JP5791095B2 - Method for detecting PNH type leukocytes - Google Patents

Description

  The present invention relates to a method for detecting PNH leukocytes.

  Bone marrow failure is a disorder that causes cytopenias because of the quantitative and qualitative abnormalities of hematopoietic stem cells, not due to increased destruction of blood cells or secondary hematopoiesis due to bone marrow occupation A state in which blood cell production is continuously reduced. Bone marrow failure includes secondary hematopoietic disorders such as drug-induced aplastic anemia (AA), myelodysplastic syndrome (MDS), refractory anemia (RA), seizures Idiopathic hematopoietic disorders such as nocturnal hemoglobinuria (PNH) are included. These diseases are not always clearly separated from each other, and may move from one to the other. These diseases include a mixture of hematopoietic disorders due to autoimmune disorders and hematopoietic disorders due to abnormalities of hematopoietic stem cells themselves, and bone marrow failure is sometimes collectively referred to as bone marrow failure syndrome.

  By the way, the most important point in primary care for bone marrow failure is to avoid carelessly toxic treatment for bone marrow failure that is likely to be improved by immunosuppressive therapy alone. For this purpose, it is necessary to accurately diagnose the immune pathology in advance in patients with bone marrow failure or patients suspected of having bone marrow failure. That is, in clinical practice, (1) bone marrow failure due to autoimmune disorder; that is, bone marrow failure that can be treated by drug therapy centered on immunosuppressive therapy, and (2) that cannot be treated by drug therapy; Therefore, it is important to correctly distinguish from bone marrow failure requiring hematopoietic stem cell transplantation, and a method capable of accurately performing this discrimination is required.

Among the diagnostic methods for immune pathology in bone marrow failure, the simplest and most reliable test is the detection of PNH trait blood cells (PNH type blood cells) in peripheral blood. PNH type blood cells are blood cells lacking GPI (Glycosylphosphatidylinositol) anchor type membrane protein, and these are derived from abnormal hematopoietic stem cells mutated in the PIGA gene.
PNH-type abnormal blood cells are present in very small amounts in the peripheral blood of healthy individuals, but their proliferation ability is not higher than that of normal hematopoietic stem cells, so it increases to a certain percentage (0.003%) or more. There is no (non-patent document 1). On the other hand, in an environment where immunological damage to normal hematopoietic stem cells exists, PNH-type stem cells are less susceptible to damage by T cells than normal stem cells, and it is considered that PNH-type blood cells increase relatively. .

It has been shown that AA positive for PNH-type blood cells has a significantly higher response rate of the ATG / cyclosporin combination therapy than that of negative AA, and also has a long-term prognosis (Non-patent Document 2). Moreover, in patients with PNH blood cell increased RA, there is a tendency that the response rate of cyclosporine therapy is higher and the rate of transition to leukemia is lower than in patients with non-increased RA (Non-patent Document 3). That is, it can be understood that PNH-type blood cells are expressed only in cases of bone marrow failure due to autoimmune disorders.
From the above, it can be seen that it is extremely important to accurately detect PNH-type blood cells in the preclinical stage in deciding the treatment policy in cases of bone marrow failure such as RA and AA.

  The presence or absence of PNH-type blood cells can be easily discriminated by detecting the blood cell surface antigen by a flow cytometry technique, usually using the remaining blood used for blood count. However, in the blood cell inspection method (hereinafter referred to as “conventional method”) by flow cytometry using an antibody against a blood cell surface antigen which is generally used at present, less than 0.01% of PNH type blood cells are accurately obtained. It is extremely difficult to detect. This is because not only those skilled in the art can detect ultra-small amounts of PNH blood cells of less than 0.01%, but they are proficient in flow cytometry at a high level in detecting PNH blood cells. Because it is necessary. In particular, as long as antibodies are used, non-specific noise is generated in flow cytometry. Therefore, in order to eliminate the noise for a small PNH blood cell signal, the original PNH blood cell signal and the noise are subjectively analyzed. In addition, there is a problem that it must be distinguished based on empirical judgment.

  An overseas report in which PNH blood cells were detected by a conventional method for AA patients is disclosed to have a low rate of about 30% of cases in which an increase in PNH blood cells is observed (Non-patent Document 4). ). However, in these reports, since the specificity of PNH type blood cell detection is low, up to 1% of PNH type blood cells are detected even in healthy individuals, and between AA patients and PALs that have increased PNH type blood cells. Does not show clear boundaries. Such low specificity was the reason why an increase in PNH type blood cells was observed only in about 30% of AA patients.

  In the past, the present inventors used an anti-CD55 antibody / anti-CD59 antibody labeled with FITC and an anti-CD11b antibody (for granulocytes) or anti-glycophorin A antibody (for erythrocytes) labeled with PE to gate cells. , It is disclosed that the boundary between the proportion of PNH blood cells in healthy subjects and the proportion of PNH blood cells in cases of increased PNH such as AA can be reduced to 0.003% (non- Patent Document 2). Since this method (hereinafter referred to as “high-sensitivity method”) has higher specificity and sensitivity than conventional methods, an increase in PNH-type blood cells could be detected in 60% of patients with aplastic anemia. However, in this method, since it is necessary to use different antibodies depending on the type of blood cells to be examined, it has been complicated to detect PNH type blood cells using many types of blood cells. Furthermore, since the binding of antibodies to blood cells decreases in specimens that have passed since blood collection, subjective and empirical judgments by skilled personnel are required to calculate only true PNH blood cells. there were. Therefore, there is a need for a method that can detect PNH-type blood cells accurately and quickly even if they are not proficient in flow cytometry, regardless of the strain of blood cells.

Araten DJ, et al. Proc Natl Acad Sci USA. 1999; 96: 5209-5214. Sugimori C, et al. , Blood. 2006; 107: 1308-1314. Wang H et al. , Blood. 2002; 100: 3897-3902. Maciejewski JP, et al. , Br J Haematol, 2002; 115: 1015-1022.

  There is a demand for a method capable of accurately diagnosing the immune pathology of bone marrow failure syndrome at a pretreatment stage. Specifically, a method capable of accurately detecting PNH-type blood cells, particularly PNH-type granulocytes and monocytes, by a simple method is desired.

