Apparatus and method for detecting rh-positive cells

Abstract

Detecting fetal maternal hemorrhage (FMH), measuring a total number of red cells in a maternal plus fetal blood samples, separating Rh+ cells from Rh- cells, and / or treating a fetal maternal hemorrhage through methods that involve labeling of red blood cells and assessment of the labeled cells in a gel card or a separation column operably connected to a detection apparatus that is configured to assess values of cells labeled in the various configurations described.

Description

[0001] APPARATUS AND METHOD FOR DETECTING RH-POSITIVE CELLS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to US Provisional Patent Application Nos. 63 / 619,275, filed January 9, 2024, and 63 / 660,627, filed June 17, 2024, the disclosures of which are hereby incorporated by reference in their entireties.

[0004] BACKGROUND OF THE INVENTION

[0005] FIELD OF THE INVENTION

[0006] The present disclosure relates to an apparatus and method for detection of Rh- positive cells, for example, in the context of assessment of fetal maternal hemorrhage. DISCUSSION OF THE BACKGROUND

[0007] Rh blood group system is one of the most clinically significant blood group systems in humans. It is important to separate Rh+ cells from Rh- cells for various applications, such as blood transfusions, diagnostics, and research. Current methods for separating Rh+ cells from Rh- cells involve complex procedures and may lack efficiency.

[0008] Fetal maternal hemorrhages (FMH) are the transplacental hemorrhage of fetal blood into the maternal circulation. Detection of FMH involves the detection of Rh- positive fetal blood in the circulation of an Rh-negative blood of the mother. In 2022, an estimated 550,000 Rh-negative women gave birth. Roughly 85% of the babies bom to an Rh-negative mother will be Rh-positive, thus requiring about 467,500 women be tested for FMH annually.

[0009] U.S. 2020 / 0408749 that generally describes that problems with detection of antigens that require antibody binding of false positives, sensitivity issues and others that are addressed by combining features in a diagnostic device using beads, hydrophilized reaction area(s) having specified properties and parameters.

[0010] The foregoing Background description is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present invention. SUMMARY OF THE ETVENTION

[0011] The foregoing paragraphs have been provided by way of general introduction and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings.

[0012] In one embodiment, the present disclosure is related to a method of assessing maternal blood for fetal cells that are Rh-positive and an apparatus sensitive enough and capable of recording and assessing the results of the method. For example, a method for separating Rh+ cells from Rh- cells, and more specifically, to a method utilizing a biotinylated antibody, a fluorescent label, and / or streptavidin-coated beads, with centrifugation in a gel card for efficient separation and retention of Rh+ cells.

[0013] The methods described herein can detect and quantitate Rh D-positive cells in the range of 0.1 %-l .6% and is suitable for the detection of and clinical management of FMH.

[0014] In another embodiment, the present disclosure is related to a method of detecting malaria in blood cells that have the parasite antigen present on the surface. In yet another embodiment, the present disclosure is related to a method of assessing blood samples for other parasites, diseases, or conditions.

[0015] In another embodiment, a method for separating Rh+ cells from Rh- cells. The method involves introducing a biotinylated antibody specifically onto the Rh-l- cells, followed by the introduction of a fluorescent label via labeled anti-human IgG. The sample is then incubated with streptavidin on either a solid support, such as Sepharose or Polystyrene bead(s). The sample is subsequently introduced to a gel card and / or column, centrifuged and provided to a gel card reader to assess the results whereby the Rh- cells move to the bottom of the gel card and / or column, while the Rh-l- cells with the associated label are trapped at the top of the gel card and / or column by the size of the bead. This methodology can provide a simple, efficient, and reliable way to separate Rh+ cells from Rh- cells, allowing for improved blood transfusions, diagnostics, and research applications such as the detection of fetal maternal hemorrhage.

[0016] In another embodiment, a method for detecting Rh-positive fetal red cells in a maternal blood sample, the method comprising: measuring a sample of maternal blood, for example, pre or post-delivery for the presence of Rh-positive fetal blood cells. In one aspect, the blood samples are labeled with fluorescent dyes and the fetal cells are agglutinated, if present, for measurement in a detector for fluorescence.

[0017] For illustration, the method can include obtaining a sample of maternal blood after delivery, washing the red blood cells in the sample into phosphate buffered saline (PBS), removing white blood cells (WBC) from the sample, incubating the sample with fluorescent labels, washing the cells with PBS three times, suspending the sample in PBS, separating the cells into three equal aliquots to be treated separately to determine the number of total cells in the sample and the number of fetal cells in the sample.

[0018] As another illustration, the method of determining the number of total cells in an aliquot obtained from a maternal blood sample comprises: incubating one aliquot derived from the maternal blood sample with BRIC 200 or other pan red blood cell antigens, such as rabbit anti-RBC, monoclonal antibody, placing the aliquot in a microfuge tube, adding PBS to the tube to suspend the cells, centrifuging the tube, removing the supernatant, suspending the remaining cells in PBS, adding Rabbit antimouse IgG to the microfuge tube, suspending the cells in the antibody with a pipette, incubating the suspended cells for a second time, loading the cell suspension into a lane of a gel card, centrifuging the gel card in a specialized gel card centrifuge, inserting the card into the laser gel card reader, obtaining a voltage that corresponds to a quantity of total red blood cells in the sample, and recording said voltage. In another embodiment, the method of determining the number of total cells in an aliquot obtained from a maternal blood sample may be carried out by another separation column rather than a gel card.

[0019] In one embodiment, a method of determining the number of total fetal cells in an aliquot obtained from a maternal blood sample is provided, the method comprising: incubating the aliquot with anti-Rh antibody, such as human IgM or the like, loading the cell suspension in a lane of a gel card, centrifuging the loaded gel card into a specialized gel card centrifuge, obtaining a voltage that corresponds to a number of fetal cells in the sample, and recording said voltage. In another embodiment, the method may be carried out by utilizing a separation column rather than a gel card.

[0020] In one embodiment, an apparatus for detecting fetal maternal hemorrhage based on a maternal blood sample obtained after delivery, the apparatus having components necessary to detect with the appropriate sensitivity the presence or absence of Rh- positive fetal cells as described herein. For illustration of such an apparatus, in one embodiment, comprises: a power source, a controller hardware and software to perform the supervisory control functions of the apparatus, an illumination source that produces the desired wavelength, a sample holder to stabilize the sample card for illumination and detection, an optics component which collects and filters the emission from the target (sample cell in the sample holder), a detection electronic comprised of a sufficiently sensitive optical detector, a software component residing in the controller HW / SW, a display to provide users with the sample results, and an enclosure to provide structure to the apparatus.

[0021] The method(s) described herein above and below embodied in this invention disclosure can be automated, in whole or in part. For instance, the supply of antibody or antibodies to the blood sample(s) can be provided in an automated system. Further, the loading of labeled cells in the sample can be automated and / or the processing of the samples in the gel card. In addition, the detection and optionally report generation can also be automated.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 are views of the card reader apparatus, according to embodiments of the present disclosure and how a gel card is inserted into one embodiment of the apparatus.

[0024] FIG. 2 is a table displaying the current parts list for a card reader apparatus, according to one embodiment of the present disclosure.

[0025] FIG. 3 is a block diagram of a FMH detection model, according to one embodiment of the present disclosure.

[0026] FIG. 4 is a view of a fetal blood sample loaded into a lane of the cell card, according to one embodiment of the present disclosure.

[0027] FIG. 5 is a graph showing an example of the quantitation of fluorescently labeled fetal cells, according to one embodiment of the present disclosure.

[0028] FIG. 6 is a block diagram of a compact fluorescent illumination and detection system, according to one embodiment of the present disclosure.

[0029] FIG. 7 is a depiction of a device for obtaining results showing an exemplary result on the screen, according to one embodiment of the present disclosure.

[0030] FIG. 8 shows results of the quantitation of Rh D-positive Cells in Contrived Mixtures, raw fluorescent data. The samples contain the indicated number of Rh+ cells in 15 million total RBCs. FIG. 9 shows a-Rh D-positive Standard Curve, according to one embodiment of the present disclosure. The samples contain the indicated number of Rh+ cells in 15 million total RBCs.

[0031] FIG. 10 shows a total Cells Standard Curve. The samples contain the indicated number of total cells.

[0032] FIG. 11 shows a measured Rh D-positive Cells vs Input Rh D-positive Cells. The X axis reflects the percentage of Rh+ cells in the contrived sample and the Y-axis reflects the percentage of Rh+ cells determined by the assay and reader.

