Patent Grant
6916658
The present invention relates to a method for determination of immature granulocytes in a blood sample. More specifically the method determines immature granulocytes using a direct current impedance measurement.
The presence of immature granulocytes (IG) in peripheral blood is potentially important information which indicates enhanced bone marrow activation. Besides the obvious significance of blasts for the diagnosis of leukaemia, the promyelocyte, myelocyte and metamyelocyte stages of myeloid maturation may indicate systemic inflammatory stress or leukaemic reactions. The determination of immature granulocytes is routinely done by visual microscopy, which requires manual review of each blood sample smear, and is a labor intensive and time consuming task.
Currently, several high end hematology analyzers which utilize optical, fluorescence and impedance measurements to provide automated determination of immature granulocytes of the blood samples. However, these instruments and their detection systems are expensive, and not suitable for low cost analyzers. Therefore, there is a need for automated and inexpensive determination of immature granulocytes and reduction of manual review rate.
On the other hand, the presence of nucleated red blood cells (NRBCs), or referred to as erythroblasts, one type of immature red blood cells, in the peripheral blood is also important information for diagnosis of certain diseases, such as anemia, and leukemia, etc. Therefore, it is of clinical importance to measure nucleated red blood cells in blood samples. Traditionally, differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide. The NRBC concentration is reported as numbers of NRBC per 100 white blood cells. Usually, 200 white blood cells and the numbers of NRBC present in the same region on a blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the numbers of NRBC/100 WBC. This approach is time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several fluorescence flow cytometry methods have been developed for differentiating NRBCs. These methods utilizes specific nuclear staining technique to distinguish NRBCs from other cell types because it is difficult to differentiate NRBCs based on their electronic or optical properties.
U.S. Pat. No. 6,410,330 (to Li et al) discloses a method for differentiation of nucleated red blood cells. The method includes the steps of lysing red blood cells of a blood sample with a lytic reagent, measuring nucleated blood cells by DC impedance measurement in a non-focused flow aperture, differentiating nucleated red blood cells from other cell types, and reporting nucleated red blood cells in the blood sample.
U.S. Pat. No. 6,472,215 (to Huo et al) teaches a method of differentiating nucleated red blood cells by lysing a first aliquot and a second aliquot of a blood sample separately with a first lysing reagent system and a second lysing reagent system; measuring the first sample mixture in a flow cell by DC impedance, radio frequency, and light scatter measurements; measuring cell distributions and counting remaining blood cells in the second sample mixture by DC impedance measurements in a non-focused flow aperture; analyzing blood cell distribution patterns obtained from measuring the first sample mixture and from measuring the second sample mixture respectively; and further performing a combined analysis to differentiate NRBCs from other cell types and determine numbers of NRBCs in the blood sample.
Furthermore, a well known problem with NRBC containing samples is erroneous white blood cell count (WBC) reported by hematology analyzers on these samples. Since the nuclear volumes of NRBC are close to those of white blood cells, and they are commonly counted as white blood cells on hematology analyzers which measure the sizes of blood cells, resulting an elevation of WBC. Therefore, correction of NRBC contribution to the WBC reported from hematology analyzer is required for samples containing NRBC. Current practice in the clinical laboratory is to subtract the numbers of NRBC obtained by manual count from the WBC count reported by the hematology analyzers. This is time consuming and error prone.
Based on foregoing, there exists a need for a simple and less costly analysis method for differentiating and enumerating nucleated red blood cells. Furthermore, it is desirable to provide automated determination of immature granulocytes together with the determination of nucleated red blood cells in one concurrent test.
In one embodiment, the present invention is directed to a method of measuring immature granulocytes. The method comprises steps of mixing a blood sample with a lytic reagent to lyse red blood cells, and to form a blood sample mixture; analyzing the blood sample mixture by a DC impedance measurement, and obtaining a blood cell distribution of the blood sample mixture from the DC impedance measurement; determining immature granulocytes from the obtained blood cell distribution; and reporting immature granulocytes in the blood sample. The immature granulocytes referred herein comprise myelocytes, promyelocytes, metamyelocytes, myeloblasts and promyeloblasts.
