DNA ploidy analysis and cell cycle analysis

During the cell cycle, the DNA content changes periodically with the intracellular phase. Under normal circumstances, most cells are in the resting phase (Go). Although the G1 phase cells have DNA synthesis, the DNA content is still 2N, which is diploid. Cells, cells in the active DNA synthesis phase (S phase) have a DNA content of 2N-4N; cells undergoing cell division (G2/M phase) contain the largest amount of DNA (4N). After the cells are fixed, they can be stained with a fluorescent dye such as PI (Propidium Iodine propidium iodide). However, specimens must be treated with RNase to exclude RNA interference. FCM can give a histogram of DNA distribution after measuring a large number of cells (105 cells can be measured in a few minutes), as shown in Figure 12.10. Schematic diagram of normal human peripheral blood DNA. The first peak (G1) in the figure is a cell with a DNA content of 2N. Peak, ? The second peak is a cell peak with a DNA content of 4N, and between the two peaks are cells with an active DNA synthesis phase (S phase) with a DNA content of 2N-4N. Using the Multicycle software provided by the manufacturer, the computer can automatically calculate G1%, S+G2M%; if using DNA/RNA two-parameter analysis, G0:G1%, G0/G1 phase DNA index can be obtained, such as apoptosis in the specimen. Cells, a sub-G1 peak appears before the G1 peak, and the software automatically calculates the % of apoptotic cells.
Figure 12.10. Schematic diagram of peripheral blood DNA in normal subjects (taken from the Coulter Training Guide)
A clinically valuable indicator of DNA ploidy analysis is DNA aneuploidy and/or hyperdiploid % and tetraploid % increase. These changes are specific changes in tumor cells. Compared with solid tumors, the incidence of aneuploidy in acute leukemia is low, about 30-40%.

Lymphocyte subset determination

Lymphocytes are responsible for the main function of immunity. The determination of lymphocyte subsets helps to understand the immune status of the body and the monitoring of some diseases. Lymphocyte subsets frequently measured clinically include T lymphocytes (CD3+), T helper cells (CD3+CD4+), T suppressor cells (CD3+CD8+), B lymphocytes (CD19+ or CD20+), NK cells (CD3-CD56+) Wait.
Monoclonal Antibody McAbs, which are commonly used to determine lymphocyte subsets, are mouse anti-human Ig, purified antibodies, and direct fluorescent antibodies. Some foreign companies now sell two-color and three-color McAbs, such as CD4-FITC. /CD8-RD1, CD3-CY5/CD4-FITC/CD8-PE, etc., at this time, the corresponding negative control should be selected according to the Ig properties of these two-color or three-color McAb, such as MsIgG1-RD1/MsIgG2-FITC. The negative control tube should be determined first when measuring on the machine. The positive cells of the negative control should be <2.0%.
The three-color assay gives more accurate subgroups: if the true T helper cell is CD3+CD4+CD8-, the true T suppressor cell should be CD3+CD4-CD8+, not only in the single-labeled CD8+ cells. There are T suppressor cells, which also contain about 30% of NK cells; while the CD3+CD4-CD8-cell population is γδT cells, which are involved in infection, CD3+CD4+CD8-cell+CD3+CD4-CD8+ cells+CD3 +CD4-CD8-cell + CD3+CD4+CD8+ cells = CD3+ cells. If singly labeled CD56 does not accurately measure NK cells, NK cells should be (CD3-CD56+) because CD56+ cells contain non-HLA-binding cytotoxic T cells (CD3+CD56+). The two-color combination can also measure B lymphocytes (CD3-CD19+ or CD20+), activated T cells (CD3+CD69+ or CD3+25+), and the like.
A subset of T helper cells can be assayed using appropriate two-color combinations such as CD4 and CD29, CD4 and CD45RA. For example, CD4+2H4 (CD45RA)+ cells are cells that induce Ts; CD4+4B4 (CD29)+ cells induce cells of Th. Similarly, a subset of T suppressor cells can be determined using CD8+CD45RA and CD8+CD29+S6F1.
T helper cells can also be divided into two subgroups of Th1 and Th2, and the intracellular cytokines IFN-γ and IL-4 can be labeled to distinguish Th1 cells (CD4+/IFN-γ+) and Th2 cells (CD4+/IL-4+). ).
The functional status of lymphocyte subsets, such as activated T8, activated B cells, etc., can also be determined using McAbs such as CD25, CD69, and other lymphocyte-labeled two- or three-color markers.
The normal range of common cell subpopulations is given below for reference. Please note that there are multiple clones of McAb in the same CD. The values ​​measured by different McAbs in the same CD are different. Each laboratory should determine the normal value of its own laboratory. .
CD3+ 70.0±12.0% +4B4+ 23.0±7.0%
CD3+CD4+CD8- 40.0±9.0% CD4+2H4+ 19.0±6.0%
CD3+CD4-CD8+ 30.0±8.0%
CD3-CD56+(NK) 12.0±8.0%

