The present invention is a flow cytometry-based hematology system useful in the analysis of biological samples, particularly whole blood or blood-derived samples. The system is capable of determining at least a complete blood count (CBC), a five-part white blood cell differential, and a reticulocyte count from a whole blood sample. The system preferably uses a laser diode that emits a thin beam to illuminate cells in a flow cell and a lensless optical detection system to measure one or more of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight measurements produced by the cells. The lensless optical detection system contains no optical components, other than photoreactive elements, and does not include any moving parts. Finally, the system uses a unique system of consumable reagent tubes that act as reaction chambers, mixing chambers, and waste chambers for the blood sample analyses. The consumable tubes incorporate reference particles, which act as internal standards to ensure that the dilutions made during processing of the samples have been carried out correctly, and to ensure that the instrument is working properly. The present invention also relates to methods for using the system.
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68. A consumable tube comprising:
a disposable tube member having two closed ends;
one or more reference particles disposed within the disposable tube member;
a reticulocyte dye disposed within the disposable tube member;
a surfactant disposed within the disposable tube member; and
a label affixed to an outer surface of the disposable tube member, said label having information relating to at least the reference particles and the reticulocyte dye.
1. A disposable vessel for use in a flow cytometry-based hematology system, comprising:
a disposable tube having two closed ends;
a known number of reference particles in said disposable tube, each of said reference particles having a predetermined diameter such that, when said reference particles are illuminated by light, said light is scattered by said reference particles such that said scattered light falls into at least one of a plurality of predetermined light scatter channels; and
at least one reagent in said disposable tube.
2. A disposable vessel for use in a flow cytometry-based hematology system, comprising:
a disposable tube having two closed ends;
a known number of reference particles in said disposable tube, each of said reference particles having one of a plurality of predetermined diameters such that when said reference particles are illuminated by light from a laser, said light is scattered by said reference particles such that said scattered light falls into one of a plurality of predetermined light scatter channels; and
at least one reagent in said disposable tube.
60. A disposable vessel for use in a flow cytometry-based hematology system, comprising:
a closed disposable tube having two closed ends;
a known number of reference particles in said disposable tube, each of said reference particles having a diameter of 4.0±0.5 microns;
a label fixable onto a surface of said closed disposable tube, said label containing information relating to the contents of said disposable tube; and
at least one reagent in said closed tube, said reagent being effective to enable said flow cytometry-based hematology system to measure at least one of a red blood cell count, a reticulocyte count, a platelet count, a two-part white blood cell differential, a five-part white blood cell differential, and hemoglobin concentration.
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a) new methylene blue, at a concentration between about 0.1 and about 0.5 grams per liter of a sample solution;
b) Plurafac-A-39-Prill, at a concentration between about 0.1 and about 0.6 grams per liter of a sample solution;
c) sodium bicarbonate, at a concentration between about 6.0 and about 10.0 grams per liter of a sample solution;
d) sodium chloride, at a concentration between about 1.0 and about 5.0 grams per liter of a sample solution;
e) Tricine, at a concentration between about 1.0 and about 5.0 grams per liter of a sample solution;
f) disodium EDTA, at a concentration between about 0.5 and about 3.0 grams per liter of a sample solution;
g) ethyl paraben at a concentration between about 0.1 and about 0.5 grams per liter of a sample solution; and
h) methyl paraben at a concentration between about 0.1 and about 0.3 grams per liter of sample solution.
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The present application is a divisional application of copending U.S. patent application Ser. No. 09/715,593, filed Nov. 17, 2000, now U.S. Pat. No. 6,784,981, which claimed the benefit of U.S. Provisional patent application Ser. No. 60/208,849, filed on Jun. 2, 2000.
This invention relates in general to bioparticle analysis, and more specifically to flow-cytometry-based methods and devices for performing automated blood cell analysis.
Mammalian peripheral blood usually contains three major classifications of blood cells—red blood cells (“RBCs”), white blood cells (“WBCs”), and platelets (“PLTs”). These cells are suspended in a solution referred to as plasma, which contains many different proteins, enzymes, and ions. The functions of the plasma components include blood coagulation, osmolality maintenance, immune surveillance, and a multitude of other functions.
Mammals usually have anywhere from 2-10×1012 RBCs per liter. RBCs are responsible for oxygen and carbon dioxide transport within the circulatory system. In many mammals, including humans, normal mature red cells have a bi-concave cross-sectional shape and lack nuclei. RBCs can range in diameter between 4 and 9 microns, depending on the species, and have a thickness that is generally less than 2 microns. The RBCs contain high concentrations of hemoglobin, a heme-containing protein which performs the dual roles of oxygen and carbon dioxide transport. Hemoglobin is responsible for the overall red color of blood, due to the presence of iron in the heme molecule. In the present application, the terms “erythrocytes”, “red blood cells”, “red , cells”, and “RBCs” are used interchangeably to refer to the hemoglobin-containing blood cells present in the circulation as described above.