[0033] FIG. 12 shows an illustration of one embodiment of the present disclosure in an example of Gel Card containing 3 samples and controls. Lane 1 -Sample 1 total cells; Lane 2-Sample 1 Rh-i- cells; Lane 3-Sample 2 total cells; Lane 4-Sample 2 Rh-i- cells; Lane 5-Sample 3 total cells; Lane 6-Sample 3 Rh+ cells; Lane 7-Negative control; and Lane 8-Positive control.

[0034] FIG. 13 depicts gel cards containing samples of 0-2.4% Rh D+ cells in 15 million Rh D- calls.

[0035] FIG. 14 shows a graph of the absorbance in green and blue channels less the blank plotted vs the % Rh D-positive cells in the sample. This signal is correlated with the number of Rh D+ cells or the percentage of Rh D+ cells in the sample. Each sample contains 15 million total RBC.

[0036] FIG. 15 shows a gel card standard curve with 0 to 28 million total cells.

[0037] FIG. 16 shows a graph illustrating that since the number of total cells in a clinical sample will be unknown by quantitating the number of Rh D+ fetal cells and dividing by the number of total cells in the sample the percent Rh D+ cells in the total sample can be determined.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The terms “a” or “an”, as used herein, are defined as one or more than one. The term “plurality”, as used herein, is defined as two or more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and / or “having”, as used herein, are defined as comprising (i.e., open language). Reference throughout this document to "one embodiment", “certain embodiments”, "an embodiment", “an implementation”, “an example” or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases or in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the features, structures, or characteristics may be combined in any suitable manner in one or more embodiments without limitation.

[0040] A maternal blood sample may be tested to detect the presence of fetal red cells; however, the sample may only contain a small number of fetal red cells. A sample may contain about 0.1 to 2.0% fetal red cells. Since detecting such a small amount of fetal red cells is a challenging, an efficient labeling of the red cells and a sensitive detection is needed. An alternative method of detecting fetal cells for FMH testing is flow cytometry which uses fluorescence.

[0041] The use of gel for the present disclosure provides substantial benefits because the gel cards require no washing of cards, easier reading, and easier to use. While gel cards may be more expensive per test, labor savings offset the higher expense. The type of gel card that may be used in any embodiment is not particularly limited. For example, a neutral gel card may be used, which may contain a resin and buffer. Another example may be a preloaded gel card which contains an antibody to a specific blood group antigen. Yet another example is a secondary gel card which may contain Rabbit anti-human IgG. Secondary cards are often used to detect RBCs with antibodies bound to them but can also be used to determine blood type in association with an IgG class antibody to a specific antigen. Unlike cards preloaded with antigen specific IgMs or loaded with RBC and antigen specific IgMs, IgG secondary cards give a broad band or smear on the gel card which is not helpful for fluorescence detection. In yet another embodiment, a separation column may be used in place of a gel card.

[0042] For some current methods, there may be issues with the reagents or assays used causing a test to show the presence of red blood cells that are both positive and negative for an antigen. This phenomenon is called a mixed field and is an undesirable outcome of a fetal maternal hemorrhage test that may be avoid with the present disclosure. A mixed field may also occur in a sample taken from a patient who just received a transfusion. The present disclosure allows using the gel card to separate and quantitate the small population of Rh-positive red blood cells. One unit of transfused RBCs is about 10-15% of the normal number of cells and they last for 60-120 days so this history of transfusion may be evident for a while in a patient’s sample. Another less common scenario in which a mixed field will be an issue is if the person is chimeric in the bone marrow or has had a bone marrow transplant. Current detection methods are expensive and require increased training and time to perform. Therefore, a minimally hands-on detection method that can be performed with minimal operator training and performed in a short period of time, at a low cost, is needed. Further, a detection method which does not require microscopy or flow cytometry, as common detections methods currently require, may help improve speed and cost of FMH detection.

[0043] The systems and methods described in the present disclosure can be especially useful when a small maternal blood sample is obtained, and a quick method is needed to detect FMH that does not utilize flow cytometry or microscopy. In one embodiment, the method can provide a detection and diagnosis of FMH by non-specifically, fluorescently labeling red blood cells in a maternal blood sample. This maternal blood sample may contain a small number of fetal red cells, where one embodiment of the present disclosure may detect up to 0.4%, or alternatively around 20,000 fetal cells, in a sample.

[0044] In one embodiment, the method of FMH detection has a wide dynamic range allowing for the quantitation of about 0.4% to about 10% fetal cells. Fetal cells are then captured by an anti-Rh antibody which may be biotinylated or attached to a particle or bead and separated on a gel card, which is a small gel filtration column unique to the blood banking industry. After a brief centrifugation non-agglutinated cells move to the bottom of the microcolumn, and antigen positive cells form a band at the top of the column. If a mixture of maternal Rh-negative and fetal Rh-positive cells are applied to a gel card with anti-Rh antiserum, the Rh-negative maternal cells are unaffected by the anti-Rh antiserum and move to the bottom of the gel card. The fetal Rh-positive cells form band at the top of the gel card which may be invisible to the naked eye. In a separate microcolumn, the total red cells in the sample can be estimated by agglutinating all the cells (maternal and fetal) with a pan-red cell antibody called BRIC 200. In yet another embodiment, a separation column may be used in place of a gel card.

[0045] A single gel card microcolumn can accommodate a sample containing about 5 to 15 million red blood cells. In one embodiment, the methods of FMH detection, can include obtaining a maternal blood sample after delivery, detecting the number of fetal red cells in the sample, and using a detected number of fetal red cells present in the sample to determine the dose of Rh immune globulin needed for treatment of FMH. A single standard 300 pg dose of Rh immune globulin is sufficient for a bleed of 30 rnL fetal blood. The sensitivity of this test, being able to quantify bleeds of 0 to 0.1% (5 mL fetal blood) opens the possibility that some women could be adequately treated with a mini-dose of Rh Immune Globulin (50 pg) rather than the standard dose. This would conserve Rh Immune Globulin which is in short supply worldwide. Once injected into the mother the Rh immune globulin apparently binds to the fetal red cells and masks the Rh antigen from the mother’s immune system preventing isoimmunization. Larger bleeds require additional doses of Rh immune globulin; thus, detection of larger bleeds is an important function of the present disclosure. In yet another embodiment, a separation column may be used in place of a gel card.

[0046] In one preferred embodiment of the described methods a biotinylated antibody specifically targeting Rh+ cells is introduced into a sample containing Rh+ and Rh- cells. The introduction of the biotinylated antibody can be performed by incubating the sample with the antibody. In one aspect, the sample is mixture of washed, in a suitable buffer, red cells (RBC) containing Rh D-negative cells and may also contain Rh D- positive cells and the biotinylated antibody can be BRAD 3, a human anti-Rh D monoclonal, subclass IgG3. Other anti-Rh D monoclonals or polyclonal antibodies would be expected to behave in a similar manner.

[0047] The BRAD 3 can be conjugated with Biotin-PEG12-NHS ester, Biotin-PEG24- NHS ester and / or Biotin-PEG106-NHS ester as well as biotin modification by photoaffinity labeling. The Biotin-PEG12-BRAD 3 binds to Rh D-positive RBC. No other cells express Rh-D antigen. Unbound Biotin-PEG12-BRAD 3 can be washed away in the cell washer leaving only the Biotin-PEG12-BRAD 3 bound to the Rh D antigen. About 15,000 Rh D antigen molecules are present on an adult RBC heterozygous for Rh D.

[0048] A fluorescent label is introduced into the Rh+ cells. This can be achieved by incubating the sample with a fluorescently labeled anti-human IgG. The fluorescent label can be used for the visualization and identification of the Rh-i- cells during the separation process.

[0049] For example, Alexa Fluor 647-Rabbit anti-human IgG (H+L) can be used as the fluorescently labeled anti-human IgG. The Alexa Fluor 647-Rabbit anti-human IgG (H+L) binds to the Biotin-PEG12-BRAD 3 bound to the Rh D-positive cells. Excess Alexa Fluor 647-Rabbit anti-human IgG (H+L) can be washed away in the cell washer leaving the Rh D-positive cells with bound Biotin-PEG12-BRAD 3 and Alexa Fluor 647-Rabbit anti-human IgG (H+L). In another example, a monoclonal Alexa Fluor 647 Labeled Mouse Anti-Human IgG3 Hinge can be used as the fluorescently labeled antibody and may be more specific for the Biotin-PEG12-BRAD 3 since only about 10% of human IgG is IgG3. As would be apparent to the skilled person, other fluorescent labels can be used as an alternative to Alexa Fluor 647.

