Classifications

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  • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
  • G01N2015/012—Red blood cells
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Definitions

• CBC Complete Blood Count
• CBC instruments currently on the market aspirate anticoagulated whole blood and divide it into several analysis streams to perform the different elements of the CBC in a "flow" system (flow cytometer). These elements include:
• Red blood cell (BBC) count hemoglobin (Hb), hematocrit (Hct), red blood cell indices (mean corpuscular volume, MCV, mean corpuscular hemoglobin, MCH and mean corpuscular hemoglobin concentration MCHC), and red blood cell morphology including reticulocyte count.
• BBC Red blood cell
• Hb hemoglobin
• Hct hematocrit
• red blood cell indices mean corpuscular volume, MCV, mean corpuscular hemoglobin, MCH and mean corpuscular hemoglobin concentration MCHC
• red blood cell morphology including reticulocyte count.
• WBC White blood cell
• CBC systems Other measurements are included in some CBC systems that are derived from the primary measurements of RBCs and platelets, such as RBC and platelet distribution widths.
• Flow based CBC instruments require extensive calibration and control, maintenance, and skilled operators, and they have substantial costs associated with reagents, consumables and disposables.
• One significant problem with these systems in routine use is that a large proportion of blood specimens require further testing to complete the morphologic components of the CBC. This involves the direct visualization of blood cells on a glass slide that is performed by a skilled technician whenever the results of the automated test includes results than need further examination. For example, a "manual" differential count is performed by direct visualization of the cells by an experienced observer when nucleated immature RBCs are found or WBCs suspicious for infection, leukernias or other hematologic diseases are found.
• the proportion of these specimens requiring further review ranges from 10 to 50%, depending on the laboratory policy, patient population and "flagging" criteria, with a median rate of around 27%.
• the most frequent reasons for retesting is the presence of abnormal cell types or cell morphology, clinical or other suspicion of viral or bacterial infections or a decreased number of WBCs, RBCs or platelets.
• the method described below makes possible a greatly simplified and more versatile CBC instrument that detennines all parameters of the CBC and include all the morphological observations of RBCs and WBCs which lead to the WBC differential count. It is based on image analysis, using a new method of preparing a monolayer for analysis.
• the preservation of cell morphology is especially important for patients with hematological malignancies, such as chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML).
• CLL chronic lymphocytic leukemia
• AML acute myeloid leukemia
• Preparation of the standard wedge smear may cause severe morphologic deformities of the malignant lymphocytes or leukocytes (blast cells) .
• some of the enlarged blast cells may burst during preparation of wedge smear due to their cytoskeletal fragility.
• the presence of these so called “smudge cells” is sufficiently frequent in CLL as to warrant notation in the differential count .
• the system of gentle monolayer creation may enable detection of a larger number of morphologically well preserved blasts to allow more accurate recognition of blasts or an earlier detection of leukemias.
• the system produces a visual appearance upon conventional staining that allows virtually every RBC, WBC, and platelet from a known volume of blood to be counted and its morphology studied. It therefore allows for both the quantitative and qualitative components of the CBC to be determined in one operation. It avoids the distribution artifacts that are known to influence WBC differential counts performed on conventional smears prepared by any current method.
• An objective of the invention is to provide a system to perform the Complete Blood Count (CBC) including a white blood cell differential count.
• CBC Complete Blood Count
• Another objective is to have the system make use of a preparation method combined with computer imaging.
• Figure 1 is a photograph of a prototype of the equipment
• Figure 2 is a two drop commercial slide having hydrophobic rings
• Figure 3 is a "screenshot" of the software used to compare manual cell counts to automated cell counts
• Figure 4 is a graph showing the correlation between the automated counts and the manual counts for the red blood cells
• Figure 5 is a graph illustrating the correlation between the automated counts and the manual counts for the white blood cells
• Figure 6 is a graph of automated counts for red blood cells plotted against a reference method (Sysmex Inc. CBC analyzer);
• Figure 7 is a graph of automated counts for white blood cells plotted against a reference method (Sysmex Inc. CBC analyzer);
• Figure 8 is a "Cell spot" picture created by tiling approximately 450 individual images together while reducing overall image size
• Figure 9 shows one of the images acquired by a digital camera for subsequent analysis.
• a system to perform the Complete Blood Count (CBC) and a white blood cell differential count makes use of a preparation method combined with computer imaging.
• a system to demonstrate the complete operation of the device to provide a total blood count, and a 5 part BC differential count is described.
