WO2019060687A1

WO2019060687A1 - Method of determining fetal red blood cells in maternal circulation using flow cytometry

Info

Publication number: WO2019060687A1
Application number: PCT/US2018/052174
Authority: WO
Inventors: Fabrice Malergue; Andreas Van Agthoven; Jean Marc BUSNEL
Original assignee: Beckman Coulter, Inc.
Priority date: 2017-09-21
Filing date: 2018-09-21
Publication date: 2019-03-28

Abstract

This invention provides methods for identifying fetal red blood cells in a blood sample that is suspected of comprising fetal blood. The invention also provides kits that can be used for this purpose.

Description

METHOD OF DETERMINING FETAL RED BLOOD CELLS IN MATERNAL CIRCULATION USING FLOW CYTOMETRY

FIELD OF THE INVENTION

[0001] This invention relates to compositions and methods for determining fetal red blood cells (RBCs) in maternal circulation using flow cytometry.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0002] The present patent application claims benefit of priority to U.S. Provisional Patent Application No.: 62/561,515, filed September 21, 2017, which is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0003] Fetal -maternal hemorrhage (FMH) is the loss of fetal blood cells into the maternal circulation. Normally the maternal circulation and the fetal circulation are kept from direct contact with each other, with gas and nutrient exchange taking place across a membrane in the placenta. FMH occurs when this membrane ceases to function as a barrier and fetal cells come in contact with and enter the maternal vessels; loss of an excess of fetal blood to the maternal circulation may result in significant morbidity and mortality to the fetus. In addition, loss of fetal blood to maternal circulation may stimulate antibody production in the pregnant female to the fetal RBCs and may have detrimental effect on the newborn, for example, causing the Rhesus disease. Therefore it is important to monitor the amount of fetal blood in the maternal blood during pregnancy.

[0004] Many of the FMH detection methods are based on the fact that fetal RBCs have a unique hemoglobin isoform, HbF, which is different from the hemoglobin isoform, HbA, in adults. The conventional assay of FMH detection is the Kleihauer assay, which involves preparing a blood smear from the maternal blood and expose the smear to an acid bath. The acid exposure removes adult hemoglobin HbA, but not fetal hemoglobin (HbF) from the respective RBCs. This allows fetal RBCs to be distinguished by color under microscope, for example, the fetal cells may appear rose-pink, while adult RBCs are only seen as "ghosts" after the staining. The drawback of this method is that in order to detect a small number of fetal erythrocytes, a large number of erythrocytes must be passed under the microscope, which represents a significant amount of handling. Also, certain pathologies can lead to a modification of the erythrocytes, resulting in a number of false positives. Finally, it can be challenging to reproduce results obtained from this assay.

[0005] Recently, flow cytometry-based methods have been developed to detect fetal RBCs in maternal blood. The method often stains the blood sample with a labeled antibody directed against HbF and identifies the population of cells that are HbF positive as fetal RBCs. These flow-based methods dispense with the manual reading of the slides, thus improving efficiencies, however, the existing methods still suffer from sensitivity issues because some adult RBCs also express HbF. For example, in diseases such as thalassemia, a number of adult erythrocytes also express fetal hemoglobin. Therefore, the need for more sensitive and convenient methods for detecting FMH remains.

BRIEF SUMMARY OF THE INVENTION

[0006] This disclosure provides a method of detecting fetal RBCs in a blood sample from a patient, wherein the blood sample is suspected of comprising fetal blood cells, comprising: contacting a blood sample with an HbF-binding agent and an HbA-binding agent to form a stained blood sample, and detecting fetal RBCs in the stained blood sample, wherein the fetal RBCs bind to the HbF-binding agent but not the HbA-binding agent. In some embodiments, the method further comprises contacting the blood sample with a permeabilizing agent before contacting the blood sample with the HbF-binding agent and the HbA-binding agent. In some embodiments, the HbA-binding agent binds to HbAlc.