Principle of leukemia immunophenotyping

Immunophenotyping of leukemia is to detect the cell membrane and cytoplasmic antigen of leukemia cells by using monoclonal antibody (McAb), and analyze its phenotype to understand the cell series of the leukemia cells to be tested and the degree of differentiation. The analysis of leukemia cell antigens helps to classify leukemia and provide a basis for diagnosis and treatment. Immunophenotyping of leukemia is an important supplement and further deepening of morphological typing. The International MIC Classification Collaboration Group believes that the immunophenotyping of each acute leukemia is essential. Leukemia immunophenotyping has a decisive role in the identification of acute lymphoblastic and acute non-leaching. It also plays a decisive role in the identification of certain subtypes such as M7 and M3. For some acute leukemias that cannot be diagnosed by morphology and histochemistry, acute Undifferentiated leukemia, mixed leukemia, etc. are of great significance. Since the advent of monoclonal antibodies, the most successful application has been to study the relationship between various cell surface antigens of the hematopoietic system and cell proliferation, differentiation and malignant transformation. Studies have found that cell surface antigens have important functions. Some antigen molecules act as receptors for cell growth factors and affect cell proliferation and differentiation. Some antigen molecules participate in cell-cell interactions as a material basis for mutual recognition between cells; some antigen molecules are cells. The material basis of specific functions.
During the process of differentiation and maturation from pluripotent hematopoietic stem cells (PHSC) into functional cells, the cell surface and cytoplasmic antigens are constantly changing along with the process of differentiation and maturation. These antigens are called hematopoietic cell differentiation antigens. Hematopoietic cell differentiation antigen is a mosaic protein encoded by a gene on the chromosome in the nucleus during hematopoietic differentiation. Its appearance, increase, decrease or disappearance are closely related to hematopoietic cell differentiation and exhibit specificity related to cell series and degree of differentiation. Sex. These antigens serve as markers for the identification and classification of hematopoietic cells. Such as myeloid cells MPO, CD33, CD13, CD14, CD15 and other antigens; megakaryocyte CD41, CD41, CD61 antigen; T lymphocytes CD2, CD3, CD4, CD5, CD7, CD8 and other antigens; B lymphocytes Antigens such as CD19, CD20, and CD22. Leukemia cell-specific antigens have not been found so far, and leukemia cells are a large accumulation of hematopoietic cells at a certain stage of differentiation, and the corresponding hematopoietic cell differentiation antigens are expressed. Therefore, hematopoietic cell differentiation antigen markers can be used to detect leukemia cells; It is not a normal hematopoietic cell, and its antigen expression is not exactly the same as normal hematopoietic cells. It is often lost to the antigens normally in a certain stage of differentiation, or to express antigens that are not normally found in a certain stage of differentiation, or to express other series of antigens, and partially lose the stringency of seriality and differentiation.
The use of FCM to detect leukemia immunophenotyping is fast, simple, and reproducible. Since FCM can distinguish cells according to FSCνSSC histogram and can delineate the cell range (Bitmap, amorphous gate), or use SSC (linear or logarithmic) and CD45 histogram to delineate the cell range (Bitmap, amorphous gate), exclude other Cell interference is therefore more accurate than optical microscopy immunofluorescence or immunohistochemistry.

Clinical significance of leukemia immunophenotyping

Currently recognized series of specific indicators are: T lymphocyte lineage - cytoplasmic CD3 (cCD3), B lymphocyte lineage - cCD22 or cCD79, myeloid - MPO or cCD13, generally can be used to distinguish cell series Further analysis of a certain series of subtypes and differentiation stages.
1. Immunological typing of ALL
Before 1986, it was divided into common ALL (cALL), undifferentiated cell ALL (Null-ALL), T cell ALL (T-ALL), pre-B cell ALL (PreB-ALL), and B-cell ALL (B-ALL). Type; from 1986 to 1994, it was divided into two categories, nine types (non-T-ALL type six, T-ALL type III). In the late 1990s, it was generally divided into B progenitor cells ALL and pre-B cells ALL according to clinical applicability. B cell ALL, T cell ALL four. Table 12.1 - Table 12.4 lists the type 5 and type 9 of ALL (B cell series type 6 and T cell series type III) and the four type classification method.

B-progenitor cells ALL: B-progenitor cells account for 65%-70% of childhood ALL, 55%-60% of adolescent ALL, 50% of adult ALL, children ALL >90% of cases CD10+, and infants CD10+ cases <50 %. FAB is classified into L1 and L2. Leukemia cells have low FS and SS; generally TdT, HLA-DR and CD19 are positive, most cases are positive for CD24 and CD34, and this type of cell membrane immunoglobulin (Ig) is negative. This type has two subtypes of CD10+ and CD10, and the prognosis of CD10+ is better than that of CD10-type.
Pre-B cell ALL: The pre-B cell ALL is later than the B progenitor ALL in the developmental stage, accounting for 25% of the childhood ALL, and the proportion in the adult ALL is still unclear. Generally, CD24, HLA-DR, CD19, CD10, cCD22 are positive, CD34 is negative, and there is a cytosolic heavy chain μ when distinguishing characteristics. The prognosis of this type is worse than that of B progenitor cells, which may be related to t(1;19), t(1;19) accounts for 25% of pre-B cell ALL, CD34-, and CD34- is independent in B-series ALL. Poor prognosis markers.
B cell ALL: B cell ALL accounts for 2%-5% of all ALL; B cell ALL is more mature, FS and SS of leukemia cells are significantly increased compared with B-progenitor ALL, in FSνSS histogram or SS (linear or logarithmic The νCD45 histogram is in the lymphocyte and monocyte regions. Typical markers are positive for cell membrane immunoglobulin (sIg), phenotypes are generally positive for CD19, CD20, CD22, CD24, and CD10+ for most cases, but the presence of sIg and mature antigen can be distinguished from earlier B-line ALL. This type of FAB classification is generally L3, rare cases have B cell ALL markers and FAB classification is generally L1, these patients have many t (1; 19) and t (14; 18).