• the system can be constructed around a robotic platform that allows movement of a substrate such as a glass or plastic microscope slide to different locations representing different stages in the preparation, staining, and measuring process.
• the device prepares the slide, dilutes a blood sample, and transfers the diluted sample to the slide.
• the machine dries the monolayer of blood cells from the droplet and immediately stains the dried blood film.
• the device uses brightfield optics and multispectral imaging to analyze the stained cells to count the red blood cells, white blood cells, and the platelets and to perform a white blood cell differential count.
• the first station includes 1) a precision automated pipettor that allows very small volumes of fluids to be handled and 2) automated movement of a glass substrate under the pipette tip to allow the volume of diluted blood to be spread over a defined area.
• a programmable precision pipettor has been interfaced to a computer, along with a X,Y, Z computer controlled stage that allows movement of a glass slide in the three orthogonal directions.
• the photograph (See Figure 1) shows the assembled unit. In the center is the microscope stage, with the pipette tip holder inserted in what is normally the optical path of a microscope. Behind the microscope is the programmable precision pipettor unit.
• a laptop computer Through the X, Y, and Z movements, and the "pipette" actions are controlled.
• a hand held dryer that is used (on low, cool) for the drying of the slide.
• a small camera On the left of the microscope stage is a small camera that is used to monitor the process and set the height of the pipette tip above the slide.
• the slide may first be "pre-treated” for example by cleaning using a corona discharge device. Pre-treating may involve other methods of changing the hydrophobicity of the slide or the "stickiness" of the slide.
• the blood sample is then transfered to the slide by the preparation system and dried.
• the blood sample may be diluted by a diluent before being transferred to the slide.
• the slide is stained.
• An external stainer may be used, but an automated analyzer product would include an integrated automated stainer.
• Software algorithms analyze digital images taken at specific wavelengths for the counting of red blood cells and white blood cells; for the white blood cell differential count and for counting platelets, and for detennining hemoglobin content and red cell morphology.
• the system When assembled as an automated device, the system will include a "specimen tube” , mixing device, a robotic mechanism to extract an aliquot of blood from the tube of blood , the hardware components described above, and a computer user interface and an interface with a standard Laboratory Information System. Such a device will be able to automatically perform the CBC and differential count from a blood sample. Ultimately, these components may be refined and assembled into a manufacturable instrument.
• the dried, stained monolayer of blood cells is analyzed using computer imaging to detennine red cell, white cell, and platelet counts and other measured and derived components of the CBC, and to perform the WBC differential count. Results of the counts, morphologic measurements, and images of RBCs and WBCs from the slide will be shown on a monitor.
• our system Unlike today's commercial systems, which only provide display of numerical data and cell population histograms or scatterplots, our system also features an additional capability of directly assessing the cellular morphology from the images of blood cells displayed on the monitor. This critical added visual capability allows the technologist to quickly establish the presence of abnormalities in cell morphology that may warrant preparing an additional slide for manual review by an experienced technician or other professional.
• the device performs the CBC and the white blood cell differential count.
• One embodiment of the device includes a robotic platform to move the glass slide through each of the several hardware stations described above.
• the stations include the preparation of the slide and droplet; staining of the slide; and brightfield imaging for the red and white blood cell counts, the white blood cell differential count, platelet counts, and additional red blood cell analyses.
• a robotic device to move the slide between the stations may be developed. Since timing is critical for the settling of cells and overall preservation of morphology, it is important that the robotic platform be efficient.
• a final commercial system will require a fully “pipelined” system that will allow slides from different patients to be processed simultaneously at the different stations, making it competitive or superior to the throughput rates of current commercial flow based CBC systems, which typically report out the first specimen out in 8 to 10 minutes, and then have a throughput rate of one specimen per minute.
• the system will aliquot blood from a patient's sample tube, dilute if necessary, and pipette an aliquot of the blood sample onto a substrate, probably in the form of a glass microscope slide.
• a typical aliquot would be approximately 1/2 ⁇ to 2 ⁇ , but may be in the range of 1/10 to 10 til.
• a single glass microscope slide (1 X 3 inches, 1mm thick) would be used for each specimen, although other substrates could be used.
• the standard microscope slide format would be a low-cost solution for a disposable substrate in a final commercial system.
• a slide from Erie Scientific (Portsmouth, NH) that contains painted (hydrophobic) rings is shown in Figure 2. The ring helps contain the blood sample and directs the hematologist's attention to the area containing cells.