The present inventors performed a flow cytometry in which an antibody against a leukocyte surface antigen such as granulocyte antigen (CD11b), monocyte antigen (CD33) and the like and FLAER are combined, thereby allowing a small amount of PNH leukocytes (PNH) in blood cells to be measured. Type granulocytes, PNH type monocytes, etc.) have been found for the first time. In particular, by digitizing the waveform processing in the flow cytometry, anyone can discriminate PNH type leukocytes that require subjective judgment when using the conventional anti-GPI-AP antibody method. I found out that
As a result of further intensive studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A method for detecting PNH-type leukocytes, comprising performing flow cytometry using FLAER and at least one anti-leukocyte surface antigen antibody;
[2] The method according to [1], wherein waveform processing in flow cytometry is digitized;
[3] The anti-leukocyte surface antigen antibody is selected from the group consisting of anti-CD11b antibody, anti-CD33 antibody, anti-CD45 antibody, anti-CD3 antibody, or anti-CD19 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody and anti-CD56 antibody. The method according to [1] or [2], selected from the group consisting of:
[4] The method according to [3], wherein two or more types of PNH leukocytes are detected simultaneously;
[5] A method for examining bone marrow failure syndrome, comprising performing flow cytometry using FLAER and an anti-leukocyte surface antigen antibody;
[6] The method according to [5], wherein the waveform processing in flow cytometry is digitized;
[7] The anti-leukocyte surface antigen antibody is a group consisting of an anti-CD11b antibody, an anti-CD33 antibody, an anti-CD45 antibody, an anti-CD3 antibody, or an anti-CD19 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody and an anti-CD56 antibody. The method according to [5] or [6], selected from the group consisting of:
[8] The method according to any one of [5] to [7], wherein bone marrow failure syndrome can be diagnosed as being autoimmune;
[9] The method according to any one of [5] to [7], which is a method for discriminating between autoimmune bone marrow failure syndrome and hematopoietic stem cell abnormal bone marrow failure syndrome;
[10] The method according to any one of [5] to [9], which is a method for determining the suitability of immunosuppressive therapy for bone marrow failure syndrome;
And so on.

  PNH type leukocytes are present in a very small amount in the peripheral blood of healthy individuals, but the ratio is usually only about 0.001% of the whole. Therefore, in order to determine whether or not PNH type leukocytes are increasing, it is necessary to accurately quantify PNH type leukocytes on the order of 0.001%. Since PNH leukocytes are blood cells in which the GPI-anchored membrane protein is “deleted”, conventionally, these blood cells have been detected by utilizing the property that anti-GPI-anchored membrane protein antibodies do not bind. However, cells damaged during the antibody treatment process have weak binding to anti-GPI-anchored membrane protein antibodies, so the position differs from true PNH leukocytes on the flow cytometry dotgram. It comes to be confused with PNH leukocytes. In order to distinguish such a false PNH leukocyte population from true PNH leukocytes, subjective judgment based on many experiences was required.

The method of the present invention solves these problems all at once and makes it possible to detect PNH type leukocytes simply and accurately. By applying the method of the present invention, (1) the generation of noise based on non-specific reaction in the detection of PNH-type leukocytes is not only suppressed, but (2) the cell population of PNH-type leukocytes is displayed on the dotgram. It is clearly depicted. This makes it possible to easily detect PNH-type leukocytes in many facilities regardless of the level of proficiency in flow cytometry of those involved in the detection of PNH-type leukocytes. Furthermore, by combining with antibodies against each surface antigen characteristic of leukocytes, (3) any kind of PNH leukocytes (PNH granulocytes, PNH monocytes, PNH lymphocytes, etc.) can be detected at once. Is possible.
By using this detection method, it is possible to diagnose the immune pathology of bone marrow failure syndrome. Since PNH leukocytes are significantly increased in autoimmune bone marrow failure cases, the method of the present invention can differentiate autoimmune bone marrow failure syndrome from bone marrow failure based on abnormalities of hematopoietic stem cells themselves.

FIG. 3 shows gating for detecting PNH type granulocytes using the method of the present invention. It is the result of having analyzed about the threshold value of PNH type | mold granulocyte for 50 healthy subjects using the method of this invention. FIG. 6 shows gating for detecting PNH type monocytes using the method of the present invention. It is the result of having analyzed about the threshold value of the PNH type | mold monocyte for 50 healthy subjects using the method of this invention. It is the result of detecting PNH type T cells (ac), PNH type B cells (d) and PNH type NK cells (e) using the method of the present invention. It is the result of having analyzed about the threshold value of a PNH type T cell (ac), a PNH type B cell (d), and a PNH type NK cell (e) using the method of the present invention. It is the figure which compared the method (C method) of this invention, the conventional method (A method), and the highly sensitive detection method (B method) about the detection accuracy of PNH type | mold leukocyte. It is the result of detecting PNH type erythrocytes by a highly sensitive detection method. It is a figure which shows that the presence or absence of PNH type | mold leukocytes influences the response rate and survival rate of immunosuppressive therapy in an AA patient.

1. Method for detecting PNH-type blood cells In the present specification, “PNH-type leukocytes” refer to GPI-anchored membrane protein-deficient leukocytes derived from hematopoietic stem cells mutated in the PIGA gene. In the PIGA gene, mutations usually occur at a certain rate, so even a healthy person has a very small number of PIGA mutant stem cells, but this mutant hematopoietic stem cell present in the bone marrow of a healthy person continues to remain stationary. This stem cell-derived PNH-type leukocyte is not detected in peripheral blood (Muchizuki K, et al., Blood, 112: 2160-2162 (2008)).
On the other hand, in an environment where immunological damage to normal hematopoietic stem cells exists (for example, autoimmune bone marrow failure cases), hematopoietic stem cells mutated in the PIGA gene lack GPI-anchored membrane proteins. It is less susceptible to damage by T cells than normal stem cells. Since PIGA mutant stem cells survive in such an environment, it is considered that PNH leukocytes increase relatively.
However, even in such cases, PNH leukocytes are present in only about 0.1% of the whole blood cells, and therefore it is necessary to use extremely sensitive flow cytometry for detection of PNH leukocytes. The detection method of the present invention provides a new means of flow cytometry particularly suitable for the detection of such a trace amount of PNH type leukocytes.

  The “GPI-anchored membrane protein” binds to a glycolipid moiety called phosphatidylinositol (PI), glucosamine (GlcN), three mannoses (Man), and ethanolamine phosphate (EtN-P), called GPI anchor. It refers to all proteins expressed on the membrane surface, and more than 100 types of molecules have been identified so far. In all GPI-anchored membrane proteins, the glycolipid moiety is conserved at a high rate. Blood cells derived from hematopoietic stem cells having a mutation in the PIGA gene cannot produce a GPI anchor, and as a result, cannot produce a GPI-anchored membrane protein.

GPI-anchored membrane proteins that are highly expressed in blood cells show different expression patterns depending on the type of blood cells. For example, complement control proteins CD55, CD59 (whole blood cells), HRF (erythrocytes, platelets); enzyme proteins acetylcholinesterase (erythrocytes), alkaline phosphatase (neutrophils); receptor CD14 (monocytes), CD16 (neutrophils); adhesion molecules CD48 (T cells, B cells, monocytes, activated platelets), CD58 (erythrocytes, granulocytes, lymphocytes), CD66b, CD66c (neutrophils); CD73 (B Cell), CD87 (monocytes, neutrophils) and the like.
“FLAER” (Flare, Fluorescent-Labeled Inactive Toxin Aerolysin) described below binds to these GPI-anchored membrane proteins.