T cell ALL: T cell ALL accounts for 15% of childhood ALL and 25% of adult ALL. Most phenotypes are thymocyte type, the most common is advanced thymic cortical cell subtype, CD1+, CD2+, CD5+, CD7+, CD4+CD8+, CD3 expression is less, TdT is often positive; another common subtype is early thymic cortical cells Subtype, CD2+, CD5+, CD7+, TdT+. The medulla subtype is less common, CD2+, CD5+, CD7+, CD3+CD4+ or CD3+CD8+, and TdT expression is less. The pre-T cell subtype has only CD7 and cCD3 and no other T-line antigen expression, and the prognosis is poor. T-line neoplastic diseases have a down-regulation of specific normal antigen expression or an antigen that should not normally occur during this differentiation stage. Adult T-cell ALL has a better prognosis, while children with T-cell ALL have a worse prognosis than children's B-progenitor cells ALL and pre-B-cell ALL. Although the prognosis of each sub-class is still unclear, CD10-negative patients have a poor prognosis.

The immunological classification of ALL was divided into five types before 1986. In 1986-1994, it was divided into two categories, nine types, and the late nineties (1997) was divided into four types. The five types before 1986 were the reflection of monoclonal antibodies and detection methods at that time; with the discovery of new monoclonal antibodies (mainly McAbs of T and B lymphocyte subsets) and the detection of a large number of clinical cases, it was not only clear The source and differentiation of T and B lymphocytes, and the more precise immunization, appeared in 1986-1994 divided into two categories, nine types; but according to the differentiation and development stages of cells, the two categories of nine The type-type method is slightly cumbersome in clinical practice, and the clinical applicability of guiding treatment and judging prognosis is not strong enough. Therefore, the above simple four-type classification method has appeared. After comparison, it is not difficult to see that the B-progenitor cell type of the four-type classification actually contains two subtypes of non-T-ALL II, III, and IV of the nine types of nine-type classification, and the V-type is the former. B cell type ALL, type VI is B cell type ALL; and T cell type type is two types of nine types of T-ALL type III combined into one type. The non-T-ALL type I of the two broad categories of nine classifications is classified as acute undifferentiated leukemia (see below).
ALL can also be classified according to DNA content. DNA content can be easily determined by flow cytometry. DNA content is divided into two sub-categories: hyperdiploid and subdiploid. The former has a good prognosis and the latter has a poor prognosis.

2. Acute myeloid leukemia (AML) stirs all monoclonal and mononuclear monoclonal antibodies to be essentially free of differentiation and developmental stage specificity. Therefore, the immunological classification of AML is not very obvious in FAB-?M0, M1, M2; Most of them do not express HLA-DR and CD34, often express CD13, CD33, CD9, CD38; GPA can help in the identification of erythroleukemia (M6); megakaryocyte leukemia (M7) has CD41, CD42, CD61? , an important indicator for the diagnosis. Due to the heterogeneity of leukemia, the expression of leukemia antigens in the same FAB classification is not identical. The immunophenotype of AML is shown in Table 12.5.

M0: M0 leukemia cells have very low FS and SS and are in the original lymphocyte region in the SSνCD45 histogram. Leukemia cells of M0 express at least one myeloid-specific marker such as MPO, CD13, CD116. MPO is more sensitive than CD13 and CD116; generally the lymphoid marker is negative, but may express CD7 or CD4; generally CD34, HLA-DR is positive. Studies have shown that AML complex expression of CD7 and CD34 has a poor prognosis.
M1: M1 leukemia cell antigen expression is similar to M0 and is not well distinguished from M0. M1 generally expresses CD13, CD33 and HLA-DR. CD34 expression is less than M0, some may express CD15, and less patients may express CD4.
M2: The main difference between M2 and M1 is that the differentiation and maturation are increased, the blast cells are decreased; CD34 expression is less than M1, CD15 expression is higher than M1, most cases are HLA-DR positive, CD13 expression is stronger than CD33; some M2 expression is CD19 and CD56 There is t(8;21), and rare M2 with t(8;21) does not express CD13, CD33 and CD14 but MPO is positive.

M3: M3 leukemia cells increase SS due to its high granularity; M3 leukemia cells generally express CD13, CD33, some patients may express CD2, but HLA-DR is negative, CD34 is generally negative, recurrent patients are positive; some patients may CD56 expression and CD56 expression should be genetically examined (APL/RARα) for extramedullary/NK cell acute leukemia (see below).
The immunophenotypes of M4 and M5: M4 and M5 are similar. The important phenotypes are CD13, CD33, CD14, CD15 and HLA-DR. Some patients express CD4 and CD7. Some patients may express CD56, and most of them express MCD. Often accompanied by chromosome 16 abnormalities, the prognosis is good.
M6: M6 is less common, generally expressing HLA-DR, CD34, CD13, CD33, and GPA is useful for determining the red line.
M7: M7 accounts for 1% of adult ANLL and 4% of children. Adult M7 is more common in secondary leukemia, ie CML.BC or MDS-RAEB or MF leukemia. The children's M7 is mostly the original. The immunophenotype of M7? is generally: CD41+, CD42+, CD61+, CD33+/-, CD13+/-, CD34+, DR+/-, CD10-. Specific markers CD41, CD42, and CD61 positive can confirm M7, but pay attention to the false positive results of extracellular platelets adhering to cells.

3. Acute undifferentiated leukemia Using flow cytometry analysis, only about 1% of acute leukemias cannot be classified. Typical acute undifferentiated leukemias have only HLA-DR and CD34 expression without series-specific antigen expression.