[0050] In one embodiment, the fluorescent label and the biotin can be incorporated into the same antibody molecule allowing a single step assay.

[0051] Biotin incorporation (biotinylation) can be accomplished any number of ways as is known in the art. In one particular embodiment, biotinylation can be accomplished as described in U.S. PG PUB 2022 / 0214337, incorporated herein by reference. Still further embodiments can utilize the methodology described in Hui et al (2015) Bioconjug Chem 26(5): 1456-1460: LASIC: Light Activated Site-Specific Conjugation of Native IgGs.

[0052] The sample is incubated with streptavidin-coated beads to allow for the specific binding of the biotinylated antibody present on the Rh+ cells. For example, the streptavidin can be coated onto either a Sepharose bead with a size range of 60-200 gm or a Polystyrene bead with a size range of 14.0-17.9pm.

[0053] The sample is applied to a gel card that comprises a column designed for the separation of cells based on their size and the specific binding between streptavidin and the biotinylated antibody. The gel card can be a commercially available product or custom-designed.

[0054] In one embodiment, neutral Gel Cards can be modified by the addition of Streptavidin- Agarose (60-120 pm particles) to one or more lanes, followed by centrifugation.

[0055] Other non-limiting examples, include Streptavidin-polystyrene, Streptavidin- Ultralink, avidin, neutravidin, Captavidin, antibodies to biotin.

[0056] The particles, e.g., Streptavidin particles, are most preferably large enough to capture the bound RBC (7 pm) so that they are retained at the top of the gel card and / or gel column. For example, polystyrene particles of in the range of greater than 10 pm are preferably used, such as 15, 40 or 105 pm. Particles less than about 10 pm are not retained by the gel and move to the bottom of the gel card and thus are not preferred.

[0057] One advantage of Streptavidin- Agarose is it captures and retains small numbers of Rh D-positive biotin labeled RBC without interfering with the passage of the much larger number (98-99.9%) of Rh D-negative cells into a through the gel card and / or gel column matrix to the bottom out of the range of the detector. For example, in a typical gel card where 100% of the cells express the antigen of interest a network of agglutinated cells is formed on the top of the column matrix indicating a positive reaction. There is no need for cells to pass through this band of agglutinated cells. When seeking to detect cells in the range of 0.1 -2.4% agglutination at the top of the gel card and / or gel column can block, in whole or in part, passage of the antigen negative cells clouding any results that can be read from the assay, particularly where the amount of Rh D-positive cells is low, e.g., in the range of 15,000-360,000 cells. With low quantities of Rh D-positive cells forming a network to be retained at the top of the gel card and / or gel column becomes more difficult but the use of Streptavidin modified beads and / or other beads as described herein that are too large to enter the gel matrix, such as in the range of 15-200 pm, permits single cell capture and retention and thus providing a useful result for detection in the assay.

[0058] In one embodiment, the gel card can be subjected to centrifugation causing the Rh- cells, which are not bound to the biotinylated antibody and fluorescent label, to move to the bottom of the gel column while the Rh+ cells with the associated fluorescent label are trapped at the top of the gel card due to the size of the streptavidin- coated bead.

[0059] After centrifugation, the separated Rh+ cells can be analyzed for further downstream applications.

[0060] In one embodiment, the detection and diagnosis of FMH, described herein can be implemented by an apparatus, such as the apparatus illustrated in FIG. 1 and FIG 7. The apparatus comprises: a power component, a controller hardware and software, an illumination source, a sample holder, an optics component, a detection electronics component, a computer algorithm, a display, and an enclosure. A parts list of the FIG. 1 embodiment is provided in FIG. 2. The apparatus is equipped with an array of optical sensors set at multiple gains to provide the apparatus with extremely high dynamic range. With proper calibration, the apparatus will output both raw sensor counts as well as the percentage of fetal cells automatically. In one embodiment, the apparatus utilizes solid state sources and detectors to be free from magnetic and microphonic problems associated with photo multiplier tubes (PMTs) found in other compact fluorescent instruments. The step-by-step method by which one embodiment of the apparatus detects the number of fetal cells within a maternal blood sample is illustrated by FIG, 3. Another embodiment of the fluorescent illumination and detection system is shown in FIG. 6. The system includes subsystem components that include power (blk 1), a microprocessor subsystem (blk 2) containing controller hardware and software (blk 2a) and computer algorithm (blk 2b), thumb wheel input (circular blk 3), bar code reader (blk 4), translational stage (blk 5), sample holder (blk 6), calibration reference (blk 7), translational stage (blk 8), illumination source (blk 9), conditioning optics / filters (blk 10), detection electronics (blk 11), thermal printer (blk 12), memory card (blk 13), display (blk 14), and enclosure (blk 15). The system illuminates labeled samples which fluoresce.

[0061] The system can be equipped with an optical sensor with multiple gains providing the instrument with extremely high dynamic range. With proper calibration, the system will output both raw sensor counts as well as the percentage of fetal cells automatically. Utilizing solid state sources and detectors, the system is mechanically robust and free from magnetic and microphonic problems associated with photo multiplier tubes (PMTs) found in other compact fluorescent instruments, but PMTs could be used if further sensitivity is required.

[0062] The power subsystem component preferably includes an on / off switch, connection to a power source via cable, battery, induction, or other connection types. In one embodiment, the power subsystem connects to the power source through an AC / DC power module but could run from DC or a battery or others) connected to standard U.S. wall power (nominal 60Hz, 1 10 / 120 VAC) with provisions for running at 50 / 60Hz and 100-260 VAC for worldwide coverage. The power module creates the internal voltages needed to power the translational stages, the illumination source, the detector and detection electronics as well as power for the microcontroller, and all input and output subsystem components (thumb wheel, barcode scanner, printer, memory, etc. In one embodiment, both positive and negative rails are provided for the high gain circuits associated with the photodiode contained in detection electronics (blk 11). If PMTs (photomultiplier tubes) or APD (avalanche photodiodes) are used, this would also provide the high voltage needed to power (drive) them accordingly.

[0063] The microprocessor subsystem component (blk 2) can contain a controller HW / SW (blk 2a) and a computer algorithm software (blk2b). Controller HW / SW (blk 2a) component performs all supervisory control functions for the system. It accepts inputs from thumb wheel (blk 3) and controls all reader functions required for the FMH test to proceed, this includes activating the bar code reader (blk 4), controlling the two translational stages (blks 5 and 8), sampling the calibration reference source (circular blk 7), controlling the illumination source (blk 9), interfacing with the detection electronics (blk 11) and outputting data to the printer (blk 12), removable memory (blk 13), and display (blk 14). Alternative embodiments would, for example, include monitoring of the illumination sources, ensuring stability. Additionally, the illumination sources could be modulated to reduce noise (unwanted light) to the detection electronics (blk 11) as well as reject ambient light that could leak into the system. Future embodiments include storage of sample identification (in this embodiment gel card number or other distinguishing nomenclature is read via the bar code scanner) and associated results for download via wire, wireless, or direct connection via media (i.e., SIM Card or other, the current embodiment uses a SD Memory Card) is envisioned. Additionally, operator identification can be envisioned which associates a specific sample and analysis with an operator for chain of custody data.

[0064] The illumination source subsystem component is activated via the system controller (blk 2). The illumination source provides precise wavelength to excite fluorophores which then emit energy some of which in the form of light wavelengths. In one embodiment, illumination is done with laser diode, but other embodiments could use LEDs, incandescent sources, traditional lasers, or any source that can produce the desired wavelength which is either spectrally pure enough to be used directly, or that is optically filtered to remove undesired wavelengths. The illumination light is then focused onto the target in the sample holder. In one preferred embodiment, the illumination and emission geometry are at hard angles (e.g., 90 degrees) to each other to minimize unwanted light from entering the detector (blk 6), but by careful design, any angle can be accommodated.