[0007] In some embodiments, the patient is a pregnant female, e.g., a pregnant female human. In some embodiments, the method further comprises determining the adult patient has fetal-maternal hemorrhage (FMH) when the presence of fetal RBCs in the blood sample are detected.

[0008] In some embodiments, the HbF-binding agent is conjugated to a first label and the HbA or HbAlc-binding agent is conjugated to a second label, wherein the first label and the second label are distinguishable, and wherein the measuring the binding of the HbF-binding agent to the RBCs in the stained blood is by detecting the signal from the first label and measuring the binding of the HbA or HbAlc-binding agent to the RBCs in the stained blood sample is by detecting the signal from the second label. [0009] In some embodiments, the step of detecting fetal RBCs includes incubating the sample with a first detection agent that specifically binds to the HbF-binding agent and a second detection agent that specifically binds to the HbA or HbAlc-binding agent, wherein the binding of the first detection agent to HbF and the binding of the second detection agent to HbAlc are detectable and distinguishable. In some embodiments, the blood sample is fixed by a fixing agent before contacting the permeabilizing agent. In some cases, the sample are permeabilized and subsequently stained without being fixed.

[0010] In some embodiments, the HbF-binding agent is an antibody. In some

embodiments, the HbA or HbAlc-binding agent is an antibody. In some embodiments, the detecting fetal RBCs is by flow cytometry. In some embodiments, the concentration of the HbF antibody and/or the concentration of the Hbl Ac antibody is in the range of 0.5-10 μg/ml. In some embodiments, the concentration of the HbF antibody and/or the

concentration of the Hbl Ac antibody is 2.5 μg/ml.

[0011] In some embodiments, the sample is first diluted with a saline solution before contacting the permeabilizing agent.

[0012] In some embodiments, the sample is diluted 10 to 100 fold before permeabilization. In some embodiments, the cells are permeabilized for 2-10 minutes. In some embodiments, the step of contacting the sample with HbF-binding agent and a HbA or HbAlc-binding agent further comprises incubating the sample with the HbF antibody and the Hbl Ac antibody for 5-30 minutes. In some embodiments, the binding of the HbF antibody and the HbAlc antibody is in the presence of 1-10 mg/ml a blocker protein, such as BSA. In some embodiments, the fixing agent contains an aliphatic aldehyde. In some embodiments, the permeabilizing agent contains a zwitter-ionic detergent. In some embodiments, the permeabilizing agent contains a N-acyl sarcosine or a salt thereof represented by the following molecular structure: Rl-CO-N(CH3)CH2COOXi, wherein Ri is an alkyl or alkylene group having 8 to 18 carbon atoms, and Xi is H, Na⁺, or K⁺; and said permeabilizing reagent has a pH between 4 and 6 and a low ionic strength defined by conductivity of less than 9 mS/cm.

[0013] Also provided is a kit comprising a labeled HbAlc-binding agent and a labeled HbF-binding agent. In some embodiments, the labeled HbAlc-binding agent and the labeled HbF-binding agent are comprised in the same vessel. The kit may further comprise one or more saline solutions, a fixing agent, a permeabilizing agent, and/or a staining solution. In some embodiments, the kit further comprising: a first vessel, wherein the first tube includes the saline solution, a second vessel, wherein the second vessel includes the fixing and permeabilizing agent, and a third vessel, wherein the third vessel contains the staining buffer. In some embodiments, the third vessel contains the a first fluor coupled to an antibody directed against HbF and a second fluor coupled to an antibody directed against HbAlc. In some embodiments, the contents in one or more of the vessels in the kit are in dry form.

[0014] Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGs. 1A and IB show the results of a flow cytometry analysis of a whole blood sample that have been stained with an HbAlc antibody and an HbF antibody. FIG. 1C shows the results of a flow cytometry analyiss of a mixed blood sample that contains a minor fraction of cord blood and whole blood sample. The mixed blood sample was also stained with an HbAlc and an HbF antibody.