4. The true double-series phenotype of leukemia in heterozygous leukemia is a patient with t (9; 22) or 11q23 MLL (myeloid/lymphoid or mixed lineage leukemia myeloid/lemphoid leukemia) gene rearrangement, previously reported Many heterozygous leukemias are mostly due to methodological problems that cannot exclude the interference of non-leukemia cells, or non-specific weak expression as specific expression. As mentioned above, leukemia cells are not normal hematopoietic cells, and their antigen expression is normal with hematopoietic cells. Not exactly the same, part of the loss of the series of specificity and the strictness of differentiation, so do not easily type heterozygous leukemia, there is no unified diagnostic criteria for internationally heterozygous leukemia.

5. Acute Chronic Myeloid Leukemia (CML BC) Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disease with a mostly acute outcome. CML BC involves all hematopoietic cell lines, and it is often difficult to determine the blast type from morphologically. 50%-60% is AML change, and about 30% is ALL change. AML changes can express all phenotypes of AML, and most ALL become B cell sources, of which cALL? and pre-B-ALL are common, and rare T-ALL changes.

6. B Lymphoid cell proliferative disease B The immunological classification of lymphoid cell proliferative diseases is shown in Table 12.6. MCL: mantle cell lymphoma; FCCL: Follicular center cell lymphoma---follicular central cell lymphoma; MZL: Marginal zone lymphoma-marginal lymphoma; SLL:small Cell lymphocyte lymphoma--small cell lymphocyte lymphoma

B Lymphoid cell proliferative diseases include B chronic lymphocytic leukemia (B-CLL), B-colony cell leukemia (B-PLL), hairy cell leukemia (HCL), and multiple myeloma (MM)/plasma leukemia and Partial lymphoma, lymphoma immunophenotyping will be described in different sections.
B-CLL: Leukemia cells of B-CLL generally express CD19, CD20, CD43 and CD79a, and CD5 is expressed in combination with the above antigens. sIgM, sIgD, CD11c and CD25 are also frequently expressed but weakly expressed, and CD10 and CD22 are negative. Patients with chromosomal abnormalities have a poor prognosis.
B-PLL: Flow cytometry is very useful in distinguishing between B-PLL and B-CLL. B-PLL usually has strong sIg expression, CD5 is negative and CD22 is positive. The difficulty is that the primary B-PLL and B-CLL are converted to The distinction between B-PLL, B-PLL converted from B-CLL is positive for CD5 and weak for CD22, similar to B-CLL.
HCL:HCL generally express mature B cell markers: CD20, CD22, CD19, CD79, sIg, CD11c, CD22 high expression, CD25 moderate expression, general CD21-, typical cases CD5-, CD10-, CD23-, but a few The case can be positive. CD103 is the most trusted marker for HCL and can be used to identify other B-cell leukemias.
Plasmacytoma: Analysis of multiple myeloma (MM)/plasma cells by flow cytometry due to the lack of myeloma cells in the bone marrow specimens of most patients with multiple myeloma and the loss of most B cell line-specific markers in tumor cells Leukemia is more difficult, and typical plasma cells are strongly expressed by CD38 and weakly expressed by CD45. Generally, plasma sIg is weakly expressed and cIg is positive.

7. T lymphoid cell proliferative disease The immunological classification of T lymphoid cell proliferative diseases is shown in Table 12.7.

T lymphoid cell proliferative diseases include T-lymphocyte leukemia (T-PLL), NK cell leukemia (T-LGL, NK-LGL), and adult T-lymphocytic leukemia (ATL), T-chronic lymphocytic leukemia (T- CLL).
T-PLL: T-PLL leukemia cells generally express CD2, CD3, CD5 and CD7. Most patients have CD4+ CD8-, but occasionally CD4+ CD8+. It is rare to express CD8 alone. This disease is often accompanied by 14q11 and 14q32 changes. The prognosis of CD4+ CD8-phenotype is better, and the disease is more malignant than B-PLL.

NK cell leukemia: NK series leukemia was first described as large granular lymphocytic leukemia (LGL). There are two types of LGL: T-LGL and NK-LGL. T-LGL expresses CD3, CD8, CD2, CD16, CD11b, CD57, CD56, CD5, CD7, CD4 and CD25 were more negative, and TCR gene rearrangement. NK-LGL expresses CD2, CD16 and CD56, while CD3 and CD4 are more negative, CD8, and CD57 are weakly expressed or negative, and there is no TCRα-β gene rearrangement. Recently, NK series leukemia has new subtypes, such as acute anterior myeloid/NK cell leukemia, acute myeloid/NK cell leukemia, primitive NK cell leukemia, and NK-like T cell leukemia. Acute anterior myeloid/NK cell leukemia is a recently recognized type of leukemia characterized by distinct extramedullary involvement, immature primitive lymphocyte-like morphology with MPO-, CD7+, CD33+/CD13+, sCD3- , cCD3-, CD56+, poor prognosis. Acute myeloid/NK cell leukemia, morphological and immunophenotype similar to M3, but no RARα gene rearrangement, generally HLA-DR-, CD33+, CD13+, CD56+, more common in elderly patients, no response to retinoic acid. Primitive NK cell leukemia, unmyelinated and lymphoid antigens, CD56+, cCD3+/-, CD2+/-, CD4+/-, CD7+/-, a few have TCRβ gene rearrangements, but no TCRγ-δ gene rearrangements. The new subtype of NK cell series leukemia is a leukemia that has been more noticeable in the past 10 years. Its clinical features, immunophenotype, chromosome and gene changes need to be further accumulated in case studies to be fully understood. About 10%-20% of acute leukemia expresses CD56, except for primitive NK cell leukemia, NK-like T cell leukemia, acute myeloid/NK cell leukemia, acute anterior myeloid/NK cell leukemia, most of the acute leukemia expressing CD56 Is the type of M2, M3, M4, M5 classified by FAB. Whether such patients are acute myeloid/NK cell leukemia or acute myeloid leukemia with NK antigen expression needs further study, in view of the partial loss of serial specificity in acute leukemia The strictness of differentiation, which may be called acute myeloid leukemia with NK cell antigen expression, is more appropriate.
Adult T-lymphocytic leukemia (ATL): Most ATL cells express activated T cell phenotypes: CD3, CD4, CD5, CD25, HLA-DR, TCR, general CD7- and CD8-, often with adhesion molecule L-selectin Abnormal expression; poor prognosis with p53 abnormalities.
T-CLL: T-CLL is less than 1% of the total number of CLL, cell morphology is similar to that of general CLL, but clinical development is faster, but most patients express CD2, CD3, CD5, CD7, CD4, and a few patients express CD8.