In this specification, “FLAER” is a toxin derived from a bacterium that specifically binds to a GPI anchor portion of a GPI-anchored protein on a cell surface, that is, a glycolipid portion. It is a kind of fluorescent bacterial protein called. Since FLAER uses the GPI anchor part as an epitope, unlike the monoclonal antibody having the protein part of the GPI anchor type protein as an epitope, it can theoretically recognize almost all GPI anchor type membrane proteins.
Therefore, leukocytes to which FLAER does not bind (not stained with FLAER) are recognized as leukocytes lacking a GPI-anchored membrane protein (for example, CD59), and thus can be determined to be PNH leukocytes.
There are two types of FLAER, liquid and powder, but liquid FLAER is desirable. In the method of the present invention, the liquid FLAER can obtain a more stable result. FLAER usually excites at 488 nm and emits fluorescence at 530/30 nm.

In the present specification, the “anti-leukocyte surface antigen antibody” is an antibody that can be used in combination with FLAER, and is preferably an antibody against a cell antigen specifically expressed in each blood cell lineage. In the present invention, for example, anti-CD11b antibody is detected when PNH type granulocytes are detected, anti-CD33 antibody is detected when PNH type monocytes are detected, and anti-CD3 antibody and anti-CD4 are detected when PNH type T cells are detected. It is preferable to use an antibody or an anti-CD3 antibody and an anti-CD8 antibody, an anti-CD19 antibody when detecting PNH type B cells, and an anti-CD56 antibody when detecting PNH type NK cells.
Also preferably, these antibodies are divided into two groups. The group consisting of anti-CD11b antibody, anti-CD33 antibody, anti-CD45 antibody, and anti-CD3 antibody is used to simultaneously detect PNH type granulocytes and PNH type monocytes. On the other hand, a group consisting of an anti-CD19 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody and an anti-CD56 antibody is used to simultaneously detect PNH type B cells, T cells and NK cells.
According to the method of the present invention, it is possible to simultaneously detect two or more types of PNH-type leukocytes by selecting one of the above two groups and using one or more antibodies thereof.
The class of these antibodies is not particularly limited, and commercially available antibodies may be used.

  In the method of the present invention, blood cell components are obtained from a biological sample before flow cytometry, and the blood cell components are further treated with FLAER and the anti-leukocyte surface antigen antibody.

  The biological sample that can be used in the method of the present invention is not particularly limited as long as it contains a blood cell component, but is preferably blood (particularly peripheral blood) or lymph, more preferably peripheral blood to which EDTA or heparin is added.

  The method for obtaining blood cell components from a biological sample is not particularly limited. For example, when obtaining blood cell components other than erythrocytes, a hemolysis reagent containing ammonium chloride, potassium bicarbonate, EDTA · 4Na in whole blood centrifuged at a constant speed And a method of lysing red blood cells.

  On the other hand, in order to obtain erythrocytes from blood, for example, plasma components are removed from the blood collected before the above centrifugation, and phosphate buffered saline (PBS) is added to the erythrocyte layer. Thereby, an erythrocyte suspension is obtained.

  The method of treating the obtained blood cells with FLAER and anti-leukocyte surface antigen antibody is not particularly limited, and various treatment conditions such as the amount of blood cells, the amount of FLAER, the treatment time of anti-leukocyte surface antibody, and the treatment temperature are detected. It can be determined appropriately according to the type of PNH-type leukocytes, flow cytometry conditions, and the like. However, in order to detect PNH type leukocytes with high sensitivity by the method of the present invention, it is desirable to carry out the treatment under the processing conditions described in the examples of the present specification.

  Anti-leukocyte surface antigen antibodies may be used for subsequent detection in flow cytometry, such as FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allophycocyanin), PerCP-Cy5.5, PE-Cy7, APC, APC-H7, etc. Those labeled with a fluorescent dye or a fluorescent substance can be used.

  Moreover, when processing with FLAER and an antibody, you may make a cell detection marker react simultaneously for the purpose of removing a dead cell in the gating process and analysis process in the histogram mentioned later. In general, a method of detecting dead cells using a specific marker and excluding them by gating is employed. Examples of dead cell detection markers include 7-aminoactinomycin (7-AAD) that binds between cytosine-guanine base pairs of dead cell DNA and DAPI that binds to AT (adenine thymine) -rich region. . By performing gating, which will be described later, using these as indices, unqualified cells can be further removed, and the object of the present invention can be achieved.

The fluorescent dye or fluorescent substance is appropriately selected depending on the type of leukocytes to be detected, the number of cells, flow cytometry condition settings, etc., but when using multiple antibodies at least for the purpose of detecting multiple types of cells simultaneously. May use different types of fluorescent dyes for each antibody, cell detection marker and the like.
For example, when FACSCanto (registered trademark) is used as a flow cytometer, detection of multiple antibodies and cells labeled with fluorescent dyes or fluorescent substances such as PE, FITC, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, etc. A marker or the like can be used at the same time.

  By these operations, a suspension of blood cells bound to FLAER and one or more anti-leukocyte surface antigen antibodies (hereinafter sometimes referred to as “cell suspension”) is obtained.

Next, flow cytometry is performed using blood cells treated with FLAER and anti-leukocyte surface antigen antibody as a specimen.
The flow cytometry technique is not particularly limited, but it is desirable to use a flow cytometer in which waveform processing is digitized (that is, adopting a “digital waveform processing method”).
That is, the preferred “flow cytometry” in the present invention means flow cytometry performed using a flow cytometer capable of digital waveform processing (“digital waveform processing method”). In this specification, an instrument used for flow cytometry is referred to as a flow cytometer.

The “digital waveform processing method” in this specification means that the fluorescence pulse signal obtained from the PMT (photomultiplier tube) detector is AD-converted by various methods and the obtained value is analyzed. AD conversion refers to converting an analog signal into a digital signal.
In particular, as will be described later, as a “digital waveform processing method” when FACSCanto (registered trademark) is adopted as flow cytometry, for example, the above fluorescence pulse signal is directly AD converted into linear data with a resolution of 262,144 channels, Thereafter, analysis is performed by a digital signal processor (DSP) by Log logarithmic conversion and fluorescence correction (inverse matrix processing).

  The method of the present invention is based on the first finding that the combination of FLAER and anti-leukocyte surface antigen antibodies with flow cytometry capable of digital waveform processing is the most suitable means for detection of PNH leukocytes. And completed.

  Examples of the flow cytometer that can realize the digital waveform processing method include FACSCanto, BD LSR2, and FACSAria. Of these, FACSCanto (registered trademark) is preferable. FACSCanto (registered trademark) is a flow cytometer that is most suitable for the method of the present invention because it is easy to handle for general clinical engineers who handle many specimens because the method of use is simplified. Hereinafter, a flow cytometer will be described using FACSCanto as an example.

The blood cell suspension attached to the flow cytometer is prepared as a single (cell non-aggregated) suspension, and each blood cell is passed through a laser beam.
The cell suspension contains dead cells and debris, which cause noise in flow cytometry. In order to obtain correct data, cells need to be flowed one by one. Therefore, before the suspension is applied to the flow cytometer, it is desirable to homogenize the cells in the cell suspension into one cell as much as possible and to remove impurities such as dead cells and debris as much as possible. For homogenization, a general method known per se can be applied. For example, cell suspension filtering or vortexing is performed according to the protocol of each operation.
The filter used for filtering is not particularly limited as long as it has a mesh size capable of removing dead cell agglomerates and debris, and examples thereof include a nylon mesh having a pore diameter (40 μm).
Before attaching to the flow cytometer, it is desirable to visually check again whether debris or cell clumps remain, and if they remain, it is desirable to pass through the filter again.