[0065] The sample holder subsystem component is a mechanical structure that acts as a stabilizing component for the optics (blk 5) and detection electronics (blk 6) and receives (holds) the sample for illumination and detection. In one embodiment the sample holder subsystem receives gel cards (containing eight (8) sample cells) in a precise and reproducible manner and contains optically clear apertures to permit both the illumination and emission wavelengths to propagate through the system. As mentioned, in one embodiment receives a gel card containing 8 sample cells, gel cards with different amounts of sample cells, or other sample cell geometries could be accommodated in future embodiments. Additionally, an alternative embodiment of the sample holder would contain light blocking apparatus(s) between adjacent sample cells to minimize light leakage. While one embodiment uses 8 sample cells, fewer or greater number of stages could be used to provide cross compatibility, to permit multiple samples to be run at once, or a wider array of samples to be run.

[0066] The optics subsystem component collects and filters the emission from the target (sample cell in the sample holder) and passes it and / or projects it onto the detector. Spectral filtering removes any of the illumination (excitation) light that scatters through the system. Failure to control this unwanted light adversely effects the dynamic range and the ultimate sensitivity of the system. Spatial filtering is also performed to ensure the light being analyzed is only coming from the area (sample cell target) to which focus is required / desired, rejecting other areas to improve contrast / sensitivity. In one embodiment the optics subsystem filters the light using both colored glass filters as well as interference filters. Alternative embodiments could employ filter mechanisms (interference, absorption, metamaterial, etc.) to reject unwanted light. A calibration source can be built into the system to both help in the initial mechanical alignment and to monitor the brightness of the illumination source to provide internal quality control as well as to provide a reference point for the detection algorithm. In one embodiment, a synthetic ruby is used as it is mechanically robust, provides a known fluorescence in the desired bands and being mineral based, rather than an organic dye, is less prone to photodegrading over time and use.

[0067] The detection electronics subsystem component can include photodiodes with amplifiers, in the current embodiment the amplifiers are built into the photodiodes to provide maximum gain and low electrical noise. Any sufficiently sensitive optical detector could be used, such as a PD (photodiode(s)), APD(s) (avalanche photodiodes), PMTs (Photomultiplier tubes), as well as various vacuum tubes, solid state PMTs, and similar devices. The optical signal is converted to an electrical signal, then amplified and subsequently sampled by an analog-to-digital converter. Another embodiment could use all analog electronics to achieve the same effect, however, working in the digital domain offers ease of manipulation and calibration of the data.

[0068] The computer algorithm subsystem component (blk 2b) is preferably a software in construction and in the one embodiment resides in the microprocessor (blk2). Other embodiments where the computer algorithm is located separately (e.g., in a desktop computer, smart phone, cloud service, etc.) is also embodied within the systems and methods described in the present disclosure. In the one embodiment the computer algorithm receives signals from the detection electronics as well as cues (signals) from the controller (blk 2) along with calibration curves (also from the controller (blk 2) to reduce the signal to a usable form. In a one embodiment, the total number of red cells is measured, along with the signal associated with the number of fetal cells. Utilizing these sampled signals (digitized total red cells and fetal cells) the computer algorithm then calculates the precise percentage of fetal to adult cells present in the sample cell being analyzed and reports this information on the display (blk 8). In one embodiment this subsystem analyzes 4 channels: Target sample, a controlled blank, and two lanes for calculating total number of cells present. By computing the total cells present, one can make a direct calculation of the percentage of cells, reducing errors in the system. Two channels for the total cells are provided in order to provide a wide dynamic range while keeping the response linear. More or less lanes could be provided as needed. As the system spatially scans the samples, the detection algorithm takes this spatial data and aligns the peaks in all the lanes. The area of all of the lanes are computed after careful subtracting the background. This data is then passed to the computer algorithm which normalizes the data for brightness (e.g., for both absolute brightness based on the calibration source as well as the relative brightness for the different sample types) and calculates the relevant percentage of target cells.

[0069] The display subsystem component provides users with the sample (test / run / analysis) results (output) and in one embodiment utilizes a liquid crystal display (LCD). This subsystem component can also provide basic status indications (power on, test status, etc.) of the instrument as well as a clear and unambiguous reading to the operator. By having all samples (reference, blanks, control samples, raw samples, treated samples, etc.), processed in an automated way, user error can be virtually eliminated and provide accurate and dependable data.

[0070] The enclosure subsystem component can be included and is mechanical in nature, to provide overall structure to the system. The enclosure provides openings for the power (blk 1) attachment and power on / off switch, slot for insertion and removal of the sample holder (blk 4), opening for display (blk 8) mounting, and a test / run / analysis push button switch for executing an analysis. The package can also contain logos and nomenclature to allow users to properly operate the system.

[0071] Future embodiments are envisioned which include sample tracking via bar code scanning, imaging camera, rf id, etc. In one embodiment, the apparatus comprises a power component. The power component consists of an on / off switch, connection to a power source via cable. In another embodiment, the power source may be provided by battery, induction, or any other suitable connection type. The apparatus connects to the power source through an AC / DC power module connected to standard United States wall power (nominal 60Hz, 110 / 120 VAC). The power component creates the internal voltages needed to run the illumination source(s), the detector(s) and the detection electronics component, as well as power the microcontroller and display components. In one embodiment, both positive and negative rails are provided for the high gain circuits associated with photodiodes contained in detection electronics. In yet another embodiment, photomultiplier tubes (PMTs) or avalanche photodiodes (APDs) may be used to provide the high voltage needed to power the circuits of the photodiodes.

[0072] The apparatus may further comprise a controller hardware with software to perform all supervisory control functions for the system, such as controlling the illumination source(s) and interfacing with the detection electronics component. In another embodiment, the controller hardware with software may further provide monitoring of the illumination sources, ensuring stability, and provide a reference point for calibration. In yet another embodiment, the controller hardware with software could modulate the illumination sources to reduce noise (unwanted light) to the detection electronics component, as well as reject ambient light that could leak into the system. In yet another embodiment, the controller hardware with software may provide storage of sample identification, such as by gel card reader number or other identification nomenclature. In another embodiment, the controller hardware with software may further provide sample results for download via wire, wireless, or direct connection via media (i.e., SIM Card or other). In yet another embodiment, the controller hardware with software may provide operator identification to associate a specific sample and analysis with an operator for chain of custody data.

[0073] The apparatus may further comprise an illumination source, which is activated via the apparatus controller hardware with software. The illumination source provides precise wavelength(s) to excite fluorophores which then emit energy. Some of the emitted energy may be in the form of light wavelengths. In one embodiment, illumination is performed by laser diodes. In yet another embodiment, illumination could be performed by LEDs, incandescent sources, traditional lasers, or any source that can produce the desired wavelength which is either spectrally pure enough to be used directly, or that is optically filtered to remove undesired wavelengths. The illumination source is focused onto the sample holder. In one embodiment, the illumination and emission geometry are at hard angles to each other to minimize unwanted light from entering the detector. In one embodiment, the hard angle is preferably 90 degrees. In yet another embodiment, the angle may be altered to any value by careful design.

[0074] The apparatus may further comprise a sample holder which is a mechanical structure that acts as a stabilizing component for the optics and detection electronics components while also holding the sample for illumination and detection. In one embodiment, the sample holder receives gel cards and contains optically clear apertures to permit both the illumination and emission wavelengths to propagate through the system. In one embodiment, the gel card held by the sample holder contains 8 sample cells. In yet another embodiment, the gel card may contain a different number of sample cells possessing various geometries. In yet another embodiment, the sample holder contains a light blocking apparatus(s) between the adjacent sample cells on the gel card to minimize light leakage. In yet another embodiment, a separation column may be used in place of a gel card.

[0075] The apparatus may further comprise an optics component which collects and filters the emission from a sample cell in the sample holder and projects it onto the detector. In one embodiment, the optics component performs spectral filtering to remove any of the illumination (excitation) light that scatters through the apparatus. Failure to control this unwanted light may adversely affect the dynamic range and the ultimate sensitivity of the system. Spatial filtering may also be performed to ensure the light being analyzed is only coming from the targeted sample cell, rejecting other areas to improve contrast / sensitivity. In one embodiment, the optics component performs spatial filtering by using both colored glass filters as well as interference filters to filter the light. In another embodiment, other filter mechanisms such as interference, absorption, or metamaterial may be used to reject unwanted light.

[0076] The apparatus may further comprise a detection electronics component consisting of a series of photodiodes with amplifiers. In one embodiment, the amplifiers are built into the photodiodes to provide maximum gain and low electrical noise. The gain settings on the two photodiodes are set differently by roughly 100-fold so the voltage signals from the two channels are similar, even when the sample contains 0.1 to 2.0% fetal cells. In another embodiment, any sufficiently sensitive optical detector such as a photodiode(s) (PD), avalanche photodiodes (APDs), photomultiplier tubes (PMTs), various vacuum tubes, solid state PMTs, or similar devices may be used. The optical signal is converted to an electrical signal, then amplified and subsequently sampled by an analog-to-digital converter. In another embodiment, all analog electronics may be used to achieve the same effect.