Lymphoma immunophenotyping

At present, the classification method of lymphoma has gradually changed from the morphological classification of LSG to the REAL classification. The REAL classification method is based on the classification method of tumor origin, and is classified according to the original morphology and immunological classification. This classification method can not only infer the source of tumors, but also have guiding significance for treatment. Therefore, the immunophenotyping of lymphoma is becoming more and more important. Like leukemia immunophenotyping, the immunophenotyping of lymphoma is also to detect the cell membrane and cytoplasmic antigen of lymphoma cells by using monoclonal antibodies, and to analyze the phenotype of the lymphoma cells to understand the cell series and the degree of differentiation of the lymphoma cells. Flow cytometry can quickly and objectively detect the cell membrane and cytoplasmic antigen of most lymphoma cells, and plays an irreplaceable role in the immunophenotyping of lymphoma.

The immunophenotyping specimens of clinical lymphoma are generally lymph nodes, spleen, pleural effusion, ascites, and the like. In the immunophenotyping work of clinical lymphoma, the following four situations are often encountered: 1B cell line lymphoma 2T/NK cell line lymphoma 3 lymphocyte lineage other than hematopoietic cell tumor 4 hematopoietic cells.
The immunophenotype of REAL-classified lymphoma is shown in Table 12.8. *: weakly expressed or negative.
BLBL: pre-B primordial lymphocytic lymphoma/leukemia; BSLL: B-small lymphocytic lymphoma; LPL: lymphoplasmacytic lymphoma; MCL: cloak cell lymphoma; FCL: follicular central lymphoma; MZL: marginal zone B cell lymphoma; SMZL: spleen MZL; HCL: hairy cell leukemia; PC: plasmacytoma; DLBL: B-diffuse large cell lymphoma; BL: Burkitts lymphoma; HBLB: high B-cell lymphoma, Burkitts-like; TLB L: pre-T primordial lymphocytic lymphoma/leukemia; TPLL: T-leukocyte leukemia; LGLT: large granular lymphocytic leukemia, T cell type; LGLNK: large granular lymphocytic leukemia, NK cell type; MF: sputum-like fungus Disease; PTL: peripheral T-cell lymphoma, non-specific; AILD: vascular immune T-cell lymphoma; ACL: vascular central lymphoma; ITCL: intestinal T-cell lymphoma; ATL: adult T-cell lymphoma/leukemia; LCL: Large cell lymphoma; LCLH: large cell lymphoma, Hodgkin-like;

1. B cell line lymphoma In most cases, B cell line lymphoma is positive for CD19 and CD20, CD20 is negative in early differentiation, CD10 and CD34 are positive; CD5 and CD23 are positive in late differentiation, and the above markers are negative after maturity. The monoclonal antibody κ, λ (normal κ: λ = 2: 1) can be used to detect the shift of the immunoglobulin light chain, and whether or not the clonal proliferation immunoglobulin is inferred. Table 12.6 lists four types of immunophenotyping of peripheral B lymphoid lymphoma.
SLL (small cell lymphocyte lymphoma)--small cell lymphocyte lymphoma: small cell lymphocyte lymphoma cells generally express CD19, CD20, CD43 and CD79, and CD5 is expressed in combination with the above antigens, and sIgM, sIgD, CD11c and CD25 are also frequently expressed. However, the expression was weak, and CD10 and CD22 were negative. Patients with chromosomal abnormalities have a poor prognosis.

MCL (mantle cell lymphoma) - cloak cell lymphoma: MCL includes previously classified as intermediate lymphocytic lymphoma (ILL), moderately differentiated lymphocytic lymphoma (IDL), central cell lymphoma, and mantle cell lymphoma (MZL) ), accounting for 2%-8% of lymphoma, λ type is more common than κ type, sIgM is moderately expressed, IgD is weak or negative, and expresses CD19, CD20, CD22, CD43, but in most cases CD5 positive CD23 is negative, CD10 is generally negative. CD5 positive CD23 negative and Ig types can help identify MCL and FCL.
FCL (Follicular center cell lymphoma)---follicular central cell lymphoma: FCL accounts for 45% of lymphoma, expresses CD19, CD20, CD22, sIg strong expression, but most cases are CD10 and CD23 positive, typical FCCL CD5, CD43 and CD11c is negative. CD10 positive, CD11c negative and strong sIg expression facilitate differentiation with MZL.
MZL (Marginal zone lymphoma)--Marginal lymphoma and related B-cell lymphoma: typical immunophenotype: CD19, CD20, CD22, HLA-DR positive, moderate expression of sIg, CD5, CD10, CD23, CD25 negative, CD21 CD24 is negative in most cases, and most of them express CD11c. CD5, CD10, CD23, CD25 negative, CD21, CD24 negative in most cases, which is helpful for identification with CLL/SLL, FCCL, MZL.