  In order to allow each cell in the cell suspension to pass through the laser beam, in flow cytometry, a sample core consisting of a sample fluid stream in which a single cell flows continuously and a sheath fluid stream surrounding it is formed. There is a need to. It is preferable to adjust the flow so that one cell passes through the center of the beam in the sample core, and to increase the intensity by focusing the laser beam as much as possible so that a plurality of cells are not irradiated at once.

  Since the intensity of the laser light becomes weaker from the beam center to the edge, it is preferable that all cells pass through the beam center along the same path in order to increase sensitivity and resolution. Therefore, the smaller the diameter of the sample core, the better. These settings can be appropriately set by those skilled in the art regardless of the flow cytometer used.

The flow rate of the sample core is not particularly limited and can be appropriately set by those skilled in the art in relation to sensitivity, resolution, processing speed, and the like. However, the present invention is intended to detect a small amount of PNH-type leukocytes. It is desirable to be as slow as possible.
In particular, when FACSCanto (registered trademark) is used as a flow cytometer, the sheath pressure for controlling the value of the sample flow (the pressure applied to the container containing the sheath liquid) is fixed at 4.5 psi. The pressure of the sample is not quantified (psi), so that the amount of the sample sucked is Low = 10 μL / min, Med = 60 μL / min, High = 120 μL / min, and the sheath liquid is 18 mL / min. Thus, the pressure in the tube during sample suction is adjusted.
The flow rate at the time of measurement is preferably from 100 to 300 cells / second, particularly preferably from 100 to 200 cells / second.

  The flow of the sample core is controlled by the sheath pressure, and the diameter of the sample core is controlled by the difference between the sheath pressure and the sample pressure. For example, while keeping the sample pressure constant, increasing the sheath pressure speeds up the flow of the sample core and decreases the sample core diameter. These settings can be appropriately set regardless of the flow cytometer used.

The laser used as the light source for flow cytometry is argon laser, helium neon laser, helium cadmium laser, semiconductor as long as it does not alter blood cells and FLAER bound to blood cells or anti-leukocyte surface antigen antibodies, and scattered light is generated from blood cells Any known laser such as a laser or a light-excited semiconductor laser can be used.
FACSCanto (registered trademark) minimizes leakage of fluorescence by using an optical gel in addition to the dual laser / separate beam spot method. In addition, when a plurality of fluorescent dyes are simultaneously detected by the conventional serial method or the branching optical method, the intensity of the fluorescent signal attenuates in proportion to the number of optical filters that pass through until reaching the detector of the optical system. However, FACSCanto (registered trademark) overcomes this problem by adopting an octagon / trigon fluorescence detection system.

  FACSCanto (registered trademark) realizes full matrix compensation by digital waveform processing. In multi-color analysis, the closer the fluorescent signals with a wavelength range are, the more the fluorescent signals are detected by the adjacent fluorescent detectors, so-called "leakage of fluorescence" occurs. It is essential. In the conventional analog method, the pulse signal is corrected by subtracting the leakage fluorescence coefficient from the pulse signal in the process of signal processing in the fluorescence correction circuit, but this method is particularly effective for correction of four or more types of multi-color analysis. Because of its limitations, FACSCanto® employs a full matrix compensation system that mathematically corrects using an inverse matrix. Specifically, the fluorescence pulse signal obtained from the PMT detector is directly AD converted into linear data with a resolution of 262,144 channels, and then converted into log data and corrected for fluorescence (inverse matrix processing) by a numerical processor. To analyze.

In the detection method of the present invention, in order to detect a target PNH-type leukocyte, the measurement parameters of individual cells are plotted and a histogram is created.
The number of parameters may be one parameter or two or more parameters, but since the method of the present invention detects at least two FLAER and anti-leukocyte surface antigen antibodies, a histogram (cytogram) of two or more parameters is obtained. It is desirable to use it. When measurements are made on many types of cells simultaneously using a plurality of antibodies, for example, list mode data may be used to change parameter combinations and obtain different histograms each time.
Examples of the measurement parameter to be analyzed include the following parameters.
(1) Forward scattered light (Forward Scatter, FSC)
(2) Side scattered light (Side Scatter, SSC)
(3) 7-ADD (DAPI)
(4) Fluorescence (FL)

In this specification, “gating” refers to selecting only a cell population of interest in a sample and creating a histogram of only those cells (also referred to as “setting a gate”). In addition to gating by the forward scattered light / side scattered light plot, gating can be performed on the cell population to be searched using the fluorescence parameter, and this can be used for narrowing down to the analysis population.
At least one of the fluorescence parameters used for gating is FLAER. As described above, FLAER is used to detect GPI-anchored membrane proteins. At least one is derived from the anti-leukocyte surface antigen antibody (labeled with a fluorescent dye or the like).

Individual cell populations are sorted by setting gates to the cell clusters on the two-dimensional screen displayed for the two or more fluorescence parameters (at least FLAER fluorescence and antibody fluorescence). In this case, for example, when analyzing granulocytes, the following operation is performed.
(1) Develop with FSC-A (Area: pulse area) and SSC-A and gate the leukocyte region consisting of granulocytes, monocytes, and lymphocytes;
(2) Expand with FSC-W (Width: pulse width) and FSC-H (High: pulse height) to exclude cells that are significantly out of the cell population in terms of electrical signals;
(3) expand with SSC-W and SSC-H and exclude cells that are significantly out of the cell population in terms of electrical signal;
(4) Exclude dead cells (gating 7-AAD positive cells and DAPI negative cells);
(5) gate the granulocyte region;
(6) gate CD11b strong positive cells;
(7) The cells derived from (1) to (6) are defined as pure mature granulocytes. FLAER expression in this cell group is analyzed.

  In the present specification, the histogram gated in this way is referred to as “gated histogram” or “gated histogram”.

Finally, statistical information about the cells in the region can be obtained by setting the region in the gated histogram.
Here, the “region” refers to a region set for narrowing down data in order to perform analysis (cluster analysis) focusing on the target cell population. Setting a region for each target cell population is called “region setting”.
The region is set using various methods and conditional expressions. These methods and conditional expressions are known to those skilled in the art, and can be set as appropriate according to the purpose.

When anti-CD11b antibody is used as an anti-leukocyte surface antigen antibody and PNH granulocytes are detected, the population of PNH granulocytes is
(1) low reactivity to FLAER;
(2) reacts with anti-CD11b antibody;
It appears in the upper left region on the two-parameter histogram of FLAER (X axis) and anti-CD11b antibody (Y axis).
Statistical information on the obtained PNH-type leukocytes can be obtained by setting a region for the cell population observed in the upper left region on this histogram.