DETAILED DESCRIPTION OF THE INVENTION

1. OVERVIEW

[0016] This invention provides compositions, kits, and methods using the HbAlc antibody and HbF antibody to stain blood samples from pregnant females in flow cytometry analysis to detect presence of fetal RBCs with desired sensitivity and specificity.

2. DEFINITIONS

[0017] As used herein, "a labeled molecule" refers to a molecule that is associated with a detectable label and can be identified using methods known in the art, e.g., in flow cytometry. In preferred embodiments, the molecule is labeled by a direct linkage to a detectable label. In some embodiments, the molecule is labeled by indirect association with a detectable label. In some embodiments, the molecule is an antibody, e.g., the HbA or HbAlc antibody and the HbF antibody. In this document, a phrase such as "HbF antibody" should be understood to mean an antibody with specificity to HbF. Under appropriate binding conditions, an HbF antibody is capable of binding to HbF. Similarly, an HbAlc antibody is capable of binding to HbAlc and an HbA antibody is capable of binding to HbA. [0018] The term "patient" refers to a human or animal. For example, the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance. In preferred embodiments, the patient is a pregnant female. For avoidance of doubt, a fetus is not a patient within the meaning of this disclosure.

[0019] The term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. A binding agent can be a small molecule compound or a protein, e.g., an antibody.

[0020] The term "an HbA-binding agent" refers to a binding agent that specifically and selectively binds to either Fib A or to HbAlc.

[0021] The term "target" refers to a molecule, compound or cell that is being detected. In certain embodiments, the target can be detected via its interaction with a binding agent. As described herein, the term "target" may refer to a protein or peptide of interest, i.e., HbF, Hb A and/or HbAlc.

[0022] The term "antibody" as used herein also includes a full-length antibody as well as an "antigen-binding portion" of an antibody. The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., HbF or HbA or HbAlc). Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 :5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG molecules or other isotypes. VH and VI can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

[0023] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof, e.g. humanized, chimeric, etc. Antibodies of the invention bind specifically or substantially specifically to one or more proteins or modified proteins, such as glycated proteins. The term "monoclonal antibodies" refer to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody molecules that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

3. SPECIFIC EMBODFMENTS Samples

[0024] The invention may be used to detect fetal RBCs in a blood sample from a patient, wherein the blood sample is suspected of comprising fetal blood. The blood sample can be obtained from a human or from another animal. The examples detailed below refer to human blood samples and to antibodies directed to human antigens, such as human HbF and human HbA or HbAlc. However, a skilled practitioner will recognize that substitution of the antibodies by others directed against corresponding proteins from other species (or use of antibodies against human HbF or human HbA or HbAlc, where there is appreciable cross- reactivity with the respective proteins in another species) may make the method applicable to determination of FMH in those other species also. In some embodiments, the blood sample comprises whole blood. In some embodiments, the blood sample comprises the whole blood and an anticoagulant, e.g., ethylene diamine tetra acetic acid (EDTA) or heparin.

FMH

[0025] The methods and kits disclosed herein can be used to detecting fetal -maternal hemorrhage (FMH). FMH is the loss of fetal blood cells into the maternal circulation. It takes place in normal pregnancies as well as when there are obstetric or trauma-related complications to pregnancy. FMH may be caused by trauma, by placental abruption or may be spontaneous with no cause found. FMH accompanied with a loss in excess of e.g., 30 ml of fetal blood to maternal circulation may result in significant morbidity and mortality to the fetus. Detecting of FMH can aid important decision-making processes during a female's pregnancy: for example, if ongoing and rapid hemorrhage is occurring then immediate delivery of the fetus may be desired if the fetus is sufficiently developed; and if the hemorrhage has already occurred and now stopped, an in utero transfusion of red cells to the fetus may be recommended.