2. T/NK cell line lymphoma Early T cells are generally positive for CD2, CD5, CD7, and cCD3. See Table 12.8 for the detailed classification and phenotype of T/NK cell line lymphoma. Cell membrane CD3 positive can be confirmed as peripheral T cell tumor, peripheral T cell tumor is characterized by CD3 and CD4 or CD8 complex expression, and often have clonal T cell receptor, or α-β type or γ-δ type;
MF (mycosis fungoides) - mycosis fungal disease and sezary syndrome: the vast majority of cutaneous lymphoma, CD4 positive, simultaneous expression of CD2, CD3, CD5, and sometimes CD7 negative, CD8 positive cases are rare but reported The presence or absence of TCRβ gene rearrangement is helpful in identifying lymphoma and inflammatory responses.
LCL (large cell lymphoma) - large cell lymphoma: large cell lymphoma (LCL) accounts for 40% of adult lymphoma, 1/3 of children, 80% of adults are B cell type, children B cell type and T cell Each type is half.
3. Hematopoietic cell tumors other than lymphocyte lineage Acute myeloid leukemia (especially M4, M5) lymph node metastasis occurs. For example, low expression of T and B lymphocyte markers should be suspected and treated according to leukemia immunophenotype.
4. Tumors other than hematopoietic cells In the case of lymph node, chest and ascites specimens, if the CD45 is negative, non-hematopoietic tumor lymph nodes, pleura, and peritoneal metastasis should be considered.

Red blood cell disease diagnosis

(I) Reticulocyte determination The number of reticulocytes in peripheral blood is counted. It is important to evaluate the transfer rate of bone marrow erythroid hematopoiesis and reticulocytes from bone marrow to peripheral blood. During the process of maturation of nucleated red blood cells, a small amount of RNA remains in the cytoplasm after the nucleus is removed, and after about one day, the residual RNA completely disappears and becomes mature red blood cells. Red blood cells with residual RNA in this cytoplasm are called reticulocytes. Under normal circumstances, a certain amount of reticulocytes appear in the peripheral blood. The normal value is 1.0 to 1.5%, and the upper limit is 3.0%. However, when the number of red blood cells is abnormal, an erroneous result may occur. Therefore, it is expressed by the absolute value of reticulocytes, and the normal value is 5 to 15×10 10 /L. Since the conventional method requires first in vitro vitrectomy (most commonly methylene blue) and then counted under a microscope, the number of cells counted is limited. The use of flow cytometry to measure reticulocytes has the following advantages: 1 avoiding the occurrence of pseudo reticulocyte elevation due to fragmentation of the nucleus or the presence of particles of iron granulocytes 2 can avoid human error 3 because it can be used in a short time Measuring tens of thousands of cells, the results are more accurate and reliable than the conventional method, and the age structure of reticulocytes can be given at the same time.
Flow cytometry is a simple method for measuring reticulocytes. It is only necessary to wash the red blood cells and then stain them with fluorescent dyes. Commonly used fluorescent dyes are Pyronin Y Acridine Orange AO, Propidium Iodine PI, Cyanine dye, and the like.

(B) PNH diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) due to the decrease or lack of anchorage protein-glycophosphoinositol (GPI) on the blood cell membrane leads to a decrease or deficiency of GPI-associated complement activation inhibitors such as CD55 and CD59 Therefore, red blood cells are abnormally sensitive to complement hemolysis. Flow cytometry showed that CD55 and CD59 were very sensitive. Normal human CD55 and CD59 double positive cells were >95%. PNH patients had significantly reduced CD55 and CD59. This reduction was not only manifested on red blood cells, but also decreased in granulocytes and lymphocytes. CD55c has been found to be more sensitive to GPI reduction or lack of CD59.

  Platelet function analysis and platelet disease diagnosis

(I) Analysis of platelet function The platelet membrane is rich in glycoprotein receptors, which is the material basis for platelets to function. The types, contents, structures and functions of platelet glycoprotein receptors in quiescent and activated phases are significantly different. Different anti-platelet monoclonal antibodies can measure and analyze platelet function status. Determination of platelet plasma membrane glycoprotein monoclonal antibodies CD41 (GPIIb/IIIa), CD61 (GPIIb/IIIa), CD42b (GPIb), CD41b (GPIIb), platelet granule membrane glycoprotein CD62p, CD63 for analysis of platelet function status, such as CD62p, CD63 normal platelets are not expressed, and the expression is significantly increased when platelets are activated, which can be used to determine activated platelets.