Similarly, a population of PNH monocytes, a population of PNH T cells, a population of PNH B cells, a population of PNH NK cells, etc.
(1) low reactivity to FLAER;
(2) reacts with antibodies to each cell specific cell surface antigen;
Therefore, it appears in the upper left area on the two-parameter histogram of FLAER (X axis) and anti-cell surface antigen antibody (Y axis). The antibody against the cell surface antigen specific to each cell is as described above.
Statistical information on the obtained PNH-type leukocytes can also be obtained by setting a region for the cell population observed in the upper left region on this histogram.

  In the dotgram obtained by the method of the present invention, the target PNH leukocyte population is clearly distinguished from other leukocyte populations.

The results obtained by the method of the present invention show remarkable effects in the following points as compared with the conventional methods for detecting PNH blood cells.
(1) Since a population of negative cells (PNH type leukocytes) of GPI-anchored membrane protein is clearly depicted on a histogram, it is easier to visually recognize than the conventional method.
(2) Cells observed in the upper left region because a population of negative cells (PNH type leukocytes) of GPI-anchored membrane protein forms a clump on the histogram and is clearly separated from the positive cell population. It is possible to determine whether or not there is an increase in PNH type leukocytes simply by confirming the number.
(3) There is very little noise due to dead cells and non-specific binding, and there is no need to omit noise by subjective judgment.

By the way, since flow cytometry capable of digital waveform processing and liquid FLAER used in the method of the present invention can be obtained at any facility, various condition settings should be studied with the aim of detecting PNH type leukocytes with high accuracy. It is thought that this can be done at other facilities. However, no examples of detecting PNH type leukocytes using flow cytometry capable of digital waveform processing and liquid FLAER have been reported so far, and according to the method of the present invention, only the setting of these conditions was examined. Then, on the histogram, a dramatic change from the conventional method is observed, which is unexpected.
In other words, the conventional high-sensitivity flow cytometry method could not detect minute PNH type leukocytes with the same sensitivity and specificity as the method of the present invention. It is expected that minute PNH type leukocytes can be detected and can be easily reproduced any number of times.
In the present invention, as a result of intensive studies by the present inventors, for the purpose of “detecting PNH-type leukocytes”, “FLAER” and “anti-leukocyte surface antigen antibody” are used in flow cytometry, particularly “ It has come to adopt very simple conditions, which are done with "digitized" flow cytometry. However, by adopting this condition, it became possible to automatically detect a very small amount of PNH-type leukocytes that could not be expected by the conventional method. In particular, the fact that the “fake PNH leukocytes” that have prevented the generalization of the conventional method from being subjectively excluded can be regarded as a breakthrough in the detection method of PNH blood cells.

  Furthermore, according to the method of the present invention, in addition to using new instrument reagents such as flow cytometry and FLAER in which waveform processing is digitized, cell processing that has been performed when detecting PNH-type blood cells in the conventional method, By adding a condition setting in gating or the like, it is possible to more accurately detect around 0.003% PNH type leukocytes. These detailed condition settings are described in detail in the embodiments of the present application.

In addition, when performing flow cytometry using FACSCanto (trademark), it is desirable to perform compensation of a flow cytometer before flow cytometry operation.
In this case, the voltage is set so that the relative fluorescence intensity of the positive cell population of each antibody is about 1 × 10 ⁴ to 1 × 10 ⁵ , and cells stained with each fluorescent antibody alone (positive control) are unstained cells. Flow the sample mixed with (negative control) in order. At that time, a positive gate is put on the positive cell population. After all the specimens for compensation have flowed, the Compensation Calculation button is pressed to perform compensation (the FACSCanto (registered trademark) automatically performs fluorescence correction, so there is no need to manually perform fluorescence correction).

2. As described above, there are two types of bone marrow failure syndromes, one caused by an autoimmune disorder and the other caused by abnormalities in the hematopoietic stem cells themselves. In the treatment of bone marrow failure syndrome, it is important to select a treatment suitable for such a disease state.
In an environment where immunological damage to normal hematopoietic stem cells exists, PNH-type stem cells are less susceptible to damage by T cells than normal stem cells, and thus PNH-type leukocytes are relatively increased. Therefore, by detecting PNH type leukocytes in the subject, it can be determined whether or not the subject is suffering from autoimmune bone marrow failure.
The present invention provides a new test method for bone marrow failure syndrome in which flow cytometry is performed using FLAER and an anti-leukocyte surface antigen antibody.

  The disease targeted by the method of the present invention is a disease belonging to bone marrow failure syndrome caused by autoimmune disorder (hereinafter referred to as “autoimmune bone marrow failure syndrome”) among bone marrow failure syndromes. This is a low-risk myelodysplastic syndrome of a type with few blasts that is difficult to distinguish between anemia and aplastic anemia. If PNH-type leukocytes are detected in these diseases, it can be diagnosed that the subject is suffering from autoimmune bone marrow failure. On the other hand, an increase in PNH type leukocytes is not observed in bone marrow failure syndrome due to abnormality of hematopoietic stem cells themselves. That is, the present invention provides a test method for autoimmune bone marrow failure syndrome.

Further, if PNH type leukocytes are detected by the method of the present invention in a subject suspected of having bone marrow failure syndrome due to a decrease in the number of reticulocytes, platelets, white blood cells, etc. in the blood, the subject is autoimmune bone marrow failure syndrome. It can be diagnosed that the possibility of bone marrow failure syndrome based on abnormal hematopoietic stem cells is low.
On the other hand, if PNH-type leukocytes cannot be detected even by the method of the present invention, the subject is unlikely to develop autoimmune bone marrow failure syndrome and bone marrow failure syndrome based on abnormal hematopoietic stem cells. It can be determined that the possibility is high.
That is, the present invention provides a method for determining whether bone marrow failure syndrome in a subject is autoimmune or hematopoietic stem cell abnormality.

In addition, if PNH-type leukocytes are detected by the method of the present invention in a subject patient suspected of having bone marrow failure syndrome due to a decrease in the number of reticulocytes, platelets, white blood cells, etc. in the blood, the subject is improved by immunosuppressive therapy. It can also be judged that there is a high possibility of doing. On the other hand, if PNH leukocytes cannot be detected by the method of the present invention, it can be determined that the response rate of the immunosuppressive therapy in the subject is not high.
That is, the present invention provides a method for determining the suitability of immunosuppressive therapy for bone marrow failure syndrome.

  As described above, a small amount of PNH-type leukocytes is detected even in a healthy person. However, according to the method of the present invention, a threshold value for the amount of PNH-type leukocyte between a healthy person and a bone marrow failure syndrome can be set. Is also possible.

In this specification, “autoimmune bone marrow failure syndrome” means bone marrow failure syndrome caused by hematopoietic stem cells being attacked and reduced by their own lymphocytes, antibodies, etc., and recovery is expected by immunosuppressive therapy This refers to bone marrow failure syndrome. Examples of such diseases include aplastic anemia and a part of low-risk myelodysplastic syndrome.
In the present specification, “hematopoietic stem cell abnormal bone marrow failure syndrome” means bone marrow failure syndrome caused by hematopoietic stem cells not proliferating / differentiating due to acquired genetic abnormality. It refers to bone marrow failure syndrome that cannot be recovered. Examples of such diseases include myelodysplastic syndrome and hypoplastic leukemia.

