution step by 15 s, the thrombocyte counts seemed to improve, in terms of being closer to the true values obtained from the same individuals using venous blood samples.
In an earlier data set (presented in
The serendipitous discovery prompted the inventors to perform similar experiments but with the added time delay for incubation. It is apparent from
Some individual donors exhibit marked differences between capillary and venous samples whereas others do not. A comparison of particle counts from an individual donor NOT exhibiting difference between capillary and venous counts shows that the added time delay does not markedly change the distribution of particle frequency counts (
In contrast, in an individual donor exhibiting large difference between capillary and venous count it can be seen that particle count in the 30-45 fL range is drastically reduced by the added 15 s time delay (
It is known from literature that platelet aggregates are detected in this range, so it was concluded that the added time delay improved the accuracy of thrombocyte counts by reducing platelet aggregation.
In order to explore the effect further, different added time delays were tested. From the results in table I it can be seen that an improvement can be discerned already with 5 s added delay. No further improvement is apparent for incubation times with more than 15 s added delay.
In another experiment, the effect of mixing was explored. The added delay was set to 60 s with mixing every 15 seconds. The results (Table II) indicated no further improvement compared to 15 s added delay.
It should be stressed that incubation of the sample in the capillary (i.e. before dilution) for several minutes results in a similar but much slower improvement effect on PLT counts as seen in Table III below.
These results depict how the PLT does improve with time though at a much slower rate than when the incubation is performed after dilution. The time taken to reach the acceptable PLT value is not optimal or acceptable on the market.
Since the standard commercial diluent used in the system contains a small amount of EDTA (0.53 mM), the inventors experimented whether a higher EDTA concentration would improve the results further (Table IV).
91%
It is to be noted that a certain amount of EDTA is carried over from the capillary with the sample. The “standard EDTA capillaries” are coated with 100 μg K2EDTA and collects 20 μl of blood. The molar mass of K2EDTA is approximately 368 g/mol, so the capillary contains about 0.27 μmol of EDTA. The concentration in the capillary, assuming all EDTA is completely eluted is maximally to 13.5 mM. The standard dilution of 1:225 would consequently amount to 0.06 mM capillary-derived EDTA at most. Since using diluent with no EDTA was not efficient, it can be concluded that an efficient EDTA concentration should be higher than that, under the present conditions.
The standard dilution buffer used in the test system contains 0.53 mM EDTA, and was shown to be an effective amount. Additional EDTA at 3.1 mM did not seem result in any further benefits even when the incubation time was very short (5 s).
In another experiment is was also noted that when the sample was diluted to 1:45000 in a diluent containing 3.1 mM EDTA, the effect on platelet count was absent. The effect of dilution ratios was further explored in Example 5.
For comparison, samples were collected in untreated capillaries and platelet counts performed. Interestingly, even in the presence of additional EDTA in diluent, the platelet counts were not improved by longer incubation (Table V). Collection in plain capillaries may cause irreversible effects on platelet aggregation.
Since the effect was no longer present at 1:45000 dilution (see Example 3), and is not present without any dilution, experiments were conducted to compare different dilution ratios. Note that for practical reasons, the same donor could not be tested at more than 3 dilutions on the same occasion.
As shown in Table VI, experiments with dilutions at 1:50 to 1:450 were all effective. The lower dilutions (1:50 to 1:225) appear somewhat better in terms of spread than 1:450, where the effect seems to be somewhat less stable.
Note: Never squeeze the finger hard, if the blood doesn't flow then warm the hand more and repeat the procedure.
Note: Refer to the user manual for a complete set of instructions for the instrument.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1651481-2 | Nov 2016 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/077998 | 11/2/2017 | WO | 00 |