HbF

[0026] HbF is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus. HbF persists in the newborn until roughly 6 months old, when HbF is nearly completely replaced by adult hemoglobin (HbA). Fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream. Adult RBCs typically do not express HbF, although in some cases, a certain percentage of adult RBCs may express HbF, for example, when the patient carries certain diseases such as thalassemia, or when the patient is taking certain medication during treatment of a disease, which reactivates HbF expression. HbF is expressed intracellularly.

HbAlc and HbA

[0027] HbA is one form of hemoglobin found in adult RBCs. Like HbF, HbA is also expressed inside the RBCs. HbA can bind to glucose, producing any of a number of glycated forms, of which HbAlc has most clinical history;HbAlc levels are routinely monitored to control blood sugar levels. Importantly, fetal RBCs do not express HbA and hence also lack glycated HbA, including HbAlc. The present invention is based on fetal RBCs' expression of HbF but not HbA. HbAlc and other glycated forms of HbA are consequently absent in fetal RBCs. Thus, fetal RBCs could be detected by identifying whether a given cell includes HbF or a variant of HbA (including HbAlc or unglycated HbA). Fetal cells include HbF but lack HbA variants.

Binding agents

[0028] The method employs HbF-binding agents and HbA or HblAc (or other glycated HbA) binding agents to detect fetal RBCs; fetal RBCs bind to HbF-binding agents but not HbA or Hbl Ac-binding agents; adult RBCs would bind to HbA or Hb l Ac-binding agents but (usually) not to HbF-binding agent. Without being bound to a particular theory, a binding agent used herein can be any molecule, i.e., a small molecule, sugars, oligosaccharides, lipids, peptides, peptidomimetics, nucleoside analog, organic compounds and the like, so long as it can specifically bind to HbF or HbA or HbAlc. In some embodiments, the binding agents are antibodies. Antibodies used for detecting fetal RBCs include any antibodies that recognize HbF and any antibodies that recognize HbA, or glycated HbA, such as HbAlc. Antibodies that can be produced by immunizing animal with the HbF or HbA or HbAlc protein or a portion thereof and produced and purified using methods are well known in the art. See, for example, Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991).

[0029] HbF, HbA, and HbAlc antibodies are also commercially available, for example, from Biocompare, South San Francisco, Calif. These antibodies can also be used in the methods and kits disclosed herein.

Labels

[0030] The present invention uses labels that can be detected, the signals of these labels can be used to indicate the binding between the HbF-binding agent, the HbA or glycated HbA (such as HbAlc)-binding agent and the RBCs. A label used in the invention can be any label that can be detected, e.g., by flow cytometry. Non-limiting examples of the labels include those listed in Table 1, below.

Table 1. Some exemplary labels used to label antibodies.

Alexa-647 647 665

R-phycoerythin PE 495-564 576

PE-Texas Red ECD 495-564 620

PE-cyanine 5 PC 5 495-564 670

Peridinin-chlorophyll PerCP 490 677

PE-cyanine-5.5 PC5.5 495-564 696

PE-cyanine 7 PC7 495-564 767

allophycocyanin APC 650 660

APC-cyanine 5.5 APC-Cy5.5 650 710

APC-cyanine 7 APC-Cy7 650 767

Note: "Absorption max." stands for maximum absorption wavelength. "Emission max." stands for maximum emission wavelength.

[0031] Other labels that can be used to label the antibodies include Quantum dots or mass labels. Quantum dots are fluorescent nanocrystals that have a wide absorption spectrum and so can be excited by a range of different wavelengths. Their emission wavelengths also vary and may range from blue to deep red. Mass labels are atoms or molecular fragments having a characteristic mass or ionization pattern detectable by, for example, a mass spectrometer. One skilled in the art can readily determine which types of labels to use based on the purpose and nature of the assay.

