Patent Grant
11841358
The present disclosure relates to methods and systems for determining a platelet concentration in a blood sample. More specifically, the present disclosure relates to determination of the platelet concentration using an impedance measurement of platelets in a diluted blood sample in combination with an optical measurement of platelets in a lysed blood sample.
In order to determine the course of treatment of a patient, it is generally mandated clinical practice to obtain an accurate platelet count. For example, if the platelet count is less than 20×109 per liter, it may become necessary to transfuse platelets. Otherwise, the patient may potentially have life threatening bleeding.
Most existing hematology analyzers count platelets using impedance measurement. Using impedance measurements of a diluted blood sample, cells can be classified as platelets and red blood cells according to their volumes. Although, in most cases, an impedance measurement system provides relatively accurate results in measuring platelet count, it has certain limitations. For example, an impedance measurement method is not able to differentiate between platelets and interfering particles, such as microcytes and schistocytes (fragmented red blood cells), which leads to falsely high platelet count. Conversely, large platelets and giant platelets may fall outside a defined threshold for platelets and be classified as red blood cells, which leads to falsely low platelet count.
With impedance measurements, typically a mathematical curve fitting is performed on the platelet volume distribution in a histogram between 2 and 20 femtoliters (fL) in order to extend a dynamic range up to 70 fL. However, an accurate platelet count cannot be obtained under certain circumstances, for example, if the platelet distribution does not fit into a logarithmic distribution, or if the upper end of the platelet distribution is not decreasing, the mean platelet volume is outside its normal range, and the fitting mode is no longer applicable. Under these circumstances, it is often that only those platelets located in the range between 2 and 20 fL are reported by the impedance measurement system with flagging.
To overcome deficiencies of impedance measurement methods, several high-end hematology analyzers have incorporated an additional optical measurement of platelets. However, although optical measurements reduce aforementioned interferences to platelet measurement, additional channel of optical measurement substantially increases complexity of the instrument, and raises the costs of instrument manufacturing and maintenance services.
Therefore, there is a need for a simple, less costly, yet reliable detection method and instrument system for accurately determining a platelet concentration in a blood sample, particularly in the presence of interference materials.
In one aspect, the present disclosure is directed to a method of determining a platelet concentration in a blood sample, which includes mixing a first aliquot of the blood sample with a diluent to form a first suspension; mixing a second aliquot of the blood sample with a lytic agent and a fluorescent dye to lyse red blood cells and stain white blood cells to form a second suspension; measuring DC impedance signals of the first suspension passing through an aperture; measuring light scatter signals and fluorescent signals of the second suspension passing through an optical flow cell; analyzing the DC impedance signals of the first suspension to obtain a first platelet distribution; analyzing the light scatter signals and the fluorescent signals of the second suspension to differentiate platelets from white blood cells and to obtain a second platelet distribution; and determining the platelet concentration of the blood sample using the first and second platelet distributions. Moreover, the method further includes differentiating the white blood cells of the blood sample using the light scatter signals and the fluorescent signals of the second suspension into white blood cell subpopulations that include monocytes, lymphocytes, neutrophils and eosinophils.
In a further aspect, the present disclosure is directed to a hematology system for determining a platelet concentration in a blood sample. The hematology system includes a first module that includes a first mixing chamber configured to mix a first aliquot of the blood sample with a diluent to form a first suspension, and a DC impedance detector equipped with an aperture in a flow path connected with the first mixing chamber, the DC impedance detector configured to measure DC impedance signals of the first suspension passing through the aperture; a second module that includes a second mixing chamber configured to mix a second aliquot of the blood sample with a lytic agent and a fluorescent dye to lyse red blood cells and stain white blood cells to form a second suspension, a light source configured with a light beam aimed at an orifice of an optical flow cell in a flow path connected with the second mixing chamber, and one or more optical detectors equipped with the optical flow cell and configured to measure light scatter signals and fluorescent signals of the second suspension passing through the optical flow cell; and a data processing module operably connecting with the DC impedance detector in the first module and the one or more optical detectors in the second module, respectively, the data processing module including a processor and a non-transitory computer readable medium programmed with computer application program that, when executed by the processor, causes the processor to generate a first platelet distribution from the DC impedance signals of the first suspension, to differentiate platelets from white blood cells using the light scatter signals and the fluorescent signals of the second suspension to generate a second platelet distribution, and to determine the platelet concentration of the blood sample using the first and second platelet distributions. Moreover, the data processing module is further configured to differentiate white blood cells of the blood sample using the light scatter signals and the fluorescent signals of the second suspension into white blood cell subpopulations that include monocytes, lymphocytes, neutrophils and eosinophils.
In yet another aspect, the present disclosure is directed to a method of determining a platelet concentration in a blood sample, which includes mixing a first aliquot of the blood sample with a diluent to form a first suspension; mixing a second aliquot of the blood sample with a lytic agent to lyse red blood cells to form a second suspension; measuring DC impedance signals of the first suspension passing through an aperture; measuring forward angle light scatter signals and side scatter signals or medium angle light scatter signals of the second suspension passing through an optical flow cell; analyzing the DC impedance signals of the first suspension to obtain a first platelet distribution; analyzing the forward angle light scatter signals, and the side scatter or the medium angle light scatter signals of the second suspension to differentiate platelets from white blood cells and to obtain a second platelet distribution; and determining the platelet concentration of the blood sample using the first and second platelet distributions. Moreover, the method further includes differentiating white blood cells of the blood sample using the forward angle light scatter signals and the side scatter signals or the medium angle light scatter signals of the second suspension into white blood cell subpopulations that include monocytes, lymphocytes, neutrophils and eosinophils.