[0077] The apparatus may further comprise a computer algorithm residing in the controller otherwise operably linked to the controller. In one embodiment, the computer algorithm may be located separately from the controller such as in a desktop computer, smart phone, or cloud service. The computer algorithm receives signals from the detection electronics component, signals from the controller, and calibration curves, also from the controller, to reduce the signal to a usable form. In one embodiment, the total number of red blood cells is measured, along with the signal associated with the number of fetal cells base on the fluorescence labeling of the red blood cells from the maternal blood sample. The computer algorithm then uses the sampled signals (digitized total red cells and fetal cells) to calculate the precise percentage of fetal to adult cells present in the sample being analyzed and reports this information on the apparatus display.

[0078] The apparatus may further comprise a display, which provides users with readings of the sample results. In one embodiment, the display utilizes a liquid crystal display (LCD) to provide said sample results. The display also can provide basic status indications (power on, test status, etc.) of the apparatus. By having all samples (reference, blanks, control samples, raw samples, treated samples, etc.), processed in an automated way, user error can be nearly eliminated to provide accurate and dependable data to the operator via the display.

[0079] The apparatus may further comprise an enclosure which is purely mechanical in nature, to provide an overall structure to the apparatus. The enclosure can provide openings for the power attachment and power on / off switch, slot for insertion and removal of the sample holder, opening for display mounting, and a push button switch for executing an analysis. The enclosure may also contain logos and nomenclature to allow operators to properly use the apparatus. In one embodiment, the enclosure may further provide openings for sample tracking via bar code scanning, imaging camera, or rf id.

[0080] FIG. 4 is a view of a fetal blood sample loaded into a lane of the cell card, according to one embodiment of the present disclosure. The first lane contains anti-A with a blue dye. The second lane contains anti-B with a yellow dye, the third lane contains anti-Rh with no dye, and the fourth lane contains BRIC 200. The fetal blood sample loaded into a lane of the cell card is type B, Rh-positive. The position of the band at the top of the column, or the bottom of the column depends on whether the cells are agglutinated. The appearance of the band varies with the nature of the antibodies and the agglutinated cells, with some appearing sharper and others appearing more diffuse. The use of fluorescence in the present disclosure enables detection of minimal fetal cells in a maternal blood sample while also tolerating the variable geometry of the band depending on the nature of the antibodies and captured cells present in the sample being labeled and read.

[0081] FIG. 5 is a graph showing the quantitation of fluorescently labeled fetal cells, according to one embodiment of the present disclosure. In one embodiment, the method of detecting FMH may include obtaining sample of maternal blood after delivery. The sample obtained may contain a small number of fetal red cells, approximately 0.1 to 2.0%. The red blood cells obtained from the maternal sample is washed into PBS, while simultaneously removing the white blood cells. The sample is then incubated with fluorescent label for 15 minutes to 2 hours at 37°C. The sample may contain 100 million red blood cells or more. In one embodiment, the fluorescent label may be CellBrite® Cytoplasmic Membrane Dye. CellBrite is a carbocyanine dye which contains a long hydrophobic chain that integrates into the plasma membrane of living or fixed cells. CellBrite, and other lipophilic carbocyanine dyes, are dye delivery solutions that may be added directly to normal culture media to uniformly label suspended or adherent cells. To label the red blood cells obtained from the maternal blood sample, the cells are suspended at a density of lxlO5 / mL in PBS or another buffer. About 5 gL of fluorescent label is added per 1 mL of cell suspension and mixed using a low speed vortexing or manually tapping the tube. After the red blood cells are fluorescently labeled, the cells are washed three times with PBS and subsequently, suspended in PBS. The cells are then separated into equal three aliquots, which are treated separately with one aliquot used to determine the total number of cells in the sample, one aliquot used to determine the number of total fetal cells in the sample, and the third aliquot used to determine background.

[0082] In one embodiment, one aliquot is incubated with BRIC 200 monoclonal antibody. BRIC 200 is a mouse antibody directed to an extracellular loop on Band 3, the most common protein in a red blood cell, outside of hemoglobin. About 30 gL of an aliquot, containing roughly 10 million red blood cells, is incubated with 20 pL of a 1.0 pg / mL solution of BRIC 200 for 30 minutes at room temperature in a 1.5 mL microfuge tube. After incubation, around 1.4 mL of PBS is added to the microfuge tube to suspend the cells. The microfuge tube is then centrifuged for 15 seconds at 6000 RPM to pellet the cells. The supernatant is removed, and the cells are suspended in 30 pL of PBS. About 20 |1L of rabbit anti-mouse IgG at a concentration of 0.12 |ig / |iL is added to the cells to suspended them in the antibody. The rabbit anti-mouse IgG is added with a pipette and a second 30-minute incubation is performed. In one embodiment, the rabbit anti-mouse IgG may be AffmiPure Rabbit Anti-Mouse IgG, Fey fragment specific. Around 40 pL of the cell suspension is loaded on a lane of a gel card. In one embodiment, the gel card may be a Grifols card. In yet another embodiment, a separation column may be used in place of a gel card. The card is centrifuged in a specialized gel card centrifuge for 9 minutes. In one embodiment, the specialized gel card centrifuge may be a Grifols centrifuge. The gel card is inserted into a laser gel card reader and a voltage is recorded which reflects the number of total cells in the sample. In one embodiment, the voltage channel is monitored on a low gain setting. The recording of a voltage is obtained by the method depicted in FIG. 3, where the sample is placed in the sample holder of the laser gel card reader and the illumination source is directed towards the sample.

[0083] The Rh-positive cell number in the sample may become too high and the Rh- negative cells may get trapped at the top of the gel card or separation column instead of dropping to the bottom of the gel card. In one embodiment, an anti-Rh antibody is attached to a bead to capture the Rh-positive cells while not impeding Rh-negative cells to drop to the bottom of the gel card. In yet another embodiment, a large bead size is used to improve the anti-impedance of the Rh-negative cells to the bottom. The beads need to be big enough to be trapped at the top of the gel card while not impeding the Rh-negative cells from going through. In one embodiment, the bead may have a size of 200 or less microns, preferably with a size of 40 microns or less. In another embodiment, Polystyrene or Sepharose may be used as the beads. Polystyrene or other beads are available commercially in a wide variety of sizes from submicron to at least 400 microns.

[0084] Using beads allows the rare Rh-positive cells to be captured and form a band within the aperture of the photodiode. To be successful, however, the band must not impede the passage of the majority Rh-negative cells to the bottom of the gel card. Normally, cells are kept on the top of the gel card gel by the formation of an immune complex or network of cells linked by antibody. This also blocks the passage of antigen negative cells to the bottom of the column. When very small amounts of Rh-positive antibodies are needed to be detected and quantitated, such as in FMH, forming an immune complex is not ideal because the 2,300-55,000 cells (per 5 million) are unable to form a complex or network large enough to prevent the Rh-positive red blood cell’s entry into the gel matrix.

[0085] Small antibody coated beads, such as 1-5 gm diameter, may not be excluded from the gel matrix. Red blood cells are generally 6-8 pm in diameter, with fetal cells being about 20-30% larger. It is preferrable to use large beads to ensure the beads are excluded from the gel matrix. This is preferred because the formation of immune complexes is not useful when the number of red blood cells to be trapped is so low. The band of cells at the top of the gel card makes laser excitation easier because the top of the gel card is open and the ability to shift either all the cells or just the Rh-positive cells to the top of the gel card column and, therefore within the aperture of the photodiode, is important.

[0086] In certain embodiments, the location of the RBC on a gel card is detected by visualization of the red color by eye or camera reader particularly when the detection sensitivity is sufficient for such a visualization or image capturing. For example, a gel card reader can be used that includes a camera, preferably one that captures color images and may include a back-lighting source to facilitate visualization of one or more bands or regions of the gel card. Preferably, the camara has high-resolution such as a Charge-Coupled Device (CCD) image sensor that is configured to capture images. In certain embodiments, the high resolution is at least 300 pixels per inch (PPI)

[0087] Examples of other devices that can be used to read the gel cards are those found in U.S. patent no. 10,107,806 and U.S. patent no. 8,574,495, both of which are incorporated herein by reference.