In a further embodiment, the present invention is directed to a method concurrently measuring immature granulocytes and nucleated red blood cells. The method comprises the steps of: mixing a blood sample with a lytic reagent to lyse red blood cells, and to form a blood sample mixture; analyzing the blood sample mixture by a DC impedance measurement and obtaining a blood cell distribution from the DC impedance measurement; determining nucleated red blood cells and immature granulocytes from the obtained blood cell distribution; and reporting nucleated red blood cells and immature granulocytes in the blood sample. The DC impedance measurement can be performed using non-focused flow aperture or focused flow cell. The non-focused flow aperture has an aperture aspect ratio of 0.7 and greater.
The present invention provides a cost effective method using DC impedance measurement for measuring immature granulocytes, as well as concurrently, measuring nucleated red blood cells of a blood sample.
In one embodiment, the present invention provides a method of measuring immature granulocytes (IG). More specifically the method measures immature granulocytes in a blood sample by a direct current (DC) impedance measurement.
The immature granulocytes referred herein include myelocytes, promyelocytes, metamyelocytes, myeloblasts and promyeloblasts.
In one specific embodiment, the method comprises the steps of mixing a blood sample with a lytic reagent to lyse red blood cells, and to form a blood sample mixture; analyzing the blood sample mixture by a DC impedance measurement, and obtaining a blood cell distribution histogram of the blood sample mixture; determining immature granulocytes from the obtained blood cell distribution; and reporting immature granulocytes in the blood sample.
To lyse a blood sample, the blood sample can be diluted first by a blood diluent, then mixed with a sufficient amount of a lytic reagent to lyse red blood cells. For the purpose of the present invention, the blood diluent contains a sufficient amount of salt or salts for impedance measurement of the sample mixture. Suitable examples of salts are alkaline metal salts.
A blood diluent is commonly used on an automated hematology analyzer to dilute a blood sample for measuring red blood cells, where the blood diluent is adjusted to isotonic by salts for maintaining the blood cell volumes. It is convenient to use commercially available isotonic blood diluents for the purpose of the present invention, although isotonicity is not required for the measurement of immature granulocytes.
The lytic reagent compositions suitable to use with a blood diluent for the present invention have been described in U.S. Pat. Nos. 6,410,330 and 6,573,102, which are hereby incorporated by reference in their entirety. Alternatively, a lytic reagent further containing a sufficient amount of a salt or salts for impedance measurement can be used for lysing a blood sample without a separate blood diluent. Suitable examples of salts are alkaline metal salts, such as sulfates, chlorides, phosphates, and citrates.
The measurement of immature granulocytes can be performed using DC impedance measurement in a non-focused flow aperture, or a focused flow cell. When a particle, such as a blood cell, passes through the aperture, an electrical signal can be measured due to conductivity or impedance change. The pulse shape, height and width, is directly related to the size of a particle, and can be converted to the size of the particles measured. When two or more particles of different sizes are measured, the histogram obtained from the measurement can represent size distribution of the particles.
The detection methods used for blood cell counting and sizing by a blood analyzer equipped with a DC impedance measurement device are generally described in U.S. Pat. No. 2,656,508 (to Coulter), and U.S. Pat. No. 3,810,011 (to Coulter, et al), which are hereby incorporated by reference in their entirety.
Example 1 illustrates an exemplary process for measurement of immature granulocytes using DC impedance measurement.
Example 2 illustrates the identification of immature granulocytes using the DC impedance measurement described above. As described in Example 2, a first test blood sample was prepared by adding a predetermined amount of promyelocytes produced by cell line in vitro into the normal whole blood sample shown in
A second test blood sample was prepared by adding a predetermined amount of promyeblasts produced by cell line in vitro into the normal whole blood sample shown in
These two test samples clearly illustrated the distribution characteristics of the two immature granulocytes on the DC histograms, which positively identified the cells of the clinical sample shown in
In a further embodiment, the present invention provides a method of concurrently measuring immature granulocytes and nucleated red blood cells. The sample preparation, the reagents and the DC measurement of the sample mixtures have been described above. In the presence of nucleated red blood cells, the obtained blood cell distribution histogram can be used for determining both immature granulocytes and nucleated red blood cells in the blood sample.