In addition to mature RBCs, immature forms of red blood cells can often be found in peripheral blood samples. A slightly immature RBC is referred to as a reticulocyte, and the very immature forms of RBCs are broadly classified as nucleated red blood cells (NRBCs). Higher level non-mammalian animals, such as birds, reptiles, and amphibians, have exclusively nucleated RBCs in their blood.
Reticulocytes are red blood cell precursors that have completed most of the normal red cell development stages in bone marrow, and have expelled their nuclei. The last portion remaining to leave the reticulocyte before it becomes a truly mature RBC is transfer RNA. Detection of reticulocytes is important in clinical evaluation of a patient's ability to produce new red blood cells. The reticulocyte count also can be used to distinguish among different types of anemia. In anemia, red cell production may be diminished to the point where it can no longer keep up with red cell removal, and as a result the overall red blood cell count and hematocrit are low. The presence of an increased number of reticulocytes in anemic patients provides evidence that their bone marrow is functioning, and attempting to make up for the red blood cell deficit. If few or no reticulocytes are detectable in these patients, the bone marrow is not adequately responding to the red blood cell deficit.
White blood cells (also called “leukocytes”) are the blood-borne immune system cells that destroy foreign agents, such as bacteria, viruses, and other pathogens that cause infection. WBCs exist in peripheral blood in very low concentrations as compared to red blood cells. Normal concentrations of these cells range from 5-15×109 per liter, which FSLtube
where
The reagents useful in the present invention are described herein in reference to counting and classification of RBCs, reticulocytes, and WBCs. It is intended that this description be illustrative and not limiting to the scope of the reagent useful in the present invention in any way. In determining the cellular composition of a blood sample, a preferred embodiment of the present invention uses at least two primary method reagents and a third system reagent. These three reagents work together to first count and classify platelets and red cells, and then, after further reagent manipulation, to count and classify white blood cells. The counting and classification take place in two discrete phases, and in each of the two discrete phases the blood-reagent mixtures are passed through a single flow cytometer and cells in the samples are identified and counted.
One method reagent, designated the RBC/Retic method reagent, is placed in a standard, capped, test tube, referred to herein as the “consumable tube”. When other tests are used, the appropriate reagents are placed in the consumable tube. A separate consumable tube is used for each test run. For a CBC test, the consumable tube containing the RBC/Retic method reagent may comprise the following:
The second method reagent is designated the “lyse”. The lyse acts to destroy red blood cells while leaving the white blood cells intact. In this fashion, the white blood cells can be analyzed, without interference from the vast numbers of red blood cells. The lyse is preferably provided in a tube separate from the consumable tube. This reagent may be packaged in a standard capped test tube, preferably in a form that allows it to be used with up to about fifty separate consumable tests.
The lyse preferably comprises:
The third reagent is designated the system reagent, or “sheath solution”. The sheath solution performs several functions, such as whole blood dilution reagent, sheath reagent for flow cytometric analysis, washing reagent for the instrument's hydraulic path, and lytic aide to assist the lyse reagent in lysing red blood cells. The sheath is preferably packaged in a container that is separate from either the consumable tube or the lyse, and preferably holds enough solution to run about fifty separate consumable tests.
The sheath preferably comprises the following:
The consumable tube may additionally or in place of the above reagents contain other reagents directed to tests that are not associated with the standard hematological analyses outlined above. For example, the system may also be used in immunoassays, or in analyses of biological fluid samples other than whole blood. Examples of such biological fluids may include, without limitation, blood products, bone marrow, cerebrospinal fluid, synovial fluid, urine, or any other fluid containing or suspected to be containing cells. In such cases, reagents useful for the desired analyses may be included in the consumable tube. Examples of such reagents include various forms of antibodies, such as monoclonal antibodies, polyclonal antibodies, and antibody derivatives, such as F(ab), F(ab′)2, scFv, and Fv fragments. Other reagents could be, for example, enzymes or enzyme substrates or inhibitors, or receptor or receptor ligands, as well as receptor bodies. The bar coded labels on the consumable tube may reflect the type of test for which the tube is intended, and may also contain information relating to the contents of the consumable tube.