[0032] In some embodiments, the methods and kits disclosed herein use a first label for detecting the binding of the HbF-binding agent to the RBCs and a second label for detecting the binding of the HbA or HbAlc-binding agent to the RBCs and the first and second labels are distinguishable. In some cases, the distinction is detected in flow cytometry based on the difference in excitation and emission wavelength of the labels. One of ordinary skill in the art can readily determine combinations of two labels that are suitable for use in detecting and distinguishing between the binding of HbF-binding agent to the RBCs and binding of HbAlc-binding agent to the RBCs in the same sample by flow cytometry analysis. In one embodiment, the first label is Alexa-488 and the second label is Alexa-647. The labels that are used to conjugated the HbF-binding agent and the HbA or HbAlc-binding agent can also be switched, for example, in some cases, the first label is Alexa-647 and the second label is Alexa-488.

Methods

[0033] In some embodiments, the present disclosure provides a method of detecting fetal RBCs from a blood sample from a patient when the blood sample is suspected of comprising fetal blood cells. The blood sample may be drawn from the patient and tested immediately. In some cases, an anticoagulant, e.g., ethylene diamine tetra-acetic acid (EDTA) or heparin is added to the blood sample before testing. One advantage of the method disclosed herein is that the amount of the blood sample needed for the test can be very small, typically no greater than 200 μΐ, no greater than 100 μΐ, no greater than 50 μΐ, no greater than 20 μΐ, or no greater than 10 μΐ.

[0034] In some cases, the whole blood sample obtained from the patient is first diluted with a saline solution. Commonly used saline solutions include phosphate buffered saline (PBS). The volume ratio between the blood and saline solution may range from 1 : 10 to 1 : 100, e.g., from 1 :20 to 1 :50, or 1 :40. The advantageously reduces the concentration of RBCs to a level low enough to permit analysis of a single RBC at a time to permit unambiguous identification, but high enough that many RBCs may be screened in a reasonable time. Many RBCs may need to be screened because the proportion of fetal cells in maternal circulation may be low even with severe FMH.

[0035] The method of detecting fetal RBCs in a blood sample from a patient comprises contacting the blood sample with a permeabilizing agent. In some embodiments, the permeabilizing agent contains a detergent, for example, a zwitter-ionic detergent. In some embodiments, the detergent may be an anionic detergent, for example, sodium dioxycholate or N-lauryl sarcoside. In one embodiment, the detergent is sodium dodecyl sulphate. The concentration of detergent can be between 0.001% and 10%, e.g., between 0.01 and 5%. In some embodiments, the permeabilizing agent contains approximately 0.03% of sodium dodecyl sulphate. In some embodiments, the permeabilizing agent comprises N-acyl sarcosine or a salt thereof that is represented by the molecular structure of Ri-CO- N(CH3)CH2COOXi, wherein the Ri is an alkyl or alkylene group having 8 to 18 carbon atoms, and Xi is H, Na⁺, or K⁺. In some embodiments, the permeabilizing agent has a pH between 3-8. In some embodiments, the permeabilizing agent has an acidic pH, e.g., a pH between 4 and 6. In some cases, the permeabilizing agent may have a low ionic strength defined by conductivity of less than 9 mS/cm. In some embodiment, the permeabilizing agent may further comprises an oligosaccharide, e.g., trehalose. The permeabilizing agent that can be used for the invention is also substantially described in US 7,541,190 and US 6,534,279, the entire contents of the patents are hereby incorporated by reference.

[0036] The permeabilizing agents used herein may cause precipitation of the hemoglobin in RBCs while the cell membranes are being permeabilized, thus advantageously dispensing the need for a separate fixation step. Thus, in some cases, the sample can be permeabilized and stained without being fixed. In some cases, the sample is fixed before permeabilization. Advantageously, the claimed method may avoid the need for a neutralization step after fixation, because the amount of fixative is relatively low and the fixing agent in the mixture is further diluted by the staining solution (see below) by at least 2 fold, e.g., at least 2.5 fold, or at least 3 fold. Therefore in some embodiments, the method does not comprise a neutralization step.