In a further aspect, the present disclosure is directed to a hematology system for determining a platelet concentration in a blood sample. The system includes a first module that includes a first mixing chamber configured to mix a first aliquot of a blood sample with a diluent to form a first suspension, and a DC impedance detector equipped with an aperture in a flow path connected with the first mixing chamber, the DC impedance detector configured to measure DC impedance signals of the first suspension passing through the aperture; a second module that includes a second mixing chamber configured to mix a second aliquot of the blood sample with a lytic agent to lyse red blood cells to form a second suspension, a light source configured with a light beam aimed at an orifice of an optical flow cell in a flow path connected with the second mixing chamber, and one or more optical detectors equipped with the optical flow cell and configured to measure forward angle light scatter signals, and side scatter or medium angle light scatter signals of the second suspension passing through the optical flow cell; a data processing module operably connecting with the DC impedance detector in the first module and the one or more optical detectors in the second module, respectively, the data processing module including a processor and a non-transitory computer readable medium programmed with computer application program that, when executed by the processor, causes the processor to generate a first platelet distribution from the DC impedance signals of the first suspension, to differentiate platelets from white blood cells using the forward angle light scatter signals and the side scatter signals or the medium angle light scatter signals of the second suspension to generate a second platelet distribution, and to determine the platelet concentration of the blood sample using the first and second platelet distributions. Moreover, the data processing module is further configured to differentiate white blood cells of the blood sample using the forward angle light scatter signals and the side scatter signals or the medium angle light scatter signals of the second suspension into white blood cell subpopulations comprising monocytes, lymphocytes, neutrophils and eosinophils.
The advantages of the present disclosure will become apparent from the following description taken in conjunction with the accompanying drawings showing exemplary embodiments.
It is noted that in the drawings like numerals or symbols refer to like components.
The present disclosure generally relates to methods and hematology systems for determining a platelet concentration in a blood sample. Embodiments are described more fully hereinafter with reference to the accompanying drawings. The various embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
In some embodiments, the present disclosure provides a method of determination of platelet concentration using an impedance measurement of platelets in a diluted blood sample in combination with an optical measurement of platelets in a lysed blood sample. Herein, platelet concentration is also often referred to as platelet count in hematology, and is reported as number of platelets per liter of blood.
In one embodiment, the method comprises mixing a first aliquot of a blood sample with a diluent to form a first suspension, mixing a second aliquot of the blood sample with a lytic agent and a fluorescent dye to lyse red blood cells and stain white blood cells to form a second suspension, measuring DC impedance signals of the first suspension passing through an aperture, measuring light scatter and fluorescent signals of the second suspension passing through an optical flow cell, analyzing the DC impedance signals of the first suspension to obtain a first platelet distribution, analyzing light scatter and fluorescent signals of the second suspension to differentiate platelets from white blood cells and to obtain a second platelet distribution, and determining a platelet concentration of the blood sample using the first and second platelet distributions.
The first suspension is a diluted blood sample. A blood diluent is commonly used on hematology analyzers to dilute a blood sample for measuring red blood cells and platelets. The diluent typically contains one or more salts, such as alkali metal salt(s), and is adjusted to be isotonic for maintaining red blood cell volumes. Commercially available blood diluents can be used for diluting the first aliquot of the blood sample to form the first suspension, for example, M-68DS diluent and M-53D diluent produced by Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China.
The direct current (DC) impedance signals of the first suspension can be measured with a DC impedance detector equipped with a non-focused flow aperture or a focused flow aperture in a flow path. When a particle or a blood cell suspended in a conductive solution passes through an aperture, an electrical signal can be measured due to impedance change. The pulse shape, height and width of the impedance signals are directly correlated to the size or volume of a particle, and can be converted to the volume of the subject particle. When two or more particles of different sizes are measured, a frequency histogram obtained from the impedance measurement may represent a size distribution of these particles. Detection methods used for blood cell counting and sizing by a blood analyzer equipped with a DC impedance measurement device are known and are generally described in U.S. Pat. Nos. 2,656,508 and 3,810,011 which are incorporated herein by reference in their entireties.
In the analysis of DC impedance signals from the first suspension according to the method disclosed herein, a frequency histogram including platelets and red blood cells in the diluted blood sample can be generated. As illustrated in
The second suspension is a lysed blood sample. Red blood cells in a blood sample can be lysed by a lytic agent, such as cationic, nonionic, anionic, or amphiphilic surfactants and combinations thereof. Various known lysing reagents used for differential analysis of white blood cells on hematology analyzers may be used for lysing red blood cells in the second suspension in the present disclosure. The lysing reagents used for differential analysis of white blood cells on hematology analyzers are typically aqueous solutions containing one or more lytic agents, which may include cationic, nonionic, anionic or amphiphilic surfactants, or combinations thereof. In some embodiments, the lysing reagent may include one or more lytic agents for lysing red blood cells and a fluorescent dye which stains nucleated blood cells for differentiation of nucleated blood cells, such as white blood cells, from other cell types by light scatter and fluorescent measurements. One suitable example is the lysing reagent formulations described in U.S. Pat. No. 8,367,358, which is incorporated herein by reference in its entirety. As described in U.S. Pat. No. 8,367,358, the lysing reagents may comprise a cationic cyanine compound (a fluorescent dye), a cationic surfactant, a nonionic surfactant, and an anionic compound, and the lysing reagents may be used for lysing red blood cells and differentiating white blood cells into their subpopulations using fluorescent and light scatter measurements. Other known fluorescent dyes may also be used in the lysing reagents, such as the fluorescent dyes described in U.S. Pat. No. 8,273,329, which is incorporated herein by reference in its entirety. Moreover, in some embodiments, the fluorescent dye may be contained in a separate staining solution, which can be used together with a lysing reagent that does not contain a dye. Such a staining solution can be added to a blood sample before, after or simultaneously with the lytic agent to stain the nucleated blood cells.
The light scatter signals and fluorescent signals of the second suspension can be measured with one or more optical detectors equipped with an optical flow cell. Herein, optical flow cell refers to a focused-flow flow cell suitable for detection of light scatter signals and fluorescent signals, such as those used in existing flow cytometers and hematology analyzers. When a particle, such as a blood cell, passes through an orifice of the optical flow cell, an incident light beam from an illumination source directed at the aperture is scattered by the particle in all directions. The scattered light or light scatter signals can be detected by a photo-detector at various angles relative to the incident light beam. Different blood cell populations have different light scattering properties, which can be utilized for differentiation of different cell populations. The light scatter signals detected near the incident light are often referred to as forward angle light scatter or small angle light scatter signals. In some embodiments, the forward angle light scatter signals may be measured from about 1° to about 10° from the incident light. In other embodiments, the forward angle light scatter signals may be measured from about 2° to about 6° from the incident light. The light scatter signals detected at about 90° from the incident light are commonly referred to as side scatter signals, and fluorescent signals emitted from blood cells stained by a fluorescent dye are typically detected at about 90° from the incident light beam. In some embodiments, the side scatter signals are measured from about 65° to about 115° from the incident light.