• a likely final slide would contain a single ring, or other containing shape such as a square or rectangle.
• the device may use disposable pipette tips or a tip that would be thoroughly washed between specimens.
• the pipetting device must be capable of pipetting microliters or portions of microliters of fluids at better than 2% accuracy . There is a desire for an overall accuracy of less than 2% in the dilutions, since the final system should be able to count the blood components with an accuracy of better than 5%.
• After the pipetting operation is complete the slide is dried. This drying may be assisted by a controlled flow of air. Other means of transferring the blood to a thin layer on the slide may be used, but it is critical that all the sampled volume of blood be transferred to the substrate.
• Diluents for the whole blood may include salt solutions or protein solutions.
• Salt solutions range from “physiological saline” (0.9N), to complex mixtures of salts, to the commercial preparation Plasmalyte that simulates virtually all the salts found in human blood serum.
• Such preparations can differ in salt concentrations, buffers, pH, osmolarity, osmalality, buffering capacity, and additives of various types.
• Protein solutions can range from simple solutions of bovine alburnin to Plasmanate, a commercial preparation with selected human plasma proteins. Such preparations can vary in protein concentrations, buffers, pH, osmolarity, osmalality, buffering capacity, and additives of various types.
• Synthetic or "substitute” versions of these solutions may also be usable, including Ficoll or Dextran or other polysaccharides. Other substitutes may be used.
• An example of a preferred diluent is Plasmanate plus Plasmalyte in the proportion of 4:1.
• the second station is the staining platform.
• the dried slide is typically flooded by, or immersed in methanol (fixation), Wright-Giemsa stain and buffer solutions. Flooding of a slide that is laying flat could be performed. Since precision pipetting is not required for this step, low cost computer driven peristaltic pumps may be used to apply the reagents to the slide. The use of the hydrophobic ring on the slides makes the transfer of the reagents relatively straightforward. Each reagent would be removed by tilting the slide. In another embodiment, the slide could be immersed into baths of the fixation and staining reagents. In another embodiment, the inverted slide could be moved across a plate that floods the slide with reagents from below. Final drying of the slide would then be performed.
• a microscope or similar optical system complete with motorized XYZ movement and automated objective lens selection may be used. Appropriate lenses are needed and an lumination subsystem that will allow a user to acquire multispectral images.
• a large field (1600 x 1200 pixel) camera with high speed interface may be used.
• the system is driven by one or more computers, which will direct the slide movement, pipetting operations, microscope movement, and image analysis.
• the system may include general purpose computer(s) and/or microprocessor boards to control the stage and focus motors and other low level operations.
• each aspect of the method must be addressed including a preparation assembly to clean a slide if necessary, aspirate (and dilute if necessary) a blood sample, and transfer a droplet of blood to the slide.
• a preparation assembly to clean a slide if necessary, aspirate (and dilute if necessary) a blood sample, and transfer a droplet of blood to the slide.
• more or less dilution may be necessary.
• a dilution of 1 part blood and 3 parts diluent may be used, where the diluent is a physiologically compatible solution.
• the dilution process and the placing of the drop on the slide is to be automated.
• the slide may first be cleaned using a coronal discharge device (Electro-Technic Products, Sawicki PA) to produce a rnaximally hydrophilic surface. This is accomplished by toning on the discharge, and moving each slide "cell spot" in a spiral motion under the device for about 15 seconds.
• a coronal discharge device Electro-Technic Products, Sawicki PA
• a disposable cuvette may be used for diluting the sample that is aspirated from the patient's vial, or the dilution may be done in the device used to aspirate the sample from the vial.
• the computer controlled pipette takes 1 microliter of blood and places it into a cuvette containing 3 microliters of the diluent.
• the pipettor is used to mix the sample, and to withdraw 1 microliter of the diluted blood from the cuvette.
• the 1 microliter of blood is dispensed on the slide, while the slide is moved. This allows the small droplet to be spread over a relatively large area uniformly.
• the pipette tip may be moved over the stationary slide.
• the method places about 900,000 red blood cells, 45,000 platelets, and 1,000 white blood cells onto the slide.
• the slide may be placed into a bath of 85% methanol (by volume).
• the reagents may consist of a single solution Wright-Giemsa stain and a rinse buffer, or other well known hematological stains may be used.
• the slide is again dried, likely by forced air.
• the stained cells must be comparable to cells on a wedge smear stained on a conventional automated stainer.