[0037] Fixing agents that can be used in the invention include but are not limited to an aliphatic aldehyde, preferably one containing C1-C5. Non-limiting examples of fixing agents includes formaldehyde, paraformaldehyde, and glutaraldehyde. In some embodiments, the sample is fixed with a solution comprises an aliphatic aldehyde, e.g., formaldehyde, in a concentration of between 0.05% and 0.5% (v/v), e.g., between 0.06% and 0.4%, between 0.1% and 0.3%, or between 0.2% and 0.5%.

[0038] The duration of the fixation and/or permeabilization may vary. In some cases, the fixation or the permeabilization may last 1-30 min, e.g., 2-20 min, 2-10 min, 3-10 min, or about 4 min. In some cases, fixation is performed before permeabilization.

[0039] The sample that has been permeabilized and/or fixed are then contacted with the HbF-binding agent and the HbA or HbAlc-binding agent. Incubating the sample with directly or indirectly labeled binding agents is commonly referred to as staining. In some embodiments, the contacting is performed by incubating the permeabilized and/or fixed sample in a staining solution containing one or both binding agents and one or more blocker proteins. Blocker proteins are those that can reduce non-specific binding, e.g., bovine serum albumin (BSA) or fetal bovine serum (FBS). In some embodiments, the staining solution contains bovine serum albumin at a concentration of 1-100 mg/ml, e.g., 1-70 mg/ml, 2-50 mg/ml, 1-10 mg/ml, 2-8 mg/ml, or about 5 mg/ml. In some cases, the permeabilized sample is contacted with both the HbF-binding agent and the HbA or HbAlc-binding agent simultaneously. In some cases, the permeabilized sample is first contacted with the HbF- binding agent and then with the HbAlc-binding agent, or vice versa. As described above, the binding of the HbF-binding agent and of the HbA or HbAlc-binding agent in the permeabilized RBCs can be detected and differentiated through the two distinguishable labels. In some cases, the labels are directly conjugated to the HbF and/or HbA or HbAlc- binding agents. In some cases, the labels are conjugated to detection agents that specifically recognize the HbF-binding agent and the HbA or HbAlc-binding agent, respectfully. [0040] The concentration of the binding agents used in the assay can be any amount that is sufficient to produce positive signal and avoid non-specific staining. In some cases, the binding agents are an HbF antibody and an HbAlc antibody. In some cases, the concentration of the HbF antibody or the HbAlc antibody is in the range of 0.5-10 μg/ml, e.g., 1-8 μg/ml, 1-5 μg/ml, 2-4 μg/ml, e.g., 2.5 μg/ml. The time period of staining the sample with the antibodies may vary, depending on the concentration of the antibody and the affinity of the antibody to the corresponding antigen. In general, the staining may last 5-60 min, e.g., 5-30 min or 10-15 minutes.

[0041] In some embodiments, a saline solution that contains a fixing agent is added to the stained sample before loading the sample to a device to detect signals from the labels.

[0042] In some embodiments, the saline solution that is used to dilute the blood sample, the staining buffer, and/or antibodies are contained in respective vessels provided for the testing in dry form, and these contents of the vessels are reconstituted with water or buffer to form solutions as described above before the assay.

Detection

[0043] The signals from the labels, which indicate whether a red blood cell binds to the HbF-binding agent or the HbA, or (glycated HbA, such as) Hbl Ac-binding agent, can be detected by a variety of methods, including but not limited to, imaging methods (e.g., fluorescent microscopy) and flow cytometry. Non-limiting examples of flow cytometers include Gallios, CytoFLEX, and Navios from Beckman Coulter, Inc.