One or more optical detectors may be used for measuring the forward angle light scatter and side scatter signals and fluorescent signals from the second suspension. Various known designs of optical detection hardware can be used for the purpose of the present disclosure.
In the analysis of the light scatter and fluorescent signals obtained from the second suspension according to the method disclosed herein, the platelets are differentiated from the white blood cells in the second suspension. As shown in
In some embodiments as described hereinafter, the second platelet distribution D2 shown in
Derived platelet volumes of the platelets in the second suspension can be calculated as a function of the light scatter signals of platelets 10b in the platelet region P. In one embodiment, a derived platelet volume Volp2 of each platelet in the platelet region P may be calculated using equation (1):
Volp2=α*FSC (1)
wherein FSC is the forward angle light scatter signal of an individual event in the platelet region, and a is a constant.
Alternatively, a derived platelet volume of each platelet in the platelet region P may also be calculated using equation (2):
Volp2a=β*exp(γ*FSC) (2)
wherein FSC is the forward angle light scatter signal of an individual event in the platelet region, and β and γ are constants.
Moreover, the derived platelet volume of each platelet in the platelet region defined above may be calculated according to Mie scatter theory using forward angle light scatter and side scatter signals obtained from the second suspension. Furthermore, in the above calculation either using equation (1) or (2) or the Mie scatter theory, the derived platelet volume of the platelets in the second suspension is correlated with the platelet volume in the DC platelet histogram according to size correlation between the platelet volume in the DC platelet histogram and respective light scatter signals obtained from the second suspension. Therefore, in the derived platelet volume histogram HPlt-L shown in
In some embodiments, after obtaining the derived platelet volume histogram HPlt-L, the method generates a composite platelet histogram HPlt-LD as a function of the platelet DC impedance histogram HPlt-D of the first suspension and the derived platelet volume histogram HPlt-L of the second suspension: HPlt-LD=f(HPlt-L, HPlt-D). The composite platelet histogram HPlt-LD incorporates information obtained from platelet measurements of both the first suspension and the second suspension.
In one exemplary embodiment, the composite platelet histogram is generated according to equation (3):
HPlt-LD(i)=ki1×HPlt-L(i)+ki2×HPlt-D(i)(i=1,2, . . . ,n) (3)
wherein HPlt-LD(i) is event (i) in the composite platelet histogram; HPlt-L(i) is event (i) in the derived platelet volume histogram of the second suspension; HPlt-D(i) is event (i) in the platelet DC impedance histogram of the first suspension; and ki1 and ki2 are coefficients.
In some embodiments, ki1 and ki2 in equation (3) may be constants. For example, in one exemplary embodiment, ki1 and ki2 are established according to the following criteria:
when Volp(i)>20 fL, ki1=1, and ki2=0; and
when Volp(i)≤20 fL, ki1=0, and ki2=1
Alternatively, ki1 and ki2 in equation (3) may also be variables determined according to a platelet valley/peak ratio (Rv/p) in the platelet DC impedance histogram HPlt-D of the first suspension which is defined hereinafter. In an alternative embodiment, ki1 and ki2 may be determined by equations (4) and (5):
ki1=1=ki2 (4)
ki2=K_Coef*Rv/p+e (5)
wherein K_Coef is a constant less than zero; e is a constant greater than zero; and if ki1<0 according to equation (4), ki1=0, and if ki2<0 according to equation (5), ki2=0, and the coefficients are substituted into the equation (3) to obtain the composite platelet histogram HPlt-LD.
As can be appreciated, the composite platelet histogram HPlt-LD can be used as a third platelet distribution obtained using the first platelet distribution from the first suspension and the second platelet distribution from the second suspension, namely the derived platelet volume histogram. The platelet concentration is obtained from this third platelet distribution.
In another exemplary embodiment, a composite platelet histogram HPlt-LDa may be generated using the platelet DC impedance histogram HPlt-D obtained from the first suspension and the derived platelet volume histogram HPlt-L obtained from the second suspension as described above according to a criterion defined by equations (6) and (7):
when Volp(i)>15 fL,HPlt-LDa(i)=min(HPlt-L(i),HPlt-D(i))(i=1,2, . . . ,n) (6)
when Volp(i)≤15 fL,HPlt-LDa(i)=HPlt-D(i)(i=1,2, . . . ,n) (7)
wherein HPlt-LDa(i) is event (i) in the composite platelet histogram; HPlt-L(i) is event (i) in the derived platelet volume histogram of the second suspension; HPlt-D(i) is event (i) in the platelet DC impedance histogram of the first suspension; and min represents for event i using the minimum number between the two histograms.
In the criterion defined by equations (6) and (7), the cut-off point of 15 fL is an empirical value, which may vary with instruments and/or reagents used with the method. Same as described above, the platelet concentration of a blood sample is then determined according to the area under the curve in the composite platelet histogram (HPlt-LDa).
In a further embodiment, instead of using equation (1) or (2), or Mie scatter theory, a derived platelet volume histogram HPlt-Lb may be generated by a curve fitting process using the light scatter signals of platelets 10b in the platelet region (P) in the SFL vs. FSC scattergram of the second suspension. In the derived platelet volume histogram HPlt-Lb, derived platelet volumes Volp2b for individual events are expressed by equation (8):
Volp2b=[1/(FSC*σ(2π)1/2)]exp(−(ln FSC−μ)2/2σ2) (8)
wherein FSC is the forward angle light scatter signal of an individual event in the platelet region in the SFL vs. FSC scattergram, μ and σ are fitting parameters of the fitted curve.
In this embodiment, a composite platelet histogram HPlt-LDb may be generated using the derived platelet volume histogram HPlt-Lb obtained using equation (8) and the platelet DC impedance histogram HPlt-Lb obtained from the first suspension according to a criterion defined by equations (9) and (10):
when Volp(i)>12 fL,HPlt-LDb=HPlt-Lb(i)(i=1,2, . . . ,n) (9)
when Volp(i)≤12 fL,HPlt-LDb(i)=HPlt-D(i)(i=1,2, . . . ,n) (10)
wherein HPlt-LDb(i) is event (i) in the composite platelet histogram; HPlt-Lb(i) is event (i) in the derived platelet volume histogram obtained with equation (8) from the second suspension; and HPlt-D(i) is event (i) in the platelet DC impedance histogram of the first suspension.