Other assays that do not require the optical detection system to count or classify cellular components or particles can also be analyzed in this invention. The consumable tube can be configured to run clottable assays, such as coagulation time assays. Prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT) can be measured by using a consumable tube containing an agonist or, less preferably, placing an agonist in the consumable tube, and then adding whole blood containing an anticoagulant, such as sodium citrate or sodium EDTA. The whole blood and the agonist will form a clot in the presence of calcium. By spinning the tube, the clot collects at the center, and is detected as a decrease in light transmission through the consumable tube.
In a similar manner, platelet aggregation tests may be run on the system. In these assays, an agonist, such as ristoceitin, is either included in or added to the consumable tube. Platelet-rich plasma is added to the consumable tube, and the tube is again spun to collect the resulting aggregates in the center. The aggregates are detected as a decrease in light transmission through the consumable tube. Both the platelet aggregation test and the clottable assays require a stable temperature for performing these assays. Therefore, a heating element is preferably included in the slot in which the consumable tube is held for these tests, in order to enable accurate measurements.
In the particular case of a CBC test, the user presents to the instrument a barcode-labeled consumable tube, as well as a properly anti-coagulated whole blood sample in a sample tube. The instrument reads the necessary information from the barcode to identify the consumable tube, then automatically aliquots a small amount of whole blood from the sample tube into the consumable tube, along with a predetermined volume of sheath solution to act as a diluent. This ideally creates a total dilution of one part blood to 100 parts solution (RBC/Retic method reagent and sheath), but the dilution can range from 1:50 to 1:5,000.
A known volume of the diluted whole blood solution is then pulled into the instrument from the consumable tube through an HGB module a first light absorption measurement is made. This measurement detects principally the amount of reticulocyte dye in the solution, but also enables determination of the amount of hemoglobin in the sample (see below). Thereafter, the red cell solution is moved to a position near the entrance to an optical flow cell. The diluted whole blood solution is then passed through an optical detector system (described above), where light scatter and light absorption for individual red blood cells, platelets and reference particles may be measured, and the number of each cell type determined. Hydrodynamic focusing is used to maintain a slow mass flow rate of the diluted whole blood solution through the optical detector system. Preferably, the mass flow rate is less than about 0.25 microliters per second.
After red cell/platelet analysis is complete, a second, larger amount of whole blood is aliquoted into the consumable tube, along with an aliquot of lyse and appropriate amounts of sheath solution. This dilution can range from 1:10 to 1:100, but is preferably a 1:20 dilution. The intent is to provide a solution that can lyse the red blood cells, but keep intact the white cells of the whole blood sample for a period of at least one minute.
A known volume of this lysed blood solution then enters the instrument from the consumable tube and moves to a position near the entrance to the optical flow cell, passing through the HGB module, where a second, more accurate HGB determination is made (see below). The solution is then passed through the optical detector system, where light scatter and light absorption for individual white blood cells, reference particles, and dye may be measured. Hydrodynamic focusing is used to maintain a slow mass flow rate of the white cell solution through the optical detector system. Preferably, the mass flow rate is about one microliter a second.
High Frequency Modulation to Reduce Diode Laser Mode-Hopping
In a preferred embodiment of the present invention, high frequency modulation is employed to obtain accurate measurements when a laser diode is used. High-frequency modulation reduces or eliminates mode hopping by having the laser exist in a multi-mode state. When temperature or current changes occur in a laser running under high-frequency modulation conditions, the primary mode of the diode laser changes, but since the diode laser is running in many modes to begin with, these changes do not produce any noise spikes. Furthermore, the frequency of modulation preferably is chosen to be much higher than the frequencies of interest for cell events, so the modulation is invisible to the system.
The use of high frequency modulation eliminates the need for temperature and/or current control systems in addressing mode hopping, thus saving the costs and complexity associated with these systems. A high frequency modulation system stabilizes the laser diode within seconds, so there is no need to control for the temperature generated by the diode laser upon activation. Thus, the useful laser life is extended. Furthermore, unlike the temperature cooling systems, high frequency-modulated diode lasers are stable over a wide range of operating temperatures. With high frequency modulation, there is no need to find a quiet temperature/current area for the laser, and no need to reset the equipment as the laser ages. Thus, the labor cost incurred in finding and maintaining a quiet temperature/current area for the diode laser is avoided.
High-frequency modulation is imposed on a diode laser in the following manner. A diode laser running in continuous wavelength (CW) mode has a built-in photodiode that is used for power feedback, so that the power does not vary with temperature. This circuit changes the current input to the diode of the laser so that the light power is kept constant. In one embodiment, the CW set point of the laser driver is set so that it produces one-half of the rated power output of the laser. Next, a modulating circuit, which uses a crystal oscillator to produce an electric current with a sine wave output, is “summed” with the CW current. As a result, the laser power output takes on a sine wave shape, with a power maximum near the maximum rated power level of the laser at the peak of the sine wave and almost zero power at the minimum of the sine wave.