The method of measuring nucleated red blood cells using DC impedance measurement has been described in U.S. Pat. No. 6,410,330, which is hereby incorporated by reference in its entirety. More specifically, it is found that the aperture aspect ratio, defined as a ratio of the aperture length versus the aperture width, affects the separation of different sizes of blood cells, in particular for the cell populations which have relatively narrow distributions, such as in the situation of separating nucleated red blood cells from lymphocytes. With the method of the present invention, separation of the NRBC population from the other cell types can be achieved by using an aperture aspect ratio of 0.7 and greater.
It has been understood that aperture aspect ratio affects the flow profile of a flow passing through the aperture, which in turn, affects trajectory of particles in the flow. In general, with a fixed aperture width, the rate of a flow at the center of the flow increases with increasing the length of the aperture. Therefore, with an increase of the aperture aspect ratio, a flow rate gradient, from the sides of the flow which interface with the wall of aperture toward center of the flow, increases. In the presence of such a flow rate gradient, particles suspended in a flow passing through the aperture tend to move to the center of the flow. Therefore, under such a condition, particles have a similar behavior to the particles passing through a focused flow aperture. A focused flow aperture can be used in the present invention for measuring nucleated blood cells, particularly for differentiating blood cells having similar sizes. However, the cost of a focused flow aperture is much higher than a non-focused flow aperture.
On the other hand, it is known that at a cross section of an aperture, an imposed electrical field has a different strength along the cross section. Consequently, the particles passing through a non-focused flow aperture can generate various pulse shapes because each particle may experience a different electrical field depending on its position along the cross section of the aperture. These pulse distortions cause distortion of the particle size distribution in the measured histogram. Historically, pulse editing has been broadly used in the art to edit out seriously distorted pulses, and improve particle size differentiation to a certain degree. It is understood that with increase of the aperture aspect ratio, electrical field gradient along the cross section, from the center to the side wall of the aperture, decreases. Consequently, electrical pulses generated from the particles not passing the center of the aperture have less distortions because of the presence of a more homogeneous electrical field along the cross section.
Therefore, with an increase of aperture aspect ratio the two effects, control of particle trajectory in the non-focused flow aperture and reduction of electrical field gradient along the cross section of the aperture, improve ability of differentiation of different sizes of particles.
It has been found using a non-focused flow aperture with an aperture aspect ratio of 0.7 and greater, the NRBC population can be differentiated from closely sized other nucleated blood cells, particularly lymphocytes. Preferably, an aperture aspect ratio of 1.0 and greater is used. More preferably, an aperture aspect ratio about 1.2 is used.
To further increase the aperture aspect ratio, the separation of NRBC from other cell types can be further improved. However, when the aperture aspect ratio is 1.5 or above, the throughput of the sample mixture passing through the aperture for measurement reduces significantly, and it can render the measurement incompatible to the throughput requirement of a hematology instrument. Therefore, it should be understood that an aperture aspect ratio about 1.2 is selected based on a balance between the population separation and throughput of the measurement for a practical reason. Theoretically, an aperture aspect ratio above 1.2 can be used for separating the NRBCs from other cell types.
The following examples are illustrative of the invention and are in no way to be interpreted as limiting the scope of the invention, as defined in the claims. It will be understood that various other ingredients and proportions may be employed, in accordance with the proceeding disclosure.
A whole blood sample was aspirated by an experimental hematology analyzer. A first aliquot of 28 μl was diluted with 6 ml of Coulter® LH Series Diluent (Beckman Coulter, Inc., Miami, Fla.), then mixed with 1 ml of a first lytic reagent composition to lyse red blood cells in a WBC bath. The first lytic reagent contained 25.0 g/L of tetradecyltrimethylammonium bromide, 15.0 g/L of Igepal SS-837 (an ethoxylated phenol from RhOne-Poulenc), 4.0 g/L of Plurofac A38 prill surfactant (from BASF Corp.), and had a pH of 6.2. The experimental hematology analyzer was a modified LH750 (product of Beckman Coulter, Inc., Miami, Fla.), which was equipped with non-focused apertures of a length of 100μ and a width of 80μ for measuring the prepared sample mixture as described above. The sample mixture was drawn through a set of three apertures (arranged in parallel) by a constant vacuum. The nucleated blood cells were counted by a DC impedance measurement, and a histogram of the blood cells, after pulse editing, was also produced (averaged from the measurements of three apertures).