In the criterion defined by equations (9) and (10), the cut-off point of 12 fL is an empirical value, which may vary with instruments and/or reagents used with the method. Same as described above, the platelet concentration of a blood sample is then determined according to the area under the curve in the composite platelet histogram HPlt-LDb.
As can be appreciated, the composite platelet histogram HPlt-LDb can be generated using the derived platelet volume histogram HPlt-Lb obtained using equation (8) and the platelet DC impedance histogram HPlt-D obtained from the first suspension according to any method related to equation (3) as described above. Similarly, the composite platelet histogram HPlt-LDa can be generated using the platelet DC impedance histogram HPlt-D obtained from the first suspension and the derived platelet volume histogram HPlt-L obtained from the second suspension according to a criterion defined by equations (9) and (10).
In yet another embodiment, the platelet DC impedance histogram HPlt-D obtained from the first suspension and the derived platelet volume histogram HPlt-L obtained using equation (1) or (2), or Mie scatter theory, or the derived platelet volume histogram HPlt-Lb obtained using equation (8) from the second suspension as described above can be used to determine a platelet concentration of a blood sample according to the following process.
As shown in
Cplt=VHD(1)+VHD(2)+ . . . +VHD(15)+VHL(16)+VHL(17)+ . . . +VHL(n) (11)
Cplt=VHD(1)+VHD(2)+ . . . +VHD(15)+VHD(16)+VHD(17)+ . . . +VHD(n) (12)
wherein VHD (1, 2, . . . n) is the value, or height, of the platelet DC impedance histogram HPlt-D at the position on the axis corresponding to the platelet volume of 1 fL, 2 fL, . . . and n fL, respectively; VHL (1, 2, . . . n) is the value, or height, of the derived platelet volume histogram HPlt-L at the position on the axis corresponding to the platelet volume of 1 fL, 2 fL, . . . and n fL, respectively; and CPlt is the platelet concentration.
In this embodiment, the cut-off point of 15 fL for calculation of Area-1 and Area-2 is determined empirically, which may vary with instruments and/or reagents used with the method. As can be appreciated, the platelet concentration CPlt obtained using equations (11) and (12) is similar to that obtained by generating a composite platelet histogram HPlt-LDc by selecting respective parts of the platelet DC impedance histogram HPlt-D and the derived platelet volume histogram HPlt-L using the composing criterion described above. In other words, when δ≤AT, the composite platelet histogram HPlt-LDc is generated using the platelet DC impedance histogram HPlt-D, and when δ>AT, the composite platelet histogram HPlt-LDc is generated using both HPlt-D and HPlt-L, with the portion corresponding to platelet volume above 15 fL using the derived platelet volume histogram HPlt-L. Then, the platelet concentration is calculated according to the area under the curve of the composite platelet histogram as described above.
The composite platelet histogram in various embodiments described above is a graphical representation of the distribution of platelet volume, which is a commonly used form of representing probability distribution of a continuous variable. Alternatively, the distribution of the platelet volume may also be expressed numerically in a form of table or list, with an equivalent or similar resolution of the histogram, or expressed by other suitable forms known in the art. Therefore, for the purpose of the present disclosure the composite platelet histogram described above can also be referred to as a composite platelet distribution which is not limited by its graphical representation. Similarly, the derived platelet volume histogram described above can also be referred to as a derived platelet volume distribution which is not limited by its graphical representation. Further, the platelet DC impedance histogram obtained from the first suspension described above can also be referred to as a DC platelet volume distribution which is not limited by its graphical representation.
Moreover, same as described above in reference to composite platelet histogram HPlt-LD, the composite platelet distribution is a third platelet distribution obtained using the first platelet distribution from the first suspension and the second platelet distribution from the second suspension, and the platelet concentration is obtained from the third platelet distribution.
Furthermore, in the methods described herein the platelet region P in the SFL vs. FSC scattergram obtained from the second suspension can be differentiated from nucleated red blood cells in the case that abnormal blood samples contain nucleated red blood cells.
In further embodiments, the platelet concentration of a blood sample can be determined using the first platelet distribution from the first suspension and the second platelet distribution from the second suspension using a method described hereinafter in reference to
In one embodiment, the method includes determining a platelet valley/peak ratio Rv/p in the platelet DC impedance histogram HPlt-D of the first suspension, and comparing the obtained platelet valley/peak ratio Rv/p with a predetermined ratio threshold RT. As illustrated in
As further illustrated in
Once the platelet valley/peak ratio Rv/p and the number of events N in the designated area PG are determined for a blood sample, the method determines a derived separation threshold Td in the valley between the platelets and the red blood cells in the platelet DC impedance histogram obtained from the first suspension for separating the platelets from the red blood cells using these parameters.
In one embodiment, the derived separation threshold Td can be determined according to equation (13):
Td=Tap+Fof (13)
wherein Tap is an apparent separation threshold determined using the conventional method for separating the platelets from the red blood cells in the platelet DC impedance histogram HPlt-D of the first suspension, which is determined according to the bottom of the valley between the two populations and known size range of platelets; and Fof is an offset that is a function of the platelet valley/peak ratio Rv/p in the platelet DC impedance histogram (HPlt-D) from the first suspension and the number of events N in the designated area PG within the platelet region P in the SFL vs. FSC scattergram from the second suspension described above.
In one exemplary embodiment, Fof can be determined using equation (14) or (15) below according to an offset criterion:
Fof=b1*Rv/p−b2*N+c (14)
wherein Rv/p is the platelet valley/peak ratio in the platelet DC impedance histogram HPlt-D of the first suspension; N is the number of events present in the designated area PG within the platelet region P in the SFL vs FSC scattergram of the second suspension; b1, b2 are constants greater than 0; and c is a constant.
Fof=b11*Rv/p+b21*N+c1 (15)
wherein Rv/p and N are the same as in equation (14); b11, b21 are constants greater than 0; and c1 is a constant.