HGB Module
In a preferred embodiment of the present invention, hemoglobin (HGB) content is measured in a unique manner. The method involves two separate measurements, which not only combine to give an accurate HGB value, but also verify the accuracy of the dilutions that the system has made. The first HGB measurement occurs after the whole blood sample is diluted with a reticulocyte staining solution. Preferably, one part of the whole blood sample is diluted with 100 parts of the reticulocyte staining solution. The resulting diluted sample is referred to as the “red cell solution”, and has a specific absorption spectra. The red cell solution is next aspirated into a manifold block. This block contains at least three light sources, configured to illuminate a cylindrical bore, where the red cell solution is held. Three light sources are used to provide a measurement of HGB concentration. The light sources are preferably LEDs, but may be any appropriate light source. Photodetectors are placed in line with the light source and the cylindrical bore containing the red cell solution. Optionally, interference filters may be employed as desired. Preferably, the three wavelengths used in measuring HGB concentration are 488 nm, 540 nm, and 580 nm. An additional light source, emitting light at a wavelength different from the other three, may be included to enable measurement of the amount of a reticulocyte dye in the red cell solution and/or the white cell solution, and thereby enable detection and quantitation of reticulocytes.
The initial absorption measurements of the red cell solution do not provide as accurate a measurement of HGB concentration as conventional techniques. However, the value of this measurement lies in its use for the determination of the accuracy of the whole blood sample dilution. As the instrument continues its cycle after making the initial “unlysed” hemoglobin measurement, total RBC and mean cell volume measurements are made in the cytometer portion of the instrument. This enables the calculation of the following:
As the instrument cycle continues, more whole blood is added to the original solution, as well a lytic agent. The lytic agent destroys the red blood cell membranes (lysis), releasing HGB free in solution. This yields a solution containing approximately 1 part whole blood to between 15 and 50 parts solution, which is referred to as the “white cell dilution”. The white cell solution contains all of the dye from the previously measured red cell solution. The dye in the red cell solution is diluted out with an optically clear fluid by a factor between 1:1 and 1:4.
The white cell solution is aspirated into the manifold block where the multiple absorption measurements are made, and a HGB value calculated. From this second HGB value, the HCT, MCHC and MCH values can be recalculated. In addition to the HGB value, dye concentration can be measured, and compared to values obtained from the red cell solution. If, within a defined tolerance, the two independent HGB (HCT, MCHC, MCH) measurements match, and the dye ratios between the two solutions match, the instrument has, by measurement, verified all of its dilutions were performed accurately.
The white cell solution HGB calculations yield an accurate measurement of the HGB concentration. The red cell solution HGB calculations are not as accurate as the white cell solution HGB calculations, because the red blood cells in the red cell solution are intact, and the intact red cell membranes scatter light. In contrast, in the white cell solution the red blood cells are lysed, and the total HGB content is released into the solution. However, the red cell solution HGB measurements permit comparison of the differences in the dye absorbances, which allow a check of the accuracy of the dilutions. In other words, the red cell solution has a certain level of light absorption which is based on the concentration of the dye in the consumable tube. Later in the cycle, the white cell solution is measured for light absorption due to the dye. Because the hydraulic pathways for the red cell solution HGB measurement and the white cell solution HGB measurement are common, the accuracy of the dilutions may be determined without the need for separate reference solutions to control for differences between pathways. The acceptable range of ratios of the HGB measurements is known, e.g., from being printed on a barcode label on the consumable tube or from a code on the consumable tube barcode label that references a value in a table of ratios stored in a linked PC. As a second check of the accuracy of the dilutions, the ratio of the HGB measurements should also be within a predetermined range. Otherwise, a dilution error should be suspected. The only case in which the dilution error might occur but be undetected is the highly unlikely situation in which identical percent dilution errors are made on both samples.
Preferably, the bar code on the consumable tube contains reference information on the expected values of dye absorption for undiluted samples (CAL). The instrument may verify this information for each lot number in use by evaluating the absorbance of the dye solution before the addition of whole blood to the solution. After the first dilution, the red cell solution should have a dye absorption value and an HGB concentration value that fall within a first set of expected ranges. These ranges should be the product of CAL and a first dilution factor D1 (i.e., CAL×D1). After the second dilution, the white cell solution should have a dye absorption value and an HGB concentration that fall within a second set of expected ranges. These ranges should be the product of CAL, D1, and a second dilution factor D2 (i.e., CAL×D1×D2). So long as both sets of measurement are within the acceptable ranges, the results of the measurements are reported without associated error messages.