A second aliquot of 31 μl of the whole blood sample was mixed with a second lytic reagent in a mixing chamber to lyse the red blood cells, and subsequently mixed with a volume of StabiLyse™ (products of Beckman Coulter, Inc., Miami, Fla.) to retard further lytic reaction in this second sample mixture. The second sample mixture was delivered to a focused flow cell with a sheath fluid, Coulter® LH Series Diluent. The second lytic reagent contained 20 g/L of ethoxylated stearyl amine with 27 moles of ethylene oxide, 1.6 ml of formic acid, and 14 g/L of solubilizer. The second lytic reagent and its use for differentiation of white blood cells are described in U.S. Pat. No. 5,843,608, which is hereby incorporated by reference in its entirety.
The second sample mixture was measured in a focused flow cell by a detector which included a DC measurement means, a radio frequency impedance (RF) measurement means, and a light scatter measurement (LS) means. The light scatter measurement means detects median angle light scatter signals from about 10° to about 70°. A three dimensional scattergram was obtained using the three measured parameters and the functions of these parameter, which was herein referred to as VCS scattergram.
For the purpose of identifying distribution characteristics of immature granulocytes under the process and detection conditions described in Example 1, two types of immature granulocytes were grown in vitro by cell lines.
The first type of immature granulocytes is human promyelocytes grown by HL-60 promyelocytic cell line (ATCC certified cell line CCL-240, HL-60). The second type of immature granulocytes is human promyeloblasts grown by KG-1a cell line (ATCC certified cell line CCL-246.1 KG-1a). The cell culture condition used is known common procedures for cell growth. More specifically, the culture medium used is RPMI 1640 (a product of Roswell Park Memorial Institute, Buffalo, N.Y.), which comprises 10% fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin. 50 ml of RPMI 1640 culture medium and 50 ml of a specific type of cells described above were added together and maintained for 24 hours at 37° C. under 5% CO2. The cell concentration upon propagation was determined by counting on a Vi-CELL™ analyzer (a product of Beckman Coulter, Inc., Fullerton, Calif.).
A first test blood sample was prepared by adding 25×106 cells of the promyelocytes described above into 2000 μL of the normal whole blood sample shown in
A second test blood sample was prepared by adding 25×106 cells of the promyeloblasts described above into 2000 μL of the normal whole blood sample shown in
A clinical sample containing both immature granulocytes and nucleated red blood cells was analyzed according to the procedure described in Example 1. The manual reference reported about 10% of NRBC and about 10% of immature granulocytes, including metamyelocytes, myelocytes and promyelocytes.
The NRBC population was differentiated from the white blood cells, and the ratio between the NRBCs and white blood cells (×100) was reported as the numbers of NRBC/100 WBC. Alternatively, the NRBC can also be reported as absolute count in the blood sample by incorporating the total count of white blood cells.
This application is a continuation-in-part of application Ser. No. 10/702,352, filed Nov. 6, 2003, now abandoned, which is a continuation of application Ser. No. 10/228,800, filed on Aug. 23, 2002, now U.S. Pat. No. 6,673,618, which is a continuation-in-part of application Ser. No. 10/165,699, filed on Jun. 7, 2002, now abandoned, which is a continuation of application Ser. No. 09/917,533, filed on Jul. 27, 2001, now U.S. Pat. No. 6,410,330. All prior applications are incorporated herein by reference in their entireties.
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| Number | Date | Country | |
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| Parent | 10226800 | Aug 2002 | US |
| Child | 10702352 | US | |
| Parent | 09917533 | Jul 2001 | US |
| Child | 10165699 | US |
| Number | Date | Country | |
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| Parent | 10702352 | Nov 2003 | US |
| Child | 10770193 | US | |
| Parent | 10165699 | Jun 2002 | US |
| Child | 10226800 | US |