• a manually prepared wedge smear stained using a laboratory stainer can be used as a control. Slides may be compared visually, and image analysis may be used to compare stain intensity, color (by comparing stain intensity at critical wavelengths), and cellular morphology.
• An automated staining device is used following the initial drying and fixation of the slide. The slide will then be ready to complete the brightfleld imaging.
• the imaging equipment may include a standard brightfleld microscope with automated stage and focus, or a functionally equivalent device.
• a microscope is attached to a Marzhauser (Germany) stage and a focus motor attachment (Prior Scientific, Rockland MA).
• the microscope has a motorized nosepiece, allowing different magnification lenses to be selected under computer control.
• a filter wheel (Prior Scientific, Rockland MA) allows one to automatically select narrow band color filters in the light path. LED illumination may be substituted for the filters.
• the use of switched LEDs will greatly reduce the image acquisition time as compared to the time required for filter wheel rotation.
• a 1600 x 1200 pixel firewire camera (Point Grey, Vancouver CA) may be used to acquire the narrow band images.
• the imaging software will use the brightfield images to determine total red cell and white cell counts.
• a single wavelength of light for example 570 nm
• two colors of iUuiniiiation may be used.
• extremely high contrast and high quality images of platelets can be obtained by subtracting a 430 nm image from a 570 nm image.
• the accuracy of the counting of the red blood cells, white blood cells, and platelets appears to be extremely high— over 98%.
• the overall accuracy for the counts will depend on how representative the slide preparation is of the sample withdrawn from the blood tube. An evaluation has shown that very few cells are lost during fixation and staining.
• the second purpose of the brightfield imaging is to acquire images for white blood cell differential counting, and for assessing platelets and the morphology of RBCs.
• white blood cells For accurate differential counting, at least 200 white blood cells are required.
• Typical computerized microscope systems using a motorized stage that is capable of scanning, tracking focus, and processing can analyze the 1600 x 1200 pixel images at a rate of better than 10 fields per second. Depending on the size of the cell deposition, there may be between 200 and 400 fields to analyze. The total analysis would take 20 - 40 seconds. At the end of this time, the computer will know the location of all white blood cell "candidates" on the cell spot.
• the computer will then return to 200 or more of those locations, refocus and digitize the white blood cells at a higher magnification (i.e. using a 40x or 60x lens), use image analysis to identify the white blood cell type, and store the images for later viewing by an operator. At least 4 narrow band colors of light may be needed to digitize the images to optimize both the analysis and the full color viewing by a hematology technician.
• the brightfield imaging red cell, white cell and platelet counting plus white blood cell location plus white blood cell differential analysis
• the timing of this imaging stage of the process is the current limiting factor for the throughput of the entire instrument. Since all of the other steps can be broken down into one minute intervals (including the individual components of the staining), a pipelined system can be achieved with a one minute per sample throughput (with "first in” to "first out” taking 8 to 10 minutes). This throughput time will be improved possibly by 1) further optimization, 2) breaking the steps down into two or three "pipelined” optical platforms or 3) using two or three parallel identical brightfleld systems witiiin the instrument.
• a goal for the WBC differential is to accurately perform a minimum 5-part differential. This is the most complex aspect of the software. Success of this software is heavily dependent on the quality of the preparation and the quality of the images. From previous work done it is known that an automated WBC differential based on image analysis is possible. More recent work has shown that image quality with dry objectives (in place of the oil objectives used in the 1980s) provides excellent quality for color cell analysis and viewing (Chromavision's ACIS), and differential counting and viewing (CellaVision's MICR021).
• the improved preparation technology described above lends itself very well to image analysis.
• cell distortion was a significant problem in achieving reproducible image analysis.
• Our method of gentle application of a monolayer of cells ensures no or nunimal distortion of the cells during the slide preparation in this system.
• the cells change morphology as they dry, but it has been shown that optimal drying conditions can be reproduced manually, and it is anticipated that even better control is available with fully automated preparation.
• the improved specimen preparation described above also eliminates the distribution artifacts that are known to occur with wedge smears prepared by any method, which introduces inaccuracy into the WBC differential count.
• At least 4 narrow-band images will be used for the image analysis, including a filter (430 nm) to acquire a hemoglobin-only image in order to assess RBC morphology and hemoglobin content, and then to digitally remove any red blood cells touching the white blood cells.
• a 570 nm filter has been shown to give excellent contrast for platelets and nuclei.