The offset criterion dictates that if Rv/p exceeds RT, and N is below GT, equation (14) is applied for determining the derived separation threshold Td defined in equation (13); and if Rv/p exceeds RT, and N also exceeds GT, equation (15) is applied for determining the derived separation threshold Td defined in equation (13). Moreover, according to the offset criterion, if Rv/p does not exceed RT, neither equation (14) nor (15) is applied, in other words, Fof is zero in equation (13).
Once the derived separation threshold Td is obtained according to equations (13)-(15) and the offset criterion, the derived separation threshold Td is used to separate the two cell populations in the platelet DC impedance histogram HPlt-D of the first suspension, namely, to separate the platelets from the red blood cells. The platelet concentration of the blood sample is then determined according to the area under the curve for the platelet population as defined by the derived separation threshold Td in the histogram.
In the example shown in
Furthermore, in some embodiments the derived separation threshold can also be determined according to equation (16):
Td′=Tap+g*(N−GT)+h*(Rv/p−RT)+s (16)
wherein N is the number of events present in the designated area PG within the platelet region P in the SFL vs FSC scattergram of the second suspension, GT is the predetermined event threshold, Rv/p is the platelet valley/peak ratio in the platelet DC impedance histogram HPlt-D of the first suspension, and RT is the predetermined ratio threshold; g, h and s are constants, wherein g, h and s=0, when Rv/p≤RT.
In determining a platelet concentration of a blood sample using equation (16), the derived separation threshold Td′ is calculated as a function of N and Rv/p obtained from the analysis of light scatter and fluorescent signals of the second suspension and the analysis of DC impedance signals of the first suspension, respectively, as described above. The platelets are separated from the red blood cells in the platelet DC impedance histogram HPlt-D of the first suspension using the derived separation threshold Td′ obtained from equation (16), in the same manner as illustrated in
As can be appreciated, in the embodiments described in reference to equations (13)-(16), the platelet DC impedance histogram from the first suspension with the derived separation threshold separating the platelets and the red blood cells is another third platelet distribution obtained using the first platelet distribution from the first suspension and the second platelet distribution from the second suspension, namely a two dimensional distribution of platelets in the platelet region in the scattergram described above. The platelet concentration is obtained from this third platelet distribution.
As can be appreciated, in any embodiment described above, platelet data of various forms can be obtained using the third platelet distribution, for example, the composite platelet histograms HPlt-LDa, HPlt-LDb, HPlt-LDc, or the curve for the platelet population as defined by the derived separation threshold Td′ in the platelet DC impedance histogram. The obtained platelet data includes but is not limited to a platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), thrombocytocrit (PCT), etc.
Moreover, in some embodiments the present method may further include differentiating white blood cells into subpopulations using the light scatter and fluorescent signals of the second suspension. Main white blood cell subpopulations include lymphocytes, monocytes, neutrophils, eosinophils and basophils.
The second module 300 includes a second mixing chamber 310 configured to mix an aliquot of a blood sample with a lytic agent and a fluorescent dye to form the second suspension, a light source 330 configured with its light beam aimed at an orifice of an optical flow cell 320 in a flow path connected with the second mixing chamber 310, and one or more optical detectors 340 equipped with the optical flow cell 320 and configured to measure light scatter signals and fluorescent signals of the second suspension passing through the orifice of the optical flow cell 320. In one exemplary embodiment, the hematology system 100 may include a red semiconductor laser with an emission wavelength of 640 nm as the light source, and three optical detectors enabling detections of forward angle light scatter, side scatter and fluorescent signals, respectively. As described above, the forward angle light scatter signals may be measured from about 1° to about 10° or from about 2° to about 6° from the incident light beam, and the side scatter signals and fluorescent signals may be measured at about 90° from the incident light beam. In one exemplary embodiment, the forward angle light scatter signals may be measured from about 1° to about 10° and the side scatter signals may be measured from about 65° to about 115° from the incident light beam. Moreover, the system 100 may include one or more optical detectors configured to detect light scatter signals at other angles.
In the embodiment shown in
The data processing module 400 is operably connecting with the DC impedance detector 230 in the first module 200 and optical detectors 340 in the second module 300, respectively. As shown in
The data processing module 400 is configured to carry out various aspects of the methods described herein. The DC impedance signals detected in the first module and the light scatter and fluorescent signals detected in the second module may be processed, respectively, in real time. In exemplary embodiments, these signals may be processed using Field-Programmable Gate Array (FPGA), Digital Signal Processing (DSP), or CPU. Then, the processed DC impedance signals, light scatter signals and fluorescent signals are analyzed automatically with the programmed computer applications to obtain the first and second platelet distributions and determine the platelet concentration of a blood sample according to the methods described herein. Moreover, the signals from the second module are also used for differentiation and enumeration of the white blood cells, or for identification of the nucleated red blood cells, the immature cells or the blast cells, according to the methods described herein.
The hematology system 100 further includes a user interface 500 which includes user interface input and output devices. The results obtained by the methods described herein may be displayed on a user interface output device, such as a computer screen. In addition to the platelet concentration and white blood cell differential results obtained using the methods described herein, graphic results may be generated on the screen display, such as a composite platelet histogram HPlt-LD shown in
In a further embodiment, the method of determining a platelet concentration of a blood sample described above can also be accomplished with multi-angle light scatter measurements of the second suspension of the blood sample without the fluorescence measurement. More specifically, the present inventors discovered that side scatter signals or medium angle light scatter signals from the second suspension could be used for obtaining the second platelet distribution in replacement of the fluorescent signals in determining the platelet concentration of a blood sample with the methods described above. Herein, the medium angle light scatter signals are detected at an angle between the forward angle light scatter and the side scatter. In one exemplary embodiment, the forward angle light scatter signals may be detected from about 1° to about 10° and the side scatter signals may be detected from about 65° to about 115° from the incident light beam. In another exemplary embodiment, the forward angle light scatter signals may be detected from about 2° to about 6° from the incident light beam, and light scatter signals at a low medium angle from about 8° to about 24° may be detected. Herein, the latter is referred to as a low medium angle light scatter. Moreover, light scatter signals at a high medium angle from about 25° to about 65° from the incident light beam may be detected, which is referred as a high medium angle light scatter.