[0088] One exemplary commercially available device to read gel cards is DG Reader Net (Grifols SA).

[0089] In other embodiments, particularly where the resolution of the captured image is not as clear or the cell count is very low such that known gel card readers would not be sufficient to assess the results on the gel card, an apparatus that includes a detection component that uses desired wavelength to detect bands on a gel card as described herein can be used. That is, as described in the present disclosure, the apparatus having components necessary to detect with the appropriate sensitivity the presence or absence of Rh-positive fetal cells as described herein.

[0090] For illustration of such an apparatus, in one embodiment, comprises: a power source, a controller hardware and software to perform the supervisory control functions of the apparatus, an illumination source that produces the desired wavelength, a sample holder to stabilize the sample card for illumination and detection, an optics component which collects and filters the emission from the target (sample cell in the sample holder), a detection electronic comprised of a sufficiently sensitive optical detector, a software component residing in the controller HW / SW, a display to provide users with the sample results, and an enclosure to provide structure to the apparatus.

[0091] Still further embodiments, include quantitation of fetal cells in maternal blood samples by image analysis. Such a method may include quantitating Rh D+ (fetal) cells in maternal post-delivery blood samples using one or more antibodies, Streptavidin agarose, a neutral gel card, camera and image analysis.

[0092] For instance, the percentage of Rh D+ cells in a post-delivery blood sample are determined by image analysis following imaging of the gel card using a digital camera. Samples are generated containing Rh D+ cells incubated with human anti-D monoclonal antibody that has been biotinylated using a photoaffinity label.

[0093] Excess unbound antibody can be removed with washes in a cell washer and the resulting cell mixture containing both Rh D+ cells and Rh D- cells is applied to one or more lanes of a neutral gel card which has been preloaded with Streptavidin-agarose. Total cells in the sample are determined from a parallel sample incubated with rabbit anti-human RBC and loaded on an untreated gel card lane. The gel cards are then centrifuged as appropriate in a gel card centrifuge. Alternatively, the total cell sample may be quantitated from imaging the same gel card lane use to determine the Rh D+ cells.

[0094] In one embodiment, the sample card is horizontal to the camera image and that the sample lanes are up / down with each sample proceeding left / right. However, this orientation is not required and may be modified.

[0095] The image can be uniformly illuminated from behind with minimal outside light to increase the clarity of the obtained image. The intensity of the image can also be assessed for the absorption by the sample by assessing inverted pixel by pixel across the image. In one preferred embodiment and for higher sensitivity (fetal blood) the green and / or blue channels are used as the red channel provides the lowest signal.

[0096] For very high concentrations (maternal blood), the red channel may be included, or used exclusively to bring the absorption back into a linear range.

[0097] A further embodiment includes the capture of an image with a monochromatic sensor, sensor with color filters, or monochrome sensor with filtered illumination. However, it is believed that using white light and a color camera sensor provides the easiest system due to availability and costs of components. Illumination that is optimized for the absorption of hemoglobin would further increase the sensitivity, but is not required.

[0098] A narrow stripe down each lane of the gel card can be isolated and horizontal stripes are combined and then read out across the area where the sample has formed a band. The area above the band provides a base line for each sample. While measuring the peaks can be focused upon as a useful signal, in one preferred embodiment, a better signal is achieved when the sample is integrated (up / down) across the band.

[0099] In a preferred embodiment, a reference “blank” channel is provided to increase the accuracy of the system and the result(s) obtained.

[0100] The cell band at the top of a gel card is highly variable, some may be thin, and some may be thicker and more diffuse. Because of this, it is not possible to use absorbance for quantitation based on the geometry of the band. Some bands may appear to have near zero thickness and near infinite density. Fluorescence solves this problem since the signal does not depend on the geometry in the same manner as absorbance. In fact, the sensitivity of fluorescence detection may be approximately 1 ,000 times greater than absorption spectrophotometric methods. The increase in sensitivity leads to lower limits of detection, which helps to detect small bleeds.

[0101] In one embodiment, the second aliquot of the maternal blood cell sample is incubated with 20 pL of anti-Rh antibody (human IgM). In one embodiment, Geyer blood typing anti-sera anti-Rh is the antibody. In yet another embodiment, a similar antibody may be used. About 40 pL of the cell suspension is loaded on a lane of a gel card. In one embodiment, the gel card may be a Grifols card. The card is centrifuged in a specialized gel card centrifuge for 9 minutes. In another embodiment, the specialized gel card centrifuge may be a Grifols centrifuge. The gel card is then inserted into the laser gel card reader and a voltage is recorded which reflects the number of fetal cells in the sample. In one embodiment, the voltage channel is monitored on a high gain setting. The recording of a voltage is obtained by the method depicted in FIG. 3, where the sample is placed in the sample holder of the laser gel card reader and the illumination source is directed towards the sample. The ratio of fetal cell signal divided by the total cell signal multiplied by 100 is the % fetal cells in the sample. The apparatus directly outputs percent fetal cells in the sample, thus eliminating operator error in reading results. In one embodiment, the result may be quantitative, giving a specific numeric value for diagnosing a fetal maternal hemorrhage.

[0102] The method(s) described herein above and below embodied in this invention disclosure can be automated, in whole or in part. For instance, the supply of antibody or antibodies to the blood sample(s) can be provided in an automated system. Further, the loading of labeled cells in the sample can be automated and / or the processing of the samples in the gel card. In addition, the detection and optionally report generation can also be automated.

[0103] In certain exemplary and non-limiting embodiments, the following examples further illustrate the invention described herein.

[0104] EXAMPLES

[0105] Example 1

[0106] Protocol:

[0107] 1. Collect a sample of maternal blood in an EDTA tube, after delivery of the fetus and about 30 min following delivery of the placenta.

[0108] 2. Wash red blood cells to remove plasma and white blood cells (WBC). Removal of plasma is not required but may be preferable as WBC contain Fc receptors that could bind the heavy chain or constant region of antibodies. If the Biotin-PEG12- BRAD 3 were to bind to the Fc receptors on any WBC that are present in the sample, it may cause false positive reactions. Adjust to 3% hematocrit with PBS or normal saline. A 3% suspension is standard for blood typing studies.

[0109] 3. Combine 3-4 drops of washed RBC with 250 pL PBS / 60% diluent A. Mix and prepare three equal 100 pl aliquots of RBCs in tubes A, B and C.

[0110] Tube A

[0111] 4. Add 100 pL Biotin-PEG12-BRAD 3 to tube A mix and incubate for 5 min at RT.

[0112] 5. Wash in automated cell washer x 3 cycles, after washing, the washer resuspends cells in about 100 pL normal saline. 6. Add 100 pL Alexa Fluor 647-Rabbit anti-human IgG in PBS / 60% diluent A, incubate 5 min at RT.

[0113] 7. Wash in automated cell washer x 3 cycles, after washing, the washer resuspends cells in about 100 pL normal saline.

[0114] 8. Add 40 pl diluent A, mix and load on lane 1 of a gel card previously loaded with 10 pL of a 50% suspension of Streptavidin- Agarose in lane 1. All of the contents of Tube A are loaded on lane 1 of the gel card.

[0115] Tube B

[0116] 9. Add 100 pL Alexa Fluor 647-Rabbit anti-human IgG, incubate 5 min at RT.

[0117] 10. Wash in automated cell washer x 3 cycles, after washing, washer resuspends cells in about 100 pL normal saline.

[0118] 11. Add 40 pl diluent A, mix and load on lane 2 of a gel card previously loaded with 10 pL 50% suspension of Streptavidin- Agarose in lane 2. All the contents of Tube are loaded on lane 2 of the gel card.

[0119] Tube C

[0120] 12. Add 100 pL Rabbit anti-human RBC, mix and load about 100 pL on lanes 3 & 4 of the gel card. All of the contents of tube C are loaded on either lane 3 or lane 4 of the gel card. Lanes 3 & 4 are not necessarily loaded precisely equally.

[0121] The gel card is centrifuged in a gel card centrifuge for the indicated time ( about 9 min).

[0122] Transfer gel card to gel card reader.