System Electronics
In a preferred embodiment, the center of the system electronics is a central processing unit (CPU) that is responsible for handling communications between the different sub-systems. A personal computer (PC) preferably acts as the user interface and data processor. Alternatively, the user interface and data processor may be built into the body of the instrument. In either case, the input issues commands to and receives data from a CPU. Data may be transmitted via any appropriate and conventional means, such as, for example, across a universal serial bus (USB). The CPU in turn translates the PC commands, and issues commands to a set of two identical motor/sensor electronic boards. These motor/sensor boards receive input from the various sensors on the system, and move the appropriate motors such that particle events are passed through the laser optics. The light signals from the scattered light detectors are amplified by a pre-amplifier, which is in close proximity to the photodetectors, and then passed to a signal processor that analyzes the electrical signals from the detectors. In one embodiment, the signal processor is an analog signal processing module (ASP), as shown in FIG. 26. Processed data from the ASP is communicated to the CPU, and then to the PC host. Alternatively, as noted above, the user interface may be contained within the instrument, obviating the need for a linked PC. The rest of the system would work as described above. A person of ordinary skill in the art would recognize that variations in this scheme are possible to create the desired level and types of control over the system.
The following description describes the system of the present invention in relation to the accompanying figures, and its use in measurement of a whole blood sample for a CBC, a five-part WBC differential, and a reticulocyte count. It will be understood by those of skill in the art that certain modifications may be made in the invention as described below without departing from the scope or spirit of the attached claims. Throughout this specification, the term “inlet side” refers to the side where a fluid enters. “Outlet side” refers to a side where fluid exits. “Sample mixture” refers to a fluid sample, usually a blood sample or blood-derived sample, that is mixed with another solution.
Referring now to
Carriage positioning assembly 76 moves carriage 70 then positions the sample tube underneath a hydraulic needle 11 and a pneumatic needle 12 (FIGS. 1 and 8). Needle 11 pulls up samples and reagents at various points during the assay, and transfers the samples and reagents among the different tubes. Needle 12 is a vent needle, used to maintain atmospheric pressure in the tubes. By moving motor 81, needles 11 and 12 pierce the sample tube septum, and continue to penetrate the sample tube until both needles 11 and 12 come in contact with the surface of the blood sample. This ensures that the orifice of hydraulic needle 11 is completely immersed, but the orifice of pneumatic vent needle 12 is not.
Referring again to FIG. 1, opening valve 3 and moving syringe 6 pulls blood into the tip of needle 11. Ideally, five microliters of whole blood are aspirated for this portion. Valve 3 is then closed. Needles 11 and 12 are then retracted from the whole blood sample. Needles 11 and 12 are wiped almost completely clean as they are withdrawn through the septum. Carriage positioning assembly 76 now positions consumable tube 60 under needles 11 and 12. Motor 81 moves needles 11 and 12 to pierce the septum of consumable tube 60 and penetrate consumable tube 60 until both needles 11 and 12 come in contact with the surface of the reagent in consumable tube 60.
Valves 5 and 3 are then opened to allow syringe 7, which is filled with sheath solution, to begin to dispense sheath solution. The movement of sheath solution from syringe 7 pushes the sample blood from the tip of needle 11, along with a volume of sheath solution, into the consumable tube, which is now represented by 61 (FIG. 6). While dispensing, motor 81 moves needles 11 and 12 upward so as to prevent the orifice of needle 12 from coming into contact with the solution in consumable tube 61. This solution is now fairly homogeneous, but to ensure homogeneity, valves 3 and 5 are closed, and needles 11 and 12 are withdrawn from consumable tube 61. Consumable tube 61 then is moved to the mix position under mixer assembly 80, and mixed by spinning, as described above. During this process valves 3 and 5 open, and syringe 7 pulls back to create an air gap at the tip of needle 11.
Consumable tube 61 is next placed under needles 11 and 12 by moving carriage assembly 76. By moving motor 81, needles 11 and 12 pierce the septum of consumable tube 61, and penetrate the tube until both needles come in contact with the surface of the solution. Syringe 7 then aspirates a predetermined volume of solution into the tip of needle 11 behind the air gap. Needles 11 and 12 are then withdrawn from the solution by motor 81, but are not removed from consumable tube 61. Syringe 7 then pulls air into needle 11. This creates a slug of the diluted whole blood in needle 11, bordered by air gaps on each side. Syringe 7 then pulls this slug through HGB module 10 (FIGS. 1 and 9), where it is evaluated for light absorption at four separate wavelengths of light. Spectral data typical of such measurements are presented in FIG. 22. The volume of the sample slug can also be evaluated at this point by evaluating the change in absorption values in the detector channels as the air gaps on each side of the slug pass through the detector channels. By evaluating the time taken for the absorption values to change, and knowing the rate of sample movement through the detectors, the volume of the sample slug can be easily calculated. If the volume of the sample slug deviates by a predetermined amount from that expected, an error message can be displayed to the user, and the sample analysis halted until the problem is fixed.