  The subject to which the method of the present invention can be applied is a human. More preferably, it is a human who exhibits pancytopenia. The biological sample that can be used in the method of the present invention is limited to human-derived peripheral blood.

  Hereinafter, the present invention will be described more specifically with reference to examples, comparative examples, and reference examples of the present invention.

(Description of equipment)
FACSCanto (registered trademark), which is a flow cytometer used in the present invention, is manufactured by Becton Dickinson.

(Example 1) Treatment of biological sample Peripheral blood was adopted as a biological sample, and the following operation was performed to collect a leukocyte cell suspension from peripheral blood.
(1) From 7 mL of EDTA-2Na peripheral blood stored at 4 ° C., 2 mL was separately transferred to another falcon tube for detection of PNH-type erythrocytes, and stored at 4 ° C. The remaining 5 mL of EDTA-2Na blood was centrifuged as it was at 2000 rpm for 10 minutes to remove the plasma portion.
(2) After adding 5-10 times amount of Lysis buffer (a solution containing 8.26 g of ammonium chloride, 1.0 g of potassium hydrogen carbonate, 0.037 g of 4NA (EDTA · 4Na) in 1 L) to the pellet and mixing by inversion. It was allowed to stand at room temperature for 8 minutes. During this time, it was confirmed that hemolysis was progressing by mixing by inversion as appropriate.
(3) Centrifugation was performed at 500 g for 1 minute (SEROMATIC II selection 3 manufactured by KUBOTA), and the supernatant was removed with an aspirator leaving a pellet.
(4) 5 to 10 times the amount of Lysis buffer was added again, and the mixture was allowed to stand at room temperature for 5 minutes while mixing by inversion. In case of insufficient hemolysis, the operation was extended to a total of 15 minutes.
(5) Centrifugation was performed at 500 g for 1 minute (SEROMATIC II selection 3 manufactured by KUBOTA), and the supernatant was removed with an aspirator, leaving a pellet.
(6) For blocking, 140 μL of 0.5% BSA + PBS was added and vortexed, and then allowed to stand at room temperature for 10 minutes.
If the specimen is within 24 hours after collection, blocking is not necessary.
Using this as a cell suspension, the following experiment was conducted.

(Example 2) Incubation with FLAER and antibody The following operation was performed to prepare a sample to be subjected to flow cytometry.
(1) Two 5 mL Falcon tubes were prepared, and 50 μL of each was taken from the cell suspension obtained in Example 1 and placed on the bottom of each tube. At this time, care was taken so that the suspended liquid did not adhere to the wall of the tube.
(2) In one tube, FLAER (10-6 M, Pinewood Scientific Services) 6 μL, anti-CD11b-PE (50 ug / mL, BD Biosciences) 4 μL, 7 AAD (50 ug / mL, BD Biosciences) 4 μL, anti-CD45-PE-Cy7 (50 μg / mL, manufactured by BD Biosciences) 4 μL, anti-CD33-APC (6.25 μg / mL, manufactured by BECKMAN COULTER) 4 μL, anti-CD3-APC-H7 (200 μg / mL) 4 μL of BD Biosciences). The other tube was used as a negative control, and antibodies other than FLAER were added. At this point, PBS used for washing later was returned to room temperature. These operations were performed in a dark room (at least in a room where the fluorescent lamp was turned off). Also, it was noted that the reagent tube was not left open.
(3) After vortexing the mixed solution, each tube was shielded from light and allowed to stand in a refrigerator at 4 ° C. for 20 minutes.
(4) 3 mL of PBS was added to each tube and centrifuged at 500 g for 1 minute (selection 3 of SEROMATIC II manufactured by KUBOTA). Next, the supernatant was removed with an aspirator leaving the pellet.
(5) Each tube was shaken by rubbing the rack knit (meaning loosening the pellet), and 400 μL of PBS was added.
(6) After light pipetting, the cells were passed through a nylon mesh filter to remove dead cell clumps, then transferred to a new tube and subjected to flow cytometry analysis.

(Example 3) Flow cytometry (I) Compensation A specimen for compensation was performed using residual blood derived from healthy subjects the day before stored at 4 ° C.
(1) Sample diluted with 400 mL of PBS (hereinafter referred to as “unstained sample”) with 50 μL of cell suspension not added for compensation, and each fluorescent antibody described in Example 2 (2) was added alone. Specimens (hereinafter referred to as “fluorescent antibody single specimen”) were prepared. Note that 50 μL each of unstained samples were added to the FLAER sample and the CD45 sample.
(2) Data was saved by attaching to FACSCanto (registered trademark) in order. The number of cells was 2 × 10 ⁴ only for 7AAD and 5 × 10 ³ for others.
(3) The compensation button of FACSCanto (registered trademark) was pressed to perform automatic correction.

(II) Gating Gating was performed on the granulocyte region by the following method.
(1) Development was performed with FSC-A and SSC-A, and a leukocyte region consisting of granulocytes, monocytes, and lymphocytes was gated.
(2) The cells were developed with FSC-W and FSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(3) The cells were developed with SSC-W and SSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(4) 7-AAD positive dead cells were excluded.
(5) The granulocyte region inferred from the complexity and size of the cell structure was gated with CD45 and SSC-A.
(6) The CD11b strong positive cells were gated.

  The cells derived by the operations (1) to (6) can be defined as pure mature granulocytes. Next, FLAER expression in this cell group was analyzed.

(III) Analysis When a histogram is developed with the leukocyte lineage marker (granulocyte: CD11b, monocyte: CD33, lymphocyte: CD3) on the vertical axis and FLAER on the horizontal axis, PNH type blood cells are Upper Left (UL) Expands to an area. Cells in the UL region were defined as PNH type granulocytes, and the ratio was calculated by (UL cell number / UR (Upper Right) cell number + UL cell number) × 100 (%). FIG. 1-1 shows the result of applying a gate to PNH type granulocytes by the above method to perform this calculation.
On the other hand, by the same flow cytometric analysis of PNH granulocytes in 50 healthy subjects, a significant increase in PNH type cells in bone marrow failure syndrome can be defined as 0.003% or more of PNH granulocytes. It was. The results are shown in FIG.

(Example 4) Detection of PNH-type monocytes After preparing a sample to be subjected to flow cytometry from peripheral blood in the same manner as in Examples 1 and 2, FACSCanto was subjected to the compensation described in Example 3 (I). The sample was attached to (registered trademark). Subsequently, gating was performed by the following method to detect PNH type monocytes.

(1) Development was performed with FSC-A and SSC-A, and a leukocyte region consisting of granulocytes, monocytes, and lymphocytes was gated.
(2) The cells were developed with FSC-W and FSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(3) The cells were developed with SSC-W and SSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(4) 7-AAD positive dead cells were excluded.
(5) A gate was put on the monocyte region.
(6) The CD33 strong positive cells were gated.