Kits

[0044] The present invention also provides a kit comprising an HbA or HbAlc-binding agent and an HbF-binding agent. In some embodiments, one or both of the HbA or HbAlc-binding agent and the HbF-binding agent are labeled. In some embodiments, the kit comprises a mixture of an HbAlc-binding agent and a HbF-binding agent. The kit may also comprise one or more additional components selected from the group consisting of a saline solution, a permeabilizing agent, and a fixing agent. In some embodiments, the HbAlc-binding agent and/or the HbF-binding agent is an antibody that can bind to the HbAlc and/or HbF, respectively. The kit may also contain vessels for carrying out the method steps as described above. In some embodiments, the kit comprises an HbAlc-binding agent, an HbF-binding agent, a first vessel that contains the saline solution, a second vessel that contains the fixing and permeabilizing agent, and a third vessel that contains the staining buffer. In some embodiments, the third vessel also contains the HbF-binding agent and the HbAlc-binding agent. These components in the vessels, e.g., the saline solution and the staining buffer, can be in liquid form or in dry form. Kits comprising components for the methods of detecting fetal RBCs that are present in dry form in the vessels have greater the stability and are more convenient during storage.

IV. EXAMPLES

[0045] The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Unless otherwise noted, standard techniques well known in the art or the techniques specifically described below were utilized.

EXAMPLE 1. REAGENTS

[0046] The composition of the permeabilizing agent contains:

[0047] To prepare the permeabilizing agent, deionized water was used to dissolve the components listed in the Table above. The solution was then filtered through a sterile nylon filter of 0.22 μπι pore size. The permeabilizing agent had a conductivity of 0.99 mS/cm and osmolality of 268 mOsm/Kg H2O.

EXAMPLE 2. DETECTING FETAL RBCS BY FLOW CYTOMETRY

[0048] At use, three tubes were provided. 400 μΐ PBS was added to tube A, and 200 μΐ of 5 mg/ml BSA, 0.5 μg of HbF antibody, and 0.5 μg of HbAlc antibody were added to tube C. 200 μΐ of the permeabilizing agent described above was added to tube B. Optionally, the contents of the tubes can be dried and reconstituted with deionized water prior to use.

[0049] Cord blood containing fetal blood was collected from a pregnant female patient at delivery of her newborn. A small amount of the cord blood containing fetal blood was added to a whole blood sample so that the mixed blood sample contained 0.03% of cord blood. 10 μΐ of mixed blood sample was added to tube A as prepared above. The tube was vortexed twice, each time 1 second, to mix the blood sample with the solution in tube A. 10 μΐ of the mixture in tube A was then added to tube B and the content of the tube B was mixed by vortexing twice immediately, 1 second each time. The mixture in tube B was incubated for about 4 minutes. 100 μΐ of mixture in tube B was then added to tube C. The content in tube C was then mixed by vortexing twice immediately, each time 1 second, followed by incubating the mixture for 10-15 min. 200 μΐ of a flow solution containing lxPBS and 0.17 M formaldehyde was added to tube C, and the mixture in tube C was vortexed and then analyzed using a flow cytometer. An aliquot of the whole blood sample (without cord blood) is treated in the same manner and serves as a control. The HbF signal from the cells was detected on the FL1 channel and the Fib Ale signal from the cells was detected on the FL6 channel.