Syringe 7 continues to pull the sample slug past valve 3, and positions it close to valve 5. Valve 3 closes while valve 2 opens, at which point syringe 7 reverses its direction and pushes the slug through valve 2 to the tip of a flow cell 22 (FIG. 2).
Valve 2 then closes, and valve 4 opens, which allows syringes 6 and 7 to be filled from a system sheath solution reservoir 8. Valve 5 then closes, valve 1 opens, and syringes 6 and 7 dispense sheath solution. Syringe 6 dispenses in a range of about 0.01-0.5 μl/sec, and preferably on the order of about 0.05 μl/sec. Syringe 7 dispenses in a range of about 50-250 μl/sec, and preferably on the order of about 100 μl/sec. The sheath solution in syringe 7 is dispensed through a feed tube 21, and the slug of diluted whole blood is pushed by syringe 6 through flow cell 22, and more specifically through flow channel 47. This forms a sheath-confined core stream of diluted whole blood in the flow cell nozzle 25, which transports the stream to the funnel of flow cell 22, and into flow channel 47, where the cells are interrogated by light sources emitting from a beam shaping assembly 41. This stream of fluid passes completely through flow channel 47, and into the tubing of outlet 23, which is affixed to flow cell 22 by flow cell cap 24. This path leads to the system waste 9.
Referring now to FIGS. 3A, 3B, and 4, beam shaping assembly 41 includes a laser diode 30, which emits light that diverges in two perpendicular axes. A collimating lens 31 eliminates the divergence in both axes, and causes the light to illuminate a lens 32. Lens 32 acts to converge the laser beam in one axis, such that nearly all of the light from laser diode 30 passes through flow cell 22. A third lens 33 is introduced between lens 32 and the flow channel 47 of flow cell 22, such that focal point 34 in flow channel 47 is produced. Focal point 34 of lens 33 is thus in flow channel 47, and a second focal point 35 of lens 32 is the preferred location at which to place photodetector array 48 (FIG. 4).
As the fluid stream passes through flow channel 47, beam shaping assembly 41 illuminates the cells of the diluted whole blood sample individually. The cells pass through first focal point 34, where the light power incident upon the cells is maximized. As cells pass through the light beam of laser 30 at first focal point 34, the quantity and quality of light incident upon photodiodes 42, 43, 44 and 45 is altered. These changes are captured by the system signal processing electronics (FIG. 26), and stored via the system electronics (FIG. 25). These system electronics assemblies 17 are shown in their context in the entire system in FIG. 27.
Photodetector array 48 includes at least two, but most preferably four, photodiode detectors. No collection or imaging lenses are required. These photodiode detectors may include two or more of an axial light loss detector 44, a low-angle forward scattered light detector 45, and a high-angle forward scattered light detector 43. In addition, a right angle scattered light detector 42 (also referred to herein as a high numerical aperture detector) may be included in the instrument, but would be separate from the rest of the photodetectors and would not be mounted onto photodetector array 48. FIG. 5 is a drawing of a photodiode mask 50, which can be used to cover photodetector diodes 43, 44, and 45 to provide three independent light measurements in the direction forward of the laser beam. In this embodiment, low-angle forward scattered light passes through mask portion 51, axial light loss is measured through mask portion 52, and high-angle forward scattered light passes through mask portion 53. Photodiode mask 50 is useful to limit the amount of light from sources other than these that passes through each of detectors 43, 44, and 45. Preferably, mask portions 51, 52, and 53 are on a single fabricated piece of semiconductor material, where only the areas of the desired geometries of mask portions 51, 52, and 53 are active portions of the semiconductor material. Alternatively, photodiode mask 50 could comprise a metal mask that is positioned atop photodetector diodes 43, 44, and 45. The entire contents of the optical system depicted in FIGS. 2 through 5, is shown in its packaged form as optical assembly 16.
FIG. 10A shows data which has been collected for the forward scatter low (FSL) signal from detector 45.