The cells derived by the operations (1) to (6) can be defined as pure mature monocytes. FLAER expression in this cell group was analyzed by the same method as in Example 3 (III). FIG. 2-1 shows the result of applying gates to PNH type monocytes by the above method to perform this calculation.
On the other hand, by the same flow cytometric analysis of PNH monocytes in 50 healthy subjects, a significant increase in PNH cells in bone marrow failure syndrome can be defined as 0.05% or more of PNH monocytes. It was. The results are shown in Fig. 2-2.

(Example 5) Detection of PNH type T lymphocytes After preparing a sample to be subjected to flow cytometry from peripheral blood in the same manner as in Examples 1 and 2, the compensation described in Example 3 (I) was performed. The sample was attached to FACSCanto (registered trademark). Subsequently, gating was performed by the following method to detect PNH type T lymphocytes.

(1) Development was performed with FSC-A and SSC-A, and a leukocyte region consisting of granulocytes, monocytes, and lymphocytes was gated.
(2) The cells were developed with FSC-W and FSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(3) The cells were developed with SSC-W and SSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(4) The lymphocyte region was gated.
(5) The CD3 strong positive cells were gated.
(6) If necessary, gates were further applied to CD8 and CD4 positive cells, respectively.

The cells derived by the operations (1) to (6) can be defined as pure mature lymphocytes. FLAER expression in this cell group was analyzed by the same method as in Example 3 (III). The results of FLAER analysis for CD3-positive T lymphocytes are shown in FIG. 2-3a, and the results of FLAER analysis by further dividing CD3-positive T lymphocytes into CD8-positive T lymphocytes and CD4-positive T lymphocytes are shown in 2-3b and 2- Each is shown in 3c.
On the other hand, according to the analysis of PNH type T lymphocytes in 50 or more healthy subjects by the same method as described in Example 3 (III), a significant increase in PNH type cells in myelogenous failure syndrome was Lymphocytes could be defined as 0.1% or more. The results are shown in FIGS. 2-4a, 2-4b and 2-4c.

(Example 6) Detection of PNH type B cells After preparing a sample to be subjected to flow cytometry from peripheral blood in the same manner as in Examples 1 and 2, FACSCanto was subjected to the compensation described in Example 3 (I). The sample was attached to (registered trademark). Next, gating was performed by the following method to detect B cells.

(1) Development was performed with FSC-A and SSC-A, and a leukocyte region consisting of granulocytes, monocytes, and lymphocytes was gated.
(2) The cells were developed with FSC-W and FSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(3) The cells were developed with SSC-W and SSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(4) The lymphocyte region was gated.
(5) CD19 strong positive cells were gated.

Cells derived by the operations of (1)-(5) can be defined as pure mature B cells. FLAER expression in this cell group was analyzed by the same method as in Example 3 (III). The results are shown in Fig. 2-3d.
In addition, the analysis of PNH type B cells in 50 or more healthy subjects revealed that a significant increase in PNH type cells in bone marrow failure syndrome could be defined as 0.05% or more of PNH type B cells. The results are shown in FIGS. 2-4d.

(Example 7) Detection of PNH-type NK cells After preparing a sample to be subjected to flow cytometry from peripheral blood in the same manner as in Examples 1 and 2, FACSCanto was subjected to the compensation described in Example 3 (I). The sample was attached to (registered trademark). Next, gating was performed by the following method to detect NK cells.

(1) Development was performed with FSC-A and SSC-A, and a leukocyte region consisting of granulocytes, monocytes, and lymphocytes was gated.
(2) The cells were developed with FSC-W and FSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(3) The cells were developed with SSC-W and SSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(4) The lymphocyte region was gated.
(5) The CD3 strong positive cells were gated.
(6) The CD56 positive cells were gated.

The cells derived by the operations (1) to (6) can be defined as pure mature NK cells. FLAER expression in this cell group was analyzed by the same method as in Example 3 (III). The results are shown in Fig. 2-3e.
Moreover, the analysis of PNH type NK cells in 50 or more healthy subjects revealed that a significant increase in PNH type cells in bone marrow failure syndrome could be defined as PNH type NK cells 0.3% or more. The results are shown in FIGS. 2-4e.

(Comparative Example 1) Detection of PNH type blood cells by conventional method (when not using FLAER and flow cytometer capable of digital waveform processing)
Conventionally, single-color flow cytometry of CD59 or two-color flow cytometry by CD55-PE and CD59-FITC has been performed for the detection of PNH type blood cells.
In this case, a model (such as FACScan or FACSCalibur) that does not employ digital waveform processing has been used as the flow cytometer.
Thus, detection of PNH-type blood cells was attempted by a conventional method not using a flow cytometer capable of FLAER and digital waveform processing. The results are shown in FIG. 3 (Method A in FIG. 3).
In the method of Comparative Example 1, the cell population in the UL region does not form an agglomerate, and the region is unclear. Therefore, the sensitivity and specificity in detecting PNH blood cells are low, and a trace amount of PNH blood cells can be detected accurately. could not.

(Comparative Example 2) Detection of PNH type leukocytes by antibody type high sensitivity method (when using flow cytometer capable of digital waveform processing but not using FLAER)
Next, detection of PNH type leukocytes was attempted using FACSCanto and an antibody without using FLAER. The results are shown in FIG. 3 (Method B in FIG. 3).
Although the method of Comparative Example 2 was relatively clearer than the method of Comparative Example 1, the cell population in the UL region still did not form agglomeration, and the region was unclear. Therefore, even in the method of Comparative Example 2, the sensitivity and specificity in detecting PNH type leukocytes are still low, and a trace amount of PNH type leukocytes could not be accurately detected.
On the other hand, according to the method of the present invention (Method C in FIG. 3), the cell population in the UL region formed an agglomeration, and the region of PNH positive cells and PNH negative cells was clear. Therefore, according to the method of the present invention, it was found that a very small amount of PNH-type leukocytes can be accurately detected with a simple procedure.

(Reference Example 1) Detection of PNH red blood cells using an antibody-type high sensitivity method (when a flow cytometer capable of digital waveform processing is used but FLAER is not used)
(I) Preparation of Erythrocyte Suspension Liquid peripheral blood was adopted as a biological sample, and a cell suspension of erythrocytes was collected from peripheral blood by performing the following operations.
(1) The Falcon tube containing 2 mL of EDTA-2Na blood separately stored in Example 1 was centrifuged at 2000 rpm for 10 minutes.
(2) A falcon tube containing 2 mL of PBS was prepared during this period.
(3) After removing plasma from the tube of (1), 50 μL of red blood cell layer was collected. This was added to the Falcon tube of (2) and the following experiment was conducted as a 3-5% red blood cell suspension.