[0050] FIGs. 1A and IB show the results of the whole blood sample and FIG. 1C shows the results of the mixed blood sample by flow cytometry analysis. The data tables below the plots in FIG. IB and 1C show the number and percentages of cells being detected in respective plots. FIG. 1A is a forward scatter and side scatter plot of the whole blood sample. FIG. IB shows that, as expected, the whole blood sample (the control) essentially did not contain cells having high expression levels of HbF but low expression levels of HbAlc, as indicated by that out of a total of 200,295 cells, only one (1) cell was detected in the boxed region adjacent the vertically-oriented axis - a 0.00%. FIG. 1C shows a small population of cells in the mixed blood sample (81 out of a total of 200,326 cells), which were visible in the boxed region adjacent the vertically-oriented axis, had high expression levels of HbF but very low or no expression of HbAlc— these cells were fetal RBCs. A large population of cells did not express HbF but expressed HbAlc to various degrees— these cells were adult RBCs. Another minor fraction of cells expressed intermediate levels of HbF and HbAlc to various degrees: these cells were adult RBCs expressing some low but significant amount of HbF, and called "F cells". These are the cells that may cause false positive results in assays using only the HbF marker, and that are better discriminated in this example, because they are mostly positive for HbA or HbAlc. The amount of the detected fetal RBCs accounts for about 0.04% of the total RBCs, indicating the method is accurate in detecting fetal RBCs in the blood sample that contains both adult blood and a minor fraction of fetal blood (0.03%) of the cord blood added to the whole blood before detection).

Claims

WHAT IS CLAIMED IS:

1. A method of detecting fetal RBCs in a blood sample, wherein the blood sample is suspected of comprising fetal blood cells, comprising: contacting a blood sample with an HbF-binding agent and an HbA-binding agent to form a stained blood sample; and detecting fetal RBCs in the stained blood sample, wherein the fetal RBCs bind to the HbF-binding agent but not the HbA -binding agent.

2 . The method of claim 1, further comprising contacting the blood sample with a permeabilizing agent.

3. The method of claim 2, wherein the HbA-binding agent binds to

HbAlc.

4. The method of any of the preceding claims, further comprising determining a fetal -maternal hemorrhage (FMH) when the fetal RBCs in the blood sample are detected.

5. The method of any of the preceding claims, wherein the HbF-binding agent is conjugated to a first label and the HbA-binding agent is conjugated to a second label, wherein the first label and the second label are distinguishable, and wherein the measuring the binding of the HbF-binding agent to the RBCs in the stained blood is by detecting the signal from the first label and measuring the binding of the HbA-binding agent to the RBCs in the stained blood sample is by detecting the signal from the second label.

6. The method of any of the claims 1-4, wherein the step of detecting fetal RBCs includes incubating the sample with a first detection agent that specifically binds to the HbF-binding agent and a second detection agent that specifically binds to the HbA - binding agent, wherein the binding of the first detection agent to HbF and the binding of the second detection agent to HbA are detectable and distinguishable.

7. The method of any of the preceding claims, wherein the blood sample is fixed by a fixing agent.

8. The method of any of the preceding claims, wherein the HbF-binding agent is an antibody.

9. The method of any of the preceding claims, wherein the HbA-binding agent is an antibody.

10. The method of any of the preceding claims, wherein the detecting fetal RBCs is by flow cytometry.

11. The method of any of the preceding claims, wherein the concentration of the HbF antibody and/or the concentration of the HbA or HbAlc antibody is in the range of 0.5-10 μg/ml.

12 . The method of any of the preceding claims, wherein the concentration of the HbF antibody and/or the concentration of the HbA or HbAlc antibody is 2.5 μg/ml.

13. The method of any of the preceding claims, wherein the sample is diluted with a saline solution.

14. A kit comprising a labeled HbA -binding agent and a labeled HbF- binding agent.

15. The kit of claim 14, wherein the labeled HbA-binding agent and the labeled HbF-binding agent are comprised in the same vessel.

16. The kit of claim 14-15, wherein the kit further comprises one or more of saline solutions, a fixing agent, a permeabilizing agent, and a staining solution.

17. A kit of claim 16, the kit further comprising: a first vessel, wherein the first tube includes the saline solution, a second vessel, wherein the second vessel includes the fixing and permeabilizing agent, a third vessel, wherein the third vessel contains the staining buffer.

18. The kit of claim 17, wherein the third vessel contains a first fluor coupled to an antibody directed against HbF and a second fluor coupled to an antibody directed against HbAlc.

19. The kit of any of the preceding claims, wherein the contents more of the vessels are in dry form.