The method in this embodiment is further described in reference to
More specifically, a platelet concentration of a blood sample may be determined using the process described above in reference to equations (1)-(3). In this embodiment, a derived platelet volume histogram HPlt-L′ can be obtained from the second suspension using forward angle scatter signals of the platelets in the platelet region P′ in the FSC vs. SSC scattergram according to equation (1) or (2) in the same manner as described above, or using the forward angle light scatter and side scatter signals of the platelets in the platelet region P′ according to the Mie scatter theory as described above. Then, a composite platelet histogram HPlt-LD′ is generated according to equation (3) using the platelet DC impedance histogram HPlt-D from the first suspension and the derived platelet volume histogram HPlt-L′ from the second suspension. In this embodiment, event (i) in the derived platelet volume histogram HPlt-L substituted into equation (3) is event (i) in the derived platelet volume histogram HPlt-L′. The platelet concentration is then determined according to the area under the curve in the composite platelet histogram HPlt-LD′.
Similarly, a platelet concentration of a blood sample may also be determined using the process described above in reference to composite platelet histogram HPlt-LDa. In this embodiment, a composite platelet histogram HPlt-LDa′ may be generated using the platelet DC impedance histogram HPlt-D obtained from the first suspension and the derived platelet volume histogram HPlt-L′ obtained from light scatter signals of the platelets in the platelet region P′ in the FSC vs. SSC scattergram described above according to equations (6) and (7). In this embodiment, event (i) in the derived platelet volume histogram HPlt-L substituted into equation (6) is event (i) in the derived platelet volume histogram HPlt-L′. The platelet concentration is then determined according to the area under the curve in the composite platelet histogram HPlt-LDa′.
Moreover, a platelet concentration of a blood sample may also be determined using the process described above in reference to composite platelet histogram HPlt-LDb. In this embodiment, a composite platelet histogram HPlt-LDb′ may be generated according to the criterion defined by equations (9) and (10), using the platelet DC impedance histogram HPlt-D obtained from the first suspension and the derived platelet volume histogram HPlt-Lb′ obtained from forward angle light scatter signals of the platelets in the platelet region P′ in the FSC vs. SSC scattergram using equation (8). In this embodiment, event (i) in the derived platelet volume histogram HPlt-L substituted into equation (9) is event (i) in the derived platelet volume histogram HPlt-L′. The platelet concentration is then determined according to the area under the curve in the composite platelet histogram HPlt-LDb′.
In addition, a platelet concentration of a blood sample may also be determined using the process described above in reference to equations (11) and (12). In this embodiment, the platelet concentration can be calculated using equations (11) and (12) according to the composing criterion described above. In this embodiment, the elements in the derived platelet volume histogram HPlt-L defined in equation (11) are corresponding elements in the derived platelet volume histogram HPlt-L′ obtained from light signals of the platelets in the platelet region P′ in the FSC vs. SSC scattergram as described above; and in the composing criterion an absolute difference δ′ is the absolute difference between the Area-1 in the platelet DC impedance histogram HPlt-D and Area-2′ in the derived platelet volume histogram HPlt-L′, which is further compared to a predetermined area threshold AT′.
As described above, in the embodiments of using multi-angle light scatter measurements of the second suspension, the composite platelet histogram such as HPlt-LDa′ or HPlt-LDb′ is a third platelet distribution obtained using the first platelet distribution from the first suspension and the second platelet distribution from the second suspension, namely, respective derived platelet volume histogram described herein. The platelet concentration is obtained from the third platelet distribution.
Furthermore, the platelet concentration may be determined using the process described above in reference to equations (13)-(15). As shown in
Furthermore, in this embodiment the derived separation threshold Td may also be determined using equation (16), in which N is the number of events in the designated area PG′ within the platelet region P′ in the FSC vs. SSC scattergram from the second suspension, and GT is the predetermined event threshold in the designated area PG′.
Corresponding to this embodiment, the hematology system carrying out the methods described above includes a second module that includes one or more optical detectors configured to detect forward angle light scatter signals and side scatter signals of the second suspension passing through the orifice of the optical flow cell, or configured to detect forward angle light scatter signals and medium angle light scatter signals of the second suspension passing through the orifice of the optical flow cell. In this case, the hematology system shown in
Moreover, in this embodiment the data processing module is configured to analyze the DC impedance signals of the first suspension from the first module and the light scatter signals of the second suspension from the second module, respectively, and to implement various aspects of the method. In the same manner as described above in reference to
As can be appreciated, in this embodiment the preparation of the second suspension does not require a fluorescent dye. The lysing reagents described above containing one or more lytic agents for lysing red blood cells in the second suspension, but without the fluorescent dye. Furthermore, various existing lysing reagents used for differential analysis of white blood cells on hematology analyzers may be used for preparing the second suspension, for example various lysing reagent formulations described in U.S. Pat. No. 7,413,905, which is incorporated herein by reference in its entirety. More specifically, as described U.S. Pat. No. 7,413,905, the lysing reagents may include one or more surfactants, as the lytic agent, for lysing red blood cells and partly damaging the cell membrane of white blood cells, an organic compound bearing an anionic group capable of binding to the cationic component present in the white blood cells to provide morphological differences among white blood cell subpopulations, and a buffer to adjust the reagent pH to 2-8. The lytic agents may include one or more cationic surfactants, one or more anionic surfactants, one or more ampholytic surfactants, a combination of one or more cationic surfactants and one or more ampholytic surfactants, or a combination of one or more anionic surfactants and one or more ampholytic surfactants.
This embodiment is advantageous in terms of instrument simplicity and low cost because no fluorescent measurement is required. This embodiment may be implemented on existing hematology analyzers that are equipped with forward angle light scatter and side scatter measurements, or forward angle light scatter and medium angle light scatter measurements. Moreover, since no fluorescent dye is needed, it substantially reduces the cost of the reagent. One skilled in the art can understand that, this embodiment is applicable for hematology analyzers with fluorescent measurement, which can simultaneously measure forward angle light scatter signals, side scatter signals and fluorescent signals.
Moreover, the methods in this embodiment may further include differentiating white blood cells into subpopulations using the light scatter signals from the second suspension.