• Other wavelengths are chosen in order to best discrii-ninate the colors of the basophils, monocytes, lymphocytes (all shades of blue), eosmophils (red), and neutrophils (neutral color). These wavelengths will also be used for the display of the color images if appropriate. Otherwise one or two additional images may need to be taken for the 200+ cells that will be analyzed for the differential count and which may be displayed on the monitor.
• cytoplasmic and nuclear segmentation algorithms are applied to locate the nucleus and cytoplasm.
• the software extracts a standard collection of color, shape, size, texture and density features from the white blood cell nuclei and cytoplasms.
• CBC instruments provide an assessment of hemoglobin content and red blood cell morphology. It is well established that hemoglobin content can be determined by measuring the integrated optical density of individual red blood cells at 430 nm (Bacus, 1985). Red blood cell morphology has also been successfully detennined on previous automated differential instruments (Bacus 1980, 1982), and the methods are well known.
• Figure 4 is a graph showing the correlation between the automated counts and the manual counts for the red blood cells. Each field contains between 200 and 1500 red blood cells. An R-squared value of 0.993 was calculated from the regression model of the data, giving an extremely good Pearson correlation coefficient of 0.996.
• Figure 5 is a graph illustrating the correlation between the automated counts and the manual counts for the white blood cells. An R-squared value of 0.960 was calculated, giving a Pearson correlation coefficient of 0.980.
• the photograph shown in Figures 8 is a cell spot from a slide generated on an automated pipetting system.
• the fields at the edges of the square cell spot contain fewer cells as the pipette tip quickly turns to lay down the next row.
• some cell spots show a "blob" of cells where the pipette tip stopped and lifted from the surface of the slide.
• the software is still able to make an estimate of the red cell counts, although it appears that the computer counts may be slightly low in crowded areas.
• the methods can be refined to produce more even distributions of cells.
• Figure 9 shows the details of one digital image from the cell spot.
• the image analysis programs analyze each individual field and sum the total red and white cell counts. To calculate the total counts per microliter in the patient vial, the number counted on the slide is multiplied by the dilution factor.
• Table 1 below shows a data summary for 34 slides.
• "Sysmex” data represents red and white blood cell counts from a commercial "flow-based” automated CBC analyzer. Automated counts, and scaled counts are provided for both the red blood cells and the white blood cells. Red blood cell counts are typically given in “million cells per microliter,” so the number of located red cells was then divided by 1,000,000 to get the “scaled” red cell count. For white blood cells the counts are given in "thousand cells per microliter,” so the number was divided by 1,000 to get the "scaled” white cell count
• specimens include very high and very low red blood cell counts and white blood cell counts.
• Table 1 Table shows the raw data from counts performed on 34 vials.
• the 2 column gives the reference (Sysmex) RBC counts, while the 3 rd column reports the automated counts from the microscope slide.
• the 4 th column scales the counts to million cells per microliter, assuming a 1:4 dilution.
• the 5 th -7 th column show the data for the WBC counts.
• At the bottom of the table are the calculated correlation coefficients (R- squared).
• the graph of Figure 6 shows the correlation between the Sysmex counts and the automated slide based counts for the red blood cells.
• the raw counts varied between 682,449 and 1,241,765 white blood cells per shde.
• An R-squared value of 97.95% was calculated.
• the graph shown in Figure 7 shows the correlation between the Sysmex counts and the automated slide counts for the white blood cells.
• the raw counts varied between 147 and 11,250 white blood cells per slide.
• An R-squared value of 99.70% was calculated.
• the data was obtained from 34 patient samples during two sessions of preparing slides.
• the data is representative of typical patients, although the tubes were selected from patients with a wide distribution of red and white cell counts. Most, if not all of the 34 samples, were obtained from specimens "archived” during the day in the refrigerator, and then pulled and prepared on our instrument in the late afternoon. Once the tubes were pulled, they were processed consecutively.
• a slide can be prepared, stained, and counted semi-automatically.
• the cells are morphologically well preserved and the counts on the slide correlate with those obtained on the state-of-the-art Sysmex CBC instrument.
• the methods described below provide the complete blood count , hemoglobin content, red blood cell morphology, and WBC differential count that reflect the true numbers of those cells and their morphology from direct observation of them in the blood specimen.
• this invention is distinguished from existing CBC instruments and methods that make measurements of electrical impedence, absorption, and scattering of light and other measures that are attributable to blood cells but not from direct observation of them.

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