[0123] The top of the gel card (7 mm) is scanned every 0.02 mm by the card reader with a 630 rnn excitation laser and fluorescence collected via a 697-75 nm band pass filter and photodiode / amplifier. The location of the band of RBC is determined by the on-board computer and the signal is integrated. Rh D-positive cells are determined by the signal in lanel less background in lane 2. The measured fluorescence is converted to number of cells by reference to a previously determined standard curve stored on the on-board computer. The standard curve is prepared with fetal RBC heterozygous for Rh D antigen diluted with adult Rh D-negative cells.

[0124] Total cells are determined by the autofluorescence signal in lanes 3 and 4. The signals in lanes 3 and 4 are combined and the fluorescence is converted to cell number by reference to a standard curve stored on the on-board computer.

[0125] The percentage fetal (Rh D-positive) cells is calculated by determining the ratio of Rh D-positive cells divided by the number of total cells x 100%. Sensitivity is very good as with 15,000,000 input cells the test can detect and quantitate 15,000 Rh D-positive cells representing 0.10% of the cells or a 5 mL FMH. This is equivalent to one sixth of a 30 mL fetal (whole blood) bleed, which should be covered by a single unit of Rh immune globulin. A bleed larger than 30 mL (0.6% or 90,000 cells) will require additional doses of Rh Immune globulin and dosing would be directed by a quantitative FMH Assay. The assay can quantitate FMH bleeds between 5 mL to 80 mL in a single step. If desired the range can be adjusted higher by changing the reagent volumes.

[0126] Example 2 Using a Maternal Post Delivery Blood Sample, divide into two equal aliquots of

[0127] -10-15 million cells one for Total Cells and one for Fetal Cells.

[0128] Example 3

[0129] Using Maternal Post Delivery Blood Sample divide into two equal aliquots of

[0130] -10-15

[0131] Fetal cell determination is performed the same as in Example 2 above.

[0132] Example 4

[0133] Using Maternal Post Delivery Blood Sample divide into two equal aliquots of

[0134] -10-15 million cells one for Total Cells and one for Fetal Cells.

[0135] Fetal cell determination same as in Example 2 above.

[0136] Example 5

[0137] Quantitation of Fetal cells in Maternal Blood Samples by Image Analysis

[0138] The percentage of Rh D+ cells in a post-delivery blood sample are determined by image analysis following imaging of the gel card using a digital camera. To demonstrate, contrived samples are generated containing 0 to 0.2.4% Rh D+ cells in 15 million total cells are incubated with BRAD 3 human anti-D monoclonal antibody that has been biotinylated using a photoaffinity label.

[0139] Excess unbound antibody is removed with 2 washes in a cell washer and the resulting cell mixture containing both Rh D+ cells and Rh D- cells is applied to a single lane of a neutral gel card which has been preloaded with 2.5 pL of Streptavidin-agarose see FIGS. 13 and 14. Total cells in the sample are determined from a parallel sample incubated with rabbit anti-human RBC and loaded on an untreated gel card lane see FIGS. 15 and 16. The gel cards are then centrifuged for 9 min in a gel card centrifuge. Alternatively, the total cell sample may be quantitated from imaging the same gel card lane used to determine the Rh D+ cells.

[0140] For this description it can be assumed that the sample card is horizontal to the camera image and that the sample lanes are up / down with each sample proceeding left / right. This orientation is not important but is described for clarity.

[0141] Ideally the image is uniformly illuminated from behind with minimal outside light. As the absorption by the sample is visualized, the intensity of the image is inverted pixel by pixel across the image. For highest sensitivity (fetal blood) the green and / or blue channels are used as the red channel provides the least signal. For very high concentrations (maternal blood), the red channel may be included, or used exclusively to bring the absorption back into a linear range.

[0142] The image could also be used with a monochromatic sensor, sensor with color filters, or monochrome sensor with filtered illumination. Using white light and a color camera sensor provides the easiest system due to availability and costs of components. Illumination that is optimized for the absorption of hemoglobin would further increase the sensitivity, but is not required.

[0143] A narrow stripe down each lane is isolated and horizontal stripes are summed up and then read out across the area where the sample has formed a band. The area above the band provides a base line for each sample. While strictly measuring the peaks can provide as useful signal, a better signal is achieved when the sample is integrated (up / down) across the band.

[0144] A reference “blank” channel can be provided to increase the accuracy.

[0145] Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

[0146] Further specific embodiments include:

[0147] A detection apparatus comprising: a. controller hardware, b. an illumination source, c. a sample holder, d. an optics component, e. a detection electronics component, f. a display, and g. an enclosure, h. wherein the detection apparatus is configured to detect the presence of fetal red cells from a maternal blood sample, i. wherein the optics component collects the emission directed from the illumination source onto the maternal sample within the sample holder, j. wherein the detection electronics component converts an optical signal received from the optics component into one or more electrical signals, and k. wherein the one or more electrical signals is / are reduced into a digitized total of red blood cells and fetal red cells.

[0148] The detection apparatus can have the controller hardware can be configured to control the illumination source and to interface with the detection electronics component; and / or the illumination source can comprise laser diodes; and / or the illumination source can be configured to focus onto the sample holder at a about a 90° angle; and / or the sample holder can be configured to receive a gel card containing the maternal blood sample to hold for illumination and detection; and / or the optics component can spatially filter the illumination source using colored glass filters and interference filters; and / or the electronics component can comprise a series of photodiodes and amplifiers; and / or the electronics component can also comprise an analog-to-digital converter configured to translate the optical signal to an electrical signal; and / or the one or more electric signals, preferably at least two electric signals, can be reduced using a computer algorithm that uses a calibration curve to produce a total number of red blood cells and fetal red cells from the electrical signal.

[0149] A method for detecting Rh-positive fetal red cells in a maternal blood sample, the method comprising: a. fluorescently labeling fetal red blood cells in a maternal blood sample, b. loading a cell suspension comprising at least a part of the labeled fetal red blood cells into a lane of a gel card or a separation column, and c. placing the gel card or separation column into an apparatus configured to detect one or more electrical signals of Rh-positive fetal red cells and a number of total blood cells in the maternal blood sample.

[0150] In this method, the maternal blood sample can be obtained after delivery; and / or the cell suspension comprising at least a part of the labeled red blood cells can be incubated with at least one antibody prior to placing into a lane of a gel card or a separation column; and / or the fluorescently labeling comprises labeling with at least one fluorescent labeled pan red cell antibody such as BRIC 200BRIC 200 an antibody directed to the extracellular domain of Band 3 (for example Sulfo-Cy5.5-BRIC 200). including BRIC 6, BRIC 71, BRAC 17, or BRAC 18; and / or total cells are quantitated by autofluorescence, direct dye labeling of the cells, fluorescent labeling, or a combination thereof; and / or the cell suspension comprising at least a part of the labeled red blood cells can be incubated with at least one rabbit anti-mouse IgG prior to placing into a lane of a gel card or a separation column.

[0151] A method of determining a total number of red cells in a maternal plus fetal blood samples, the method comprising: a. fluorescently labeling red blood cells in a maternal and fetal blood samples, b. loading a cell suspension comprising the labeled red blood cells into a lane of a gel card, and c. analyzing one or ore electric signals generated by illuminating the gel card to determine the number of total red cells in the blood samples.

[0152] In this method, the fluorescently labeling can comprise a fluorescent label comprising a carbocyanine dye containing a long hydrophobic chain; and / or further adding an anti-Rh antibody to the cell suspension; and / or the number of total fetal red cells in the maternal blood sample is represented by a percentage according to Formula

[0153] 1:

[0154] A method for separating Rh+ cells from Rh- cells, comprising a. introducing a biotinylated antibody specifically onto the Rh+ cells; b. b. introducing a fluorescent label via Alexa Fluor 647 labeled rabbit antihuman IgG; c. c. incubating the sample with Streptavidin, Neutravidin, anti-biotin or avidin on - Sepharose (45-165 pm), agarose, or Ultralink or polystyrene (14.0-105 pm) bead; d. d. introducing the sample to a gel card; and e. e. centrifuging the gel card, wherein the Rh- cells move to the bottom of the gel column, while the Rh+ cells with the associated fluorescent label are trapped at the top of the gel column by the size of the bead.

[0155] In this method, the biotinylated antibody can be introduced into the Rh+ cells by incubation; and / or the fluorescent label can be introduced into the Rh+ cells via Alexa Fluor 647 labeled rabbit anti-human IgG by incubation; and / or the Streptavidin, Neutravidin, anti-biotin or avidin can be coated onto a Sepharose bead; and / or the Streptavidin, Neutravidin, anti-biotin or avidin can be coated onto a Polystyrene bead; and / or the gel card can comprise a column for separation of cells; and / or the gel card can be centrifuged at a predetermined speed and duration; and / or can further comprise analyzing the separated Rh+ cells.