Examples 1 to 7 further illustrate the methods of determining a platelet concentration of a blood sample described above. As shown in Example 1, a hematology analyzer enabling detections of forward angle light scatter, side scatter and fluorescent signals was used to determine platelet concentrations of 25 blood samples including 5 normal and 20 abnormal blood samples which contained fragment red blood cells, microcytes, or large platelets. The platelet concentrations of the same blood samples were also measured by a flow cytometer as a reference method, as well as by a conventional DC impedance measurement method for comparison.
However, as shown in
Similarly, as shown in Example 2, the platelet concentrations of these 25 blood samples obtained using a composite platelet histogram HPlt-LDa generated according to the criterion defined in equations (6) and (7) correlated closely with the results obtained by the flow cytometer reference method with a correlation coefficient (R2) of 0.9739, see
Example 5 further illustrates measurements of platelet concentrations of the blood samples using equations (13)-(15) and the offset criterion described above. The same 25 blood samples in Example 1 were used for the measurements. As shown in
The methods for measuring platelets in Examples 1-5 were applied in the steps for differentiating white blood cells after lysing red blood cells. The method for measuring platelets as described in Example 6 was applied in the steps for differentiating nucleated red blood cell after lysing red blood cells. Examples 6 and 7 shows the composite platelet histogram HPlt-LD generated using equation (3) by the methods described above.
It has been found that the platelet region can also be differentiated in the fluorescent (SFL) vs. side light scatter (SSC) scattergram, as shown in
Similarly, in Example 7, the platelet concentration of a sample may be determined using the process described above in reference to equations (13)-(15).
The embodiments described above provide accurate measurements of platelet concentration of blood samples, which are particularly effective in the presence of an interference to the impedance measurement methods, such as with blood samples containing fragmented red blood cells, microcytes, or large platelets. As such, the present methods have overcome the difficulties of existing impedance measurement methods and met a long felt need of accurate measurements of platelet concentration in in-vitro diagnostic analysis. Moreover, as discussed above some existing high end hematology analyzers use a separate optical measurement of platelets in addition to the conventional impedance measurement in order to differentiate interference substances and remove the effect of interferences. However, this substantially increases instrument complexity and manufacturing cost. In contrast, the instant methods can be implemented with existing complete blood count (CBC) and white blood cell differential measurements on various commercial hematology analyzers without increasing instrument cost. Therefore, the present methods are particularly advantageous in their broad applications in improving accuracy of platelet measurements on existing instruments in the field of in-vitro diagnostic industry.
The following examples are illustrative and are in no way to be interpreted as limiting the scope of the present disclosure, as defined in the claims. It will be understood that various modifications and changes can be made in accordance with the proceeding disclosure.
Whole blood samples were measured on a commercial hematology analyzer BC-6800 (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) and the data collected from measurements of the first and second suspensions were post analyzed using the method in one embodiment described above.
BC-6800 hematology analyzer has a CBC module and a differential module. The CBC module includes a mixing chamber configured to mix one aliquot of a blood sample with a diluent to form a first suspension and a DC impedance detector configured to measure DC impedance signals of the first suspension passing through an aperture in a flow path. The differential module includes another mixing chamber configured to mix another aliquot of the blood sample with a lytic agent and a fluorescent dye to form a second suspension, a red semiconductor laser with an emission wavelength of 640 nm as the light source which is configured with its light beam aimed at an orifice of an optical flow cell, and optical detectors enabling detection of forward angle light scatter signals from about 1° to about 10° and side scatter signals from about 65° to about 115° from the incident light beam, and fluorescent signals of the second suspension passing through the orifice of the optical flow cell.
In the CBC module, 4 μL of an anticoagulated whole blood sample was mixed with 1.5 mL of M-68DS diluent (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) to form the first suspension. In the differential module, 20 μL of the same whole blood sample was mixed with 1 mL of M-68LD Lyse and 20 μL of M-68FD Dye (both are products of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) to lyse red blood cells and stain nucleated blood cells and to form the second suspension. The M-68LD Lyse was an aqueous solution containing a cationic surfactant, a nonionic surfactant, and an anionic compound for lysing red blood cells in a blood sample. The M-68FD Dye was an aqueous solution containing a cationic cyanine compound for staining nucleated blood cells in a blood sample.
The data collected from the measurement of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D, as that illustrated in
25 whole blood samples were analyzed as described above, including five normal blood samples, ten abnormal blood samples which contained fragmented red blood cells or microcytes, and ten abnormal blood samples which contained large platelets. The presence of fragmented red blood cells, microcytes, or large platelets in these abnormal blood samples was confirmed by manual examination of blood smears under a microscope. These blood samples were further analyzed on a BriCyte E6 flow cytometer (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) using the RBC/Platelet Ratio Method, a reference method as recommended by The International Council for Standardization in Hematology (ICSH) and the International Society of Laboratory Hematology (ISLH). Moreover, platelet concentrations of these blood samples reported on the BC-6800 hematology analyzer using a conventional DC impedance measurement method were also obtained for comparison.
The data collected on the BC-6800 hematology analyzer from measurements of the first and second suspensions of the blood samples, as described above in Example 1, were further post analyzed to determine platelet concentrations of the blood samples using composite platelet histogram HPlt-LDa described above.
More specifically, same as in Example 1, the data collected from the measurements of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D. The data collected from the measurements of forward angle light scatter signals and fluorescent signals of the second suspension were post analyzed to differentiate the platelet region (P) from the white blood cell region (W) in the SFL vs. FSC scattergram. A derived platelet volume histogram HPlt-L and derived platelet volumes were obtained using equation (1). Then, a composite platelet histogram HPlt-LDa was generated according to the criterion defined in equations (6) and (7) as described above. The platelet concentration of each blood sample was determined according to the area under the curve in the composite platelet histogram HPlt-LDa.
The same 25 blood samples used in Example 1 were analyzed with the process described above in this example.
The data collected on the BC-6800 hematology analyzer from measurements of the first and second suspensions of the blood samples, as described above in Example 1, were further post analyzed to determine platelet concentrations of blood samples using composite platelet histogram HPlt-LDb described above.
More specifically, the data collected from the measurement of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D. The data collected from the measurements of forward angle light scatter signals and fluorescent signals of the second suspension were post analyzed to differentiate the platelet region P from the white blood cell region W in a SFL vs. FSC scattergram. In this example, a derived platelet volume histogram HPlt-Lb was generated with light scatter signals of platelets in the platelet region P using the curve fitting process defined by equation (8) as described above. Then, a composite platelet histogram HPlt-LDb was generated according to the criterion defined by equations (9) and (10) as described above. The platelet concentration of each blood sample was determined according to the area under the curve in the composite platelet histogram HPlt-LDb.