(B) the diagnosis of platelet disease
1. Platelet-associated antibody assays Platelet-associated antibodies can be produced by different mechanisms. Antiplatelet autoantibodies (including primary, drug-induced, combined with other autoimmune diseases such as systemic lupus erythematosus) can cause severe thrombocytopenia. Most patients with primary (immune) thrombocytopenic purpura have platelet-associated antibodies PAIgG, PAIgA, PAIgM against autologous platelets on platelets and/or serum. The antigens of platelet-associated antibodies are still not well understood and may be diverse, such as GPIb, GPIIb, GPIIIa, Pa, or ?HLA antigens. Anti-platelet autoantibodies may also occur after multiple transfusions or during pregnancy. These autoantibodies are mostly antibodies against HLA class I epitopes, typically IgG, which rapidly destroy and clear platelets from random donors. . Therefore, a method for rapidly determining the presence or absence of these antibodies is necessary. Many different methods for the determination of platelet antibodies using radioimmunoassay, fluorescence, enzymes, and SPA have been established, but these methods are complicated and time consuming; and the results are only given in a certain amount of platelets (105). Platelet antibodies are indicated. The method for measuring platelet antibodies by FCM developed in recent years has the advantages of quickness and simplicity, and can also measure antibodies on platelets and serum. In the determination of FCM, the presence or absence of antibodies on the surface of each platelet can be given in the platelet population. How many platelets have antibodies (in %); and can give a histogram of platelet-associated antibody and platelet counts? Let us know the distribution of platelet-associated antibodies in the platelet population.

FCM assay for platelet antibodies: platelets labeled with fluorescent anti-human IgG, IgA, IgM or anti-platelet GPIb, IIb, IIb/IIIa, respectively, and platelets from washed patients and normal O incubated in patient sera The type of platelets is measured by FCM, the former is the antibody on the patient's platelets and the latter is the antibody in the patient's serum.
2. Platelet membrane GPIIb/IIIa was significantly reduced in patients with platelet weakness, and platelet membrane glycoprotein monoclonal antibodies CD41, CD61 and flow cytometry were not only able to detect GPIIb/IIIa reduction, CD42a, CD42b was normal or high. It can also measure the degree of GPIIb/IIIa reduction and classify platelet weakness.
3. Giant platelet syndrome (BSS): platelets are large, CD42a is reduced, CD42b is decreased, and CD61 is increased.

Minimal residual leukemia test

Minimal Residual Leukemia (MLL) refers to the state in which leukemia cells remain in the body after complete remission (Complete Remisson CR). There is no clear boundary between minimal residual leukemia and obvious leukemia (Overt leukemia). Only the amount of leukemia cell load is different. Generally, leukemia cells can reach 1012 in adults with obvious leukemia, and ≤1010 after treatment, CR. Therefore, the number of leukemia cells in MRL state may be It is 100-10. MRL is the root cause of leukemia recurrence, so the detection of MRL is of great significance: 1 Guide clinical treatment 2 predict leukemia recurrence 3 evaluate the purification effect of autologous bone marrow transplantation. The advantage of using FCM? to detect MRL is that it can analyze a large number of specimens in a short time, which has a fast feature.
Due to the lack of specific markers in leukemia cells, the effect of FCM in detecting MRL is not satisfactory, and the sensitivity is not high enough, generally only 1/103-104. At present, there are mainly two types of detection methods: 1 Using FCM to analyze the nucleic acid amount or chromosome of CR phase bone marrow cells, which can be used for MRL analysis of some AL patients, but the sensitivity is only 1-5%. 2 Immunophenotypic analysis, because there is no leukemia-specific marker, it can only be used as a basis for comparison of differences in certain antigenic quantities and distribution sites compared with normal cells; for example, MRD is detected by TdT and CD10 double labeling, but sensitivity is also Not high, about 0.1-1%.
The false negative results may be due to: 1 Leukemia cells in the specimen <10-4 or <10-5; 2 specimens can not represent the systemic bone marrow; 3 patient leukemia cell phenotype conversion.

Determination of leukocyte phagocytosis

Granulocyte-mononuclear-macrophages are important members of the body's immune response and immune regulatory cells. They not only have phagocytic function, but also engulf foreign microorganisms, tumor cells, etc., and secrete a variety of biological factors to participate in the immune response. In addition, mononuclear-macrophage cells take up, modify, and present antigenic substances, which are lymphocytes. The immune function is essential. Therefore, detecting leukocyte phagocytosis is important for understanding the immune status of the body. The following is a brief overview of FCM? The assessment of leukocyte phagocytosis.

When measuring the phagocytosis of leukocytes by optical microscopy and fluorescence microscopy, the granules and monocytes should be isolated and the cell activity should be maintained as much as possible. When using FCM, it is not necessary to separate cells, and whole blood can be applied. Therefore, it can be measured under natural conditions, and the results are accurate and reliable.

The target particles are generally yeast, bacteria and other artificial particles that can stimulate natural phagocytosis or pre-treatment with antibodies; and are labeled with fluorescence, commonly used as FITC (fluorescein isothiocyanate) or RA (Rodamin).

Peripheral blood was taken by heparin or sputum acid (avoiding EDTA), 200 μl of whole blood and 200 μl of target particles (target particle concentration 4×107/ml) were incubated at 37 ° C, and immediately after the incubation, the ice water was placed. As a control, the same specimen was not placed in ice water at 37 ° C.目标粒子不同温育时间不一,FITC标记的金黄色葡萄球菌25?分钟?,FITC标记的粒子15分钟即可达到吞噬饱和;此外,粒子与白细胞的比例也很重要,一般为10:1。温育结束后以溶血剂溶去红细胞即可上机检测。

应用FCM全血法测定白细胞吞噬功能,对白细胞的丢失、损伤最小;?此外用本法可测定血清中的调理素(Opsonine)水平及血清中有无吞噬作用抑制因子。如果用FITC标记的?单克隆抗体 CD13、CD14、CD15标记粒、单核细胞,用红荧光标记目标粒子,即可测定吞噬功能与细胞表面标记的关系,对白细胞吞噬功能作进一步的研究。