[0156] A method of treating a fetal maternal hemorrhage conducting a method of any one of described herein above to detect fetal blood cells and / or with the apparatus of anyone of described herein above and administering a dose of Rh immune globulin to the subject according to the size of the maternal fetal hemorrhage detected. One or more of the following steps can also be conducted: a. fluorescently labeling red blood cells taken from a maternal blood sample, b. inserting the red blood cells into a lane of a gel card, c. utilizing a fetal maternal hemorrhage detection apparatus to read the gel card, d. wherein the fetal maternal hemorrhage detection apparatus emits a light source through the gel card to produce an optical signal, e. wherein the fetal maternal hemorrhage detection apparatus converts the optical signal into an electrical signal to produce a digitized total of fetal red cells present in the maternal blood sample, f. wherein the total of fetal red cells present in the maternal blood sample corresponds with a size of a maternal fetal hemorrhage in a subject from which the maternal blood sample was taken, and g. wherein a dose of Rh immune globulin is administered to the subject according to the size of the maternal fetal hemorrhage detected.

[0157] A method for quantitating fetal Rh D+ cells in a maternal post-delivery blood sample, the method comprising a. contacting the sample with one or more antibodies with at least one of the antibodies is a human anti-D antibody that is biotinylated with a photoaffinity label, b. optionally washing the contacted sample to remove excess unbound antibody, c. loading the washed contacted sample to a neutral gel card comprising streptavidin agarose, and d. analyzing one or more images obtained from the neutral gel card after centrifugation of the loaded gel card, optionally with one or more controls and / or blank channels

Claims

CLAIMS1. A detection apparatus comprising: a. controller hardware, b. an illumination source, c. a sample holder, d. an optics component, e. a detection electronics component, f. a display, and g. an enclosure, h. wherein the detection apparatus is configured to detect the presence of fetal red cells from a maternal blood sample, i. wherein the optics component collects the emission directed from the illumination source onto the maternal sample within the sample holder, j. wherein the detection electronics component converts an optical signal received from the optics component into one or more electrical signals, and k. wherein the one or more electrical signals is / are reduced into a digitized total of red blood cells and fetal red cells.

2. The detection apparatus of Claim 1 , wherein the controller hardware is configured to control the illumination source and to interface with the detection electronics component.

3. The detection apparatus of Claim 1, wherein the illumination source comprises laser diodes.

4. The detection apparatus of Claim 1 , wherein the illumination source is configured to focus onto the sample holder at a about a 90° angle.

5. The detection apparatus of Claim 1, wherein the sample holder is configured to receive a gel card containing the maternal blood sample to hold for illumination and detection.

6. The detection apparatus of Claim 1 , wherein the optics component spatially filters the illumination source using colored glass filters and / or interference filters.

7. The detection apparatus of Claim 1 , wherein the electronics component comprises a series of photodiodes and amplifiers.

8. The detection apparatus of Claim 7, wherein the electronics component further comprises an analog-to-digital converter configured to translate the optical signal to an electrical signal.

9. The detection apparatus of Claim 1 , wherein the one or more electric signals, preferably at least two electric signals, is / are reduced using a computer algorithm that uses a calibration curve to produce a total number of red blood cells and fetal red cells from the electrical signal.

10. A method for detecting Rh-positive fetal red cells in a maternal blood sample, the method comprising: a. fluorescently labeling fetal red blood cells in a maternal blood sample, b. loading a cell suspension comprising at least a part of the labeled fetal red blood cells into a lane of a gel card or a separation column, and c. placing the gel card or separation column into an apparatus configured to detect one or more electrical signals of Rh-positive fetal red cells and a number of total blood cells in the maternal blood sample.

11. The method of Claim 10, wherein the maternal blood sample is obtained after delivery.

12. The method of Claim 10, wherein the cell suspension comprising at least a part of the labeled red blood cells is incubated with at least one antibody prior to placing into a lane of a gel card or a separation column.

13. The method of Claim 10, wherein the fluorescently labeling comprises labeling with at least one fluorescent labeled pan red cell antibody such as BRIC 200, anantibody directed to the extracellular domain of Band 3 (for example Sulfo- Cy5.5-BRIC 200). including BRIC 6, BRIC 71, BRAC 17, or BRAC 18.

14. The method of Claim 13, wherein total cells are quantitated by autofluorescence, direct dye labeling of the cells, fluorescent labeling, or a combination thereof.

15. The method of Claim 10, wherein the cell suspension comprising at least a part of the labeled red blood cells is incubated with at least one rabbit anti-mouse IgG prior to placing into a lane of a gel card or a separation column.

16. A method of determining a total number of red cells in a maternal plus fetal blood samples, the method comprising: a. fluorescently labeling red blood cells in maternal and fetal blood samples, b. loading a cell suspension comprising the labeled red blood cells into a lane of a gel card, and c. analyzing one or more electric signals generated by illuminating the gel card to determine the number of total red cells in the blood samples.

17. The method of Claim 16, wherein the fluorescently labeling comprises a fluorescent label comprising a carbocyanine dye containing a long hydrophobic chain.

18. The method of Claim 16, further comprising adding an anti-Rh antibody to the cell suspension.

19. The method of Claim 16, wherein the number of total fetal red cells in the maternal blood sample is represented by a percentage according to Formula 1 :

20. A method for separating Rh+ cells from Rh- cells, comprising: a. introducing a biotinylated antibody specifically onto the Rh+ cells; b. introducing a fluorescent label via Alexa Fluor 647 labeled rabbit antihuman IgG; c. incubating the sample with Streptavidin, Neutravidin, anti-biotin or avidin on - Sepharose (45-165 pm), agarose, or Ultralink or polystyrene (14.0-105 pm)bead; d. introducing the sample to a gel card; and e. centrifuging the gel card, wherein the Rh- cells move to the bottom of the gel column, while the Rh+ cells with the associated fluorescent label are trapped at the top of the gel column by the size of the bead.

21. The method of Claim 20, wherein the biotinylated antibody is introduced into the Rh+ cells by incubation.

22. The method of Claim 20, wherein the fluorescent label is introduced into the Rh+ cells via Alexa Fluor 647 labeled rabbit anti-human IgG by incubation.

23. The method of Claim 20, wherein the Streptavidin, Neutravidin, anti-biotin or avidin is coated onto a Sepharose bead.

24. The method of Claim 20, wherein the Streptavidin, Neutravidin, anti-biotin or avidin is coated onto a Polystyrene bead.

25. The method of Claim 20, wherein the gel card comprises a column for separation of cells.

26. The method of Claim 20, wherein the gel card is centrifuged at a predetermined speed and duration.

27. The method of Claim 20, further comprising analyzing the separated Rh+ cells.

28. A method of treating a fetal maternal hemorrhage conducting a method of any one of Claims 10-27 to detect fetal blood cells and / or with the apparatus ofanyone of Claims 1-9 and administering a dose of Rh immune globulin to the subject according to the size of the maternal fetal hemorrhage detected:

29. The method of Claim 28, wherein one or more of the following steps is / are conducted: a. fluorescently labeling red blood cells taken from a maternal blood sample, b. inserting the red blood cells into a lane of a gel card, c. utilizing a fetal maternal hemorrhage detection apparatus to read the gel card, d. wherein the fetal maternal hemorrhage detection apparatus emits a light source through the gel card to produce an optical signal, e. wherein the fetal maternal hemorrhage detection apparatus converts the optical signal into an electrical signal to produce a digitized total of fetal red cells present in the maternal blood sample, f. wherein the total of fetal red cells present in the maternal blood sample corresponds with a size of a maternal fetal hemorrhage in a subject from which the maternal blood sample was taken, and g. wherein a dose of Rh immune globulin is administered to the subject according to the size of the maternal fetal hemorrhage detected.

30. A method for quantitating fetal Rh D+ cells in a maternal post-delivery blood sample, the method comprising a. contacting the sample with one or more antibodies with at least one of the antibodies is a human anti-D antibody that is biotinylated with a photoaffinity label, b. optionally washing the contacted sample to remove excess unbound antibody, c. loading the washed contacted sample to a neutral gel card comprising streptavidin agarose, and d. analyzing one or more images obtained from the neutral gel card after centrifugation of the loaded gel card, optionally with one or more controls and / or blank channels

Images