Should the reagents used in creating this dilution use an RNA staining dye, such as new methylene blue, the data can be further analyzed by measuring the red blood portions (designated C) in FIGS. 10A-10D to evaluate reticulocyte counts. Histogram of such analysis are shown in FIGS. 14A and 14B, and a scatter plot is shown in FIG. 15. FIG. 15 shows a calculated reticulocyte population of 1.72%. This compares favorably with a 1.60% reticulocyte count, as determined from the same sample by a manual method.
After successfully counting and classifying the red blood cell, reticulocyte, platelet and latex particle populations, needles 11 and 12 are withdrawn from consumable tube 61. Carriage positioning assembly 76 moves the whole blood sample tube so it is again positioned under needles 11 and 12. Needles 11 and 12 are then moved by motor 81 to pierce the whole blood sample tube's septum, and continue to penetrate the tube until both needles 11 and 12 come in contact with the surface of the blood sample. This ensures that the orifice of hydraulic needle 11 is completely immersed in the blood sample, but the orifice of pneumatic vent needle 12 is not. Opening valve 3, and moving syringe 6, causes a portion of the blood sample to be pulled into the tip of needle 11. Ideally, 100 microliters will be aspirated for this portion. Valve 3 is then closed. Needles 11 and 12 are then retracted from the whole blood sample tube. Needles 11 and 12 are wiped almost completely clean as they are withdrawn through the septum. Carriage positioning assembly 76 now positions consumable tube 61 under needles 11 and 12. By moving motor 81, needles 11 and 12 pierce the septum of consumable tube 61, and penetrate the tube until both needles 11 and 12 come in contact with the surface of the solution.
Valves 5 and 3 are opened, while syringe 7, which is filled with the sheath solution, begins to dispense sheath solution. This pushes the 100 microliters of blood sample solution through the tip of needle 11. A volume of sheath fluid follows the whole blood into the consumable tube, which is now represented by 62 in FIG. 6. As the sheath fluid is dispensed into consumable tube 62, motor 81 moves needles 11 and 12 upward, to prevent the orifice of needle 12 from coming into contact with the solution in consumable tube 62. Needles 11 and 12 are next removed from consumable tube 62, and carriage positioning assembly 76 moves a tube of lyse reagent under needles 11 and 12. Motor 81 moves needles 11 and 12 downward to pierce the lyse tube's septum and continues until needles 11 and 12 come in contact with the surface of the lyse. Opening valve 3 and moving syringe 6 pulls lyse into the tip of needle 11. Ideally, 100 microliters of lyse will be aspirated for this portion, which is followed by an air gap created by removing needle 11 from the solution, then moving syringe 6 to pull air into needle 11. Valve 3 is then closed. Needles 11 and 12 are then retracted from the lyse tube. Needles 11 and 12 are wiped almost completely clean as they are withdrawn through the septum. Carriage positioning assembly 76 positions the consumable tube 62 under needles 11 and 12. Valve 5 and 3 are then opened, while syringe 7, which is filled with the system diluent, begins to dispense diluent. This pushes the 100 microliters of lyse through the tip of needle 11. A volume of sheath fluid follows this into the consumable tube, now represented by 63 in FIG. 6. As the sheath fluid is dispensed, motor 81 moves needles 11 and 12 upward, so as to prevent the orifice of needle 12 from coming into contact with the solution.
The solution in consumable tube 63 is fairly homogeneous, but to ensure homogeneity, valves 3 and 5 are closed, and the needles are withdrawn from consumable tube 63. Consumable tube 63 is then moved to the mix position under mixer assembly 80, and mixed by spinning, as describe above. During this process, valves 3 and 5 are opened, and syringe 7 creates an air gap at the tip of needle 11.
Carriage positioning assembly 76 moves consumable tube 63 under needles 11 and 12. Motor 81 moves needles 11 and 12 downward to pierce the septum of consumable tube 63 until needles 11 and 12 come in contact with the surface of the solution. Syringe 7 then aspirates a predetermined volume of solution into the tip of needle 11. Needles 11 and 12 are withdrawn by motor 81 from the solution, but not completely from consumable tube 63. This creates a slug of the lysed whole blood in needle 11. Syringe 7 pulls this slug of lysed whole blood through HGB module 10, where absorption at four separate wavelengths of light is measured. Red blood cells have been lysed in this slug, freeing hemoglobin from the cells. FIG. 19A shows absorption data, where cyanide was used in the lysing agent to label the HGB. FIG. 19B shows absorption data where only a lytic agent was used. FIG. 21 shows absorption data where cell lysis occurred in the presence of a reticulocyte dye. The volume of the lysed sample slug can also be evaluated at this point by evaluating the change in absorption values in the detector channels as the air gaps on each side of the slug pass through the detector channels. By evaluating the time taken for the absorption values to change, and knowing the rate of sample movement through the detectors, the volume of the lysed sample slug can be easily calculated. If the volume of the lysed sample slug deviates by a predetermined amount from that expected, an error message can be displayed to the user, and the sample analysis halted until the problem is fixed.