The same 25 blood samples used in Example 1 were analyzed with the process described above in this example.
The data collected on the BC-6800 hematology analyzer from measurements of the first and second suspensions of blood samples, as described above in Example 1, were further post analyzed to determine platelet concentrations of blood samples using equations (11) and (12) and the composing criterion described above.
More specifically, the data collected from the measurement of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D. The data collected from the measurements of forward angle light scatter signals and fluorescent signals of the second suspension were post analyzed to differentiate the platelet region P from the white blood cell region W in a SFL vs. FSC scattergram. A derived platelet volume histogram HPlt-L and derived platelet volumes were obtained using equation (1). Then, the platelet concentration of each blood sample was calculated using equations (11) and (12) according to the composing criterion as described above.
The same 25 blood samples used in Example 1 were analyzed with the process described above in this example.
The data collected on the BC-6800 hematology analyzer from measurements of the first and second suspensions of blood samples, as described above in Example 1, were further post analyzed using the method in a further embodiment described above.
More specifically, same as described in Example 1, the data collected from the measurement of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D, as those illustrated in
The same 25 blood samples used in Example 1 were analyzed with the process described above in this example.
A whole blood sample containing nucleated red blood cells was measured on a commercial hematology analyzer BC-6800 (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) and the data collected from measurements of the first and second suspensions were post analyzed using the method in one embodiment described above.
BC-6800 hematology analyzer has a CBC module and a differential module. The CBC module includes a mixing chamber configured to mix one aliquot of a blood sample with a diluent to form a first suspension and a DC impedance detector configured to measure DC impedance signals of the first suspension passing through an aperture in a flow path. The differential module is a differential module for nucleated red blood cell and includes another mixing chamber configured to mix another aliquot of the blood sample with a lytic agent and a fluorescent dye to form a second suspension, a red semiconductor laser with an emission wavelength of 640 nm as the light source which is configured with its light beam aimed at an orifice of an optical flow cell, and optical detectors enabling detection of forward angle light scatter signals from about 1° to about 10° and side scatter signals from about 65° to about 115° from the incident light beam, and fluorescent signals of the second suspension passing through the orifice of the optical flow cell.
In the CBC module, 4 μL of an anticoagulated whole blood sample was mixed with 1.5 mL of M-68DS diluent (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) to form the first suspension. In the differential module, 20 μL of the same whole blood sample was mixed with 1 mL of M-68LN Lyse and 20 μL of M-68FN Dye (both are products of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) to lyse red blood cells and stain nucleated blood cells and to form the second suspension. The M-68LN Lyse was an aqueous solution containing a cationic surfactant and an anionic compound for lysing red blood cells in a blood sample. The M-68FN Dye was an aqueous solution containing a cationic cyanine compound for staining nucleated blood cells in a blood sample.
The data collected from the measurement of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D, as that illustrated in
As shown in
The blood sample was confirmed to contain fragmented red blood cells by manual examination of blood smears under a microscope. The reference value obtained by the flow cytometry reference method was 86×109/L, the platelet count result obtained by the impedance method was 110×109/L, and the platelet count result obtained by the present disclosure was 91×109/L, which was closer to the reference value.
A whole blood sample containing nucleated red blood cells was measured on a commercial hematology analyzer BC-6800 (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) and the data collected from measurements of the first and second suspensions were post analyzed using the method in one embodiment described above.
BC-6800 hematology analyzer has a CBC module and a differential module. The CBC module includes a mixing chamber configured to mix one aliquot of a blood sample with a diluent to form a first suspension and a DC impedance detector configured to measure DC impedance signals of the first suspension passing through an aperture in a flow path. The differential module is a differential module for nucleated red blood cell and includes another mixing chamber configured to mix another aliquot of the blood sample with a lytic agent and a fluorescent dye to form a second suspension, a red semiconductor laser with an emission wavelength of 640 nm as the light source which is configured with its light beam aimed at an orifice of an optical flow cell, and optical detectors enabling detection of forward angle light scatter signals from about 1° to about 10° and side scatter signals from about 65° to about 115° from the incident light beam, and fluorescent signals of the second suspension passing through the orifice of the optical flow cell.
In the CBC module, 4 μL of an anticoagulated whole blood sample was mixed with 1.5 mL of M-68DS diluent (a product of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) to form the first suspension. In the differential module, 20 μL of the same whole blood sample was mixed with 1 mL of M-68LN Lyse and 20 μL of M-68FN Dye (both are products of Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) to lyse red blood cells and stain nucleated blood cells and to form the second suspension. The M-68LN Lyse was an aqueous solution containing a cationic surfactant and an anionic compound for lysing red blood cells in a blood sample. The M-68FN Dye was an aqueous solution containing a cationic cyanine compound for staining nucleated blood cells in a blood sample.
The data collected from the measurement of DC impedance signals of the first suspension were post analyzed to generate a platelet DC impedance histogram HPlt-D, as that illustrated in
The blood sample was confirmed to contain fragmented red blood cells by manual examination of blood smears under a microscope. The reference value obtained by the flow cytometry reference method was 86×109/L, the platelet count result obtained by the impedance method was 110×109/L, and the platelet count result obtained by the present disclosure was 95×109/L, which was closer to the reference value.
While the present disclosure has been described in detail and pictorially shown in the accompanying drawings, these should not be construed as limitations on the scope of the present disclosure, but rather as an exemplification of preferred embodiments thereof. It will be apparent, however, that various modifications and changes can be made within the scope of this disclosure as described in the above specification and defined in the appended claims and their legal equivalents. All patents and other publications cited herein are expressly incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/085197 | Apr 2018 | WO | international |
This application is a continuation of International Application No. PCT/CN2019/084687, filed Apr. 26, 2019, which claims priority benefit of International Application No. PCT/CN2018/085197, filed Apr. 28, 2018, the contents of which are incorporated herein by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20210033593 A1 | Feb 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2019/084687 | Apr 2019 | US |
| Child | 17075603 | US |