(II) Incubation of red blood cells and antibody The following operation was performed to prepare a sample to be subjected to flow cytometry.
(1) Two new falcon tubes were prepared, and 50 μL of the red blood cell suspension obtained in (I) was allowed to stand at the bottom of each tube.
(2) Anti-CD55-FITC (200 μg / mL, manufactured by BD Biosciences) and the same amount of CD59-FITC (200 μg / mL, manufactured by BD Biosciences) were added to prepare an antibody mixture (hereinafter referred to as anti-CD55 / FITC). Described as anti-CD59-FITC).
(3) Add 4 μL of CD55 / CD59-FITC and 4 μL of glycophorin A-PE to one erythrocyte suspension, add 4 μL of mouse IgG-FITC and 4 μL of glycophorin A-PE to the other erythrocyte suspension (negative control), and add each mixture to vortex did. Next, each tube was shielded from light and allowed to stand in a refrigerator at 4 ° C. for 25 minutes.
(4) 3 mL of PBS was added to each tube and stirred gently. Subsequently, it was centrifuged at 500 g for 4 minutes, and the supernatant was removed with an aspirator, leaving a pellet.
(5) After adding 600 μL of PBS to the pellet and pipetting with a dropper several times, dead cell clumps were removed through a nylon mesh filter, and then the specimen was transferred to a new tube and subjected to flow cytometry analysis.

(III) Flow cytometry Compensation is performed in the same manner as in Example 3 (I), after flowing an unstained specimen and each fluorescent antibody single specimen sequentially in FACSCanto (registered trademark), and then pressing the compensation button to perform automatic correction. It was.

  Next, gating on the erythrocyte region was performed by the following method.

(1) Development was performed with FSC-A and SSC-A, and the erythrocyte region was gated.
(2) The cells were developed with FSC-W and FSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(3) The cells were developed with SSC-W and SSC-H, and cells that were significantly deviated from the cell population in terms of electrical signals were excluded.
(4) Glycophorin strong positive cells were gated.

  The cells derived by (1)-(4) can be defined as pure mature erythrocytes. Next, CD55 / CD59 in this cell group was analyzed.

When the histogram is developed with the erythrocyte lineage marker (Glycophorin A) on the vertical axis and CD55 / CD59 on the horizontal axis, PNH-type red blood cells are developed in the Upper Left (UL) region. Cells in this UL region were PNH type red blood cells, and the ratio was calculated by (UL / UR + RL) × 100 (%). FIG. 4 shows the result of gating PNH type erythrocytes by the above method to perform this calculation. In positive cases of PNH blood cells, 1.3% of PNH red blood cells were present in the UL region.
On the other hand, by analyzing this experiment in detail for 50 healthy subjects, a significant increase in PNH type cells in bone marrow failure syndrome could be defined as 0.005% or more of PNH type erythrocytes.

(Reference Example 2) Difference in response rate and survival rate of immunosuppressive therapy depending on the presence or absence of PNH type blood cells. 2006; 107: 1308-1314 In the same way as described in PNP type blood cells, the response rate of immunosuppressive therapy and the subsequent survival rate in patients with aplastic anemia were examined. . The results are shown in FIG.
It was found that PNH blood cell positive aplastic anemia patients are more likely to respond to ATG + cyclosporine therapy (FIG. 5A) and have a significantly higher survival rate (FIG. 5B) than negative patients.

  From these results, it can be understood that the presence of PNH-type leukocytes can be detected by the method of the present invention. In addition, by applying the method of the present invention, it is possible to diagnose the immune pathology of bone marrow failure syndrome. Therefore, by adopting the examination method of the present invention as one of the judgment materials for the treatment policy of bone marrow failure cases such as RA and AA, it is possible to determine whether or not the subject patient is a target for immunosuppressive therapy. As a result, it can be seen that in the treatment of bone marrow failure cases, it is possible to select an optimal treatment for each individual patient.

The present invention is a method that enables simple and accurate detection of extremely small amounts of PNH-type leukocytes. By applying the method of the present invention,
(1) Generation of noise based on non-specific reaction in detection of PNH type leukocytes is suppressed.
(2) A cell population having no GPI-anchored membrane protein is clearly depicted on the histogram. This makes it possible to easily detect PNH-type leukocytes in many facilities regardless of the level of proficiency in flow cytometry of those involved in the detection of PNH-type leukocytes.
(3) Furthermore, any type of PNH type leukocytes (PNH type granulocytes, PNH type monocytes, etc.) can be detected at a time by combining with antibodies against each surface antigen characteristic of leukocytes.

  In addition, by using this detection method, it is possible to diagnose bone marrow failure syndrome. In particular, since PNH leukocytes are significantly increased in autoimmune bone marrow failure cases, autoimmune bone marrow failure syndromes and bone marrow failure syndromes due to abnormalities of hematopoietic stem cells themselves can be distinguished by the method of the present invention.

Claims (6)

A method for detecting PNH-type leukocytes, comprising performing flow cytometry using FLAER and at least one anti-leukocyte surface antigen antibody, wherein the waveform processing in the flow cytometry is digitized, and at least one anti-leukocyte is detected. A method wherein the surface antigen antibody comprises an anti-CD11b antibody and the PNH leukocytes are PNH granulocytes . The method according to claim 1 , wherein PNH leukocytes other than PNH granulocytes are detected simultaneously with PNH granulocytes . Anti leukocyte surface antigen antibody, anti-CD11b antibody, anti-CD33 antibodies, including anti-CD45 antibody and anti-CD3 antibody, method according to claim 1 or 2.   A method for examining bone marrow failure syndrome, comprising detecting PNH-type leukocytes by the method according to claim 1. A method for detecting PNH type granulocytes using a flow cytometer in which waveform processing is digitized, including the following operations (1) to (7), wherein the waveform processing is performed on a blood cell suspension: A method comprising attaching to a digitized flow cytometer and performing the following operations (1) to (7):
(1) Developed with a forward scattered light pulse area ( FSC-A ) and a side scattered light pulse area ( SSC-A ) , and gating to a leukocyte region composed of granulocytes, monocytes, and lymphocytes.
(2) Developing with forward scattered light pulse width ( FSC-W ) and forward scattered light pulse height ( FSC-H ) , and gating so as to exclude cells that are significantly deviated from the cell population in terms of electrical signals.
(3) Expand with side scattered light pulse width ( SSC-W ) and side scattered light pulse height ( SSC-H ) , and perform gating to exclude cells that are significantly deviated from the cell population in terms of electrical signals.
(4) Gating to exclude 7-AAD positive cells,
(5) Gating with CD45 and SSC-A to the granulocyte region inferred from the complexity and size of the cell structure ;
(6) gating on CD11b strong positive cells; and
(7) Expand the histogram with FLAER expression in the cell group derived by (1) to (6) , CD11b on the vertical axis and FLAER on the horizontal axis, and confirm the number of cells in the Upper Left (UL) region to analysis by,
Here, the blood cell suspension is treated with anti-leukocyte surface antigen antibody including FLAER antibody, anti-CD11b antibody and anti-CD45 antibody, and 7-aminoactinomycin (7-AAD) .   A method for detecting bone marrow failure syndrome, wherein a significant increase in PNH type granulocytes detected by the method according to claim 5 is defined as 0.003% or more.