Syringe 7 continues to pull the lysed sample slug past valve 3 and close to valve 5. Valve 3 closes and valve 2 opens, at which point syringe 7 reverses its direction and pushes the slug through valve 2 to the tip of flow cell assembly 20.
Valve 2 then closes, and valve 4 opens, which allows syringes 6 and 7 to be filled from a system diluent reservoir 8. Valve 5 then closes, valve 1 opens, and syringes 6 and 7 dispense sheath solution. Syringe 6 dispenses in a range of about 0.1-1.0 μl/sec, and preferably on the order of about 0.5 μl/sec. Syringe 7 dispenses in a range of about 50-250 μl/sec, and preferably on the order of about 100 μl/sec. The sheath solution in syringe 7 is dispensed through feed tube 21, and the slug of lysed whole blood is pushed by syringe 6 through flow cell 22. This forms a sheath-confined core stream of lysed whole blood in flow cell nozzle 25, which transports the stream to the funnel of flow cell 22, where the cells are interrogated by light sources emitting from optical assembly 41. This stream of fluid passes completely through flow cell 22, and into the tubing of outlet 23, which is affixed to flow cell 22 by flow cell cap 24. This path leads to the system waste 9.
As the fluid stream passes through flow cell 22, optical assembly 41 illuminates the cells of the lysed whole blood individually. The cells pass through first focal point 34 of laser 30. At first focal point 34, the light power incident upon the cells is maximized. This narrow focus at first focal point 34 is generated by lens 33. As cells pass through the light beam of laser 30 at first focal point 34, the quantity and quality of light incident upon photodiodes 42, 43, 44 and 45 is altered. These changes are captured by the system signal processing electronics (FIG. 26), and stored via the system electronics (FIG. 25). Examples of data collected in this manner are shown FIGS. 12A through 12F.
FIG. 12A presents data which has been collected for the extinction or axial light loss (EXT) signal from detector 44 in the absence of latex particles. FIG. 12B shows the same sample with latex present. FIG. 12C presents data collected for the forward scatter low signal (FSL) from detector 45 in the absence of latex particles. FIG. 12D shows the same sample in the presence of latex particles. FIG. 12E presents data collected for the right angle scatter (RAS) signal from detector 42 in the absence of latex particles, and FIG. 12F shows the same sample with latex particles present.
For FIGS. 12A through 12F, the peaks shown represent lymphocyte events (A), monocyte events (B), granulocyte events (C), and latex particle events (D). This same data is shown in a scatter plot form in FIGS. 13A and 13B. For FIGS. 11A and 11B, the dots in circle D represent the latex particle events. Scatter plot data may be represented by any pair derived from the set of EXT, FSL, FSH, RAS, and TOF measurements.
For samples which contain eosinophils in the granulocyte population, eosinophil detection is achieved in the right angle scatter channel of detector 42.
After the white blood cells have been counted and classified, syringe 7 is charged with fluid from reagent reservoir 8, by opening valve 4. Valve 4 closes and valves 3 and 5 open, which allows syringe 7 to clean the sample lines by back-flushing fluid into consumable tube 63 through needle 11. Needle 12 acts to vent the tube through filter 13, to atmosphere. This yields a consumable tube of the configuration shown in 64 in FIG. 6. Carriage positioning assembly 76 now moves to its home position, where the user can open the system door, and remove the tubes from the carriage slots. Consumable tube 64 is now a waste tube, and is discarded. The lyse solution tube remains in the instrument until its contents are depleted.
Other modes of operation, along the same lines as described above would allow for immunoassay to be performed by latex agglutination techniques. Here the consumable would contain latex particles that are coated with an antibody, such that the particles would clump together in the presence of the proper analyte. This latex particle mixture would be run through the optical channel described in
Various patents and publications are cited herein, and their disclosures are hereby incorporated by reference in their entireties. The present invention is not intended to be limited in scope by the specific embodiments described herein. Although the present invention has been described in detail for the purpose of illustration, various modifications of the invention as disclosed, in addition to those described herein, will become apparent to those of skill in the art from the foregoing description. Such modifications are intended to be encompassed within the scope of the present claims.
Crews, Harold R., Roche, John W., Hansen, Peter W., Coleman, Michelle L.
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