NK 和 LAK 细胞活性测定

NK细胞存在于外周血大颗粒淋巴细胞中,它对靶细胞的细胞毒活性不依赖于抗体,无MHC限制性,它们的数量及细胞毒活性是机体免疫系统的重要指标。人NK细胞一般表达CD56、CD16、CD57部分表达CD2、CD8、CD11而不表达CD3。

LAK(Lymphokine Activated Killer cells)细胞主要存在于LGL的高密度群体中,?LAK前体细胞表达NK细胞标志CD16而不表达T细胞标志如CD3、CD4、CD5等;?它们不需要抗原刺激就能杀伤NK细胞不能杀伤的肿瘤细胞,并且无MHC限制性;从表型上看大多数LAK细胞来自NK细胞,但严格讲LAK细胞对K562细胞的杀伤活性不能称作LAK活性,测定LAK?活性的靶细胞应为NK抵抗的实体瘤细胞,如HL-60细胞、HeLa细胞等。

常用的测定NK和LAK细胞活性的方法较多,如攩51Cr释放法、[3H]?TdR?参入法或释放法、LDH释放法、ATP发光法等;应用放射性同位素对人体和环境不利,利用FCM测定NK和LAK细胞活性从1986年就开始摸索,方法已趋于成熟,以下介绍其中一种:

肝素抗凝取外周血后分离PBMC后洗净,调整细胞浓度为106/ml,为效应细胞;?靶细胞分别为K562和HL-60或HeLa细胞,在对数生长期受集细胞,调整浓度为106。用3mMDiO(3,3-Diacradecyloxacarbocyanine Perchlorate)标记靶细胞,效应细胞:靶细胞(E:T)为40:1、20:1、10:1和5:1,孵育后加入PI(10μg/ml)染死细胞,洗净后即可上机检测。DiO发绿荧光,PI发红荧光,利用双参数(FL1vFL2)直方图可清楚地了解靶细胞中的死亡细胞,?计算出靶细胞%溶解率。

造血干 / 祖细胞测定

造血干/祖细胞移植已广泛应用于血液肿瘤、实体瘤、某些遗传性疾病、免疫缺陷病等，因此检测造血干/祖细胞已成为临床必不可少的手段。应用流式细胞仪多色技术测定外周血造血干/祖细胞，具有快速、准确的优点，不仅能确定造血细胞数量，而且能对造血干祖细胞的质量进行评价，为临床干细胞移植治疗提供重要数据。目前多色组合一般包括CD34、CD38、HLA-DR等McAb。

CD34抗原是一种分子量约11万的糖蛋白，选择性地表达于早期造血干/祖细胞、小血管内皮细胞和胚胎纤维母细胞表面。该抗原在原始造血细胞上表达，以后随细胞分化、成熟逐渐减少直至消失，因此一直作为造血干/祖细胞表面的一种特征性标志而广泛应用于造血干细胞基础研究和临床。CD34+细胞是一群不均一的群体，依其是否表达CD38、HLA-DR、CD33、c-kit等分出的亚群表现出了不同的造血性能。最近随着造血理论的深入研究关于造血干细胞究竟是否都是CD34+细胞出现一些争论,实验研究证明, CD34-造血干细胞较CD34+造血干细胞更具造血潜能,有人经实验研究提出CD34的表达与造血干细胞的细胞动力学相关,细胞激活时CD34+,细胞处于稳定状态时CD34-;也有人提出CD34-造血干细胞较CD34+造血干细胞发育阶段更早。我们认为：成人体内可能仍保留着一小部分原始间充质细胞，他们仍保留着多向分化能力，也能向造血干细胞分化，因此体内有CD34-造血干细胞和CD34+造血干细胞是正常的，CD34-造血干细胞较CD34+造血干细胞发育阶段更早、更具造血潜能；但目前的用CD34+细胞代表造血干细胞的检查方法已足以满足临床需要，因为临床实践已证明这一点。理论研究往往能推动学术发展，开发新技术，我们期待着造血理论的不断发展给临床带来新技术，不断提高临床疗效。

我们用CD34、CD38、HLA-DR三色组合测定了151份外周血经动员后标本，CD34+细胞占单个核细胞（MNC）的（0.954±0.466）%，含量为（3.55±2.41）×109/L；其中CD34+CD38-亚群含量为（0.253±0.24）×109/L，占CD34+细胞的7.13%；CD34+HLA-DR-亚群含量为（0.273±0.310）×109/L，占CD34+细胞的7.61%；随着采集次数的增加，CD34+细胞及其亚群数量逐渐减少（P＜0.05）；随着供者年龄增加，其外周血CD34+细胞数逐渐减少，在≥40岁供者CD34+细胞百分比和含量比＜20岁供者分别降低了47%和50%；动员后外周血CD34+细胞数存在性别差异，男性供者外周血CD34+细胞数较女性高23%。

应用流式细胞仪测定造血干细胞虽具有快速、准确的特点，目前被广泛采用。但应用中要特别重视质量控制，要建立一套完整质控方法以确保检测的准确性。

  细胞凋亡研究

细胞凋亡是细胞在基因控制下的有序死亡，在疾病发生、发展中有重要作用，因而研究细胞凋亡有重要意义。细胞凋亡检测方法很多，应用流式细胞仪技术可根据细胞在凋亡过程中发生一系列形态、生化变化从多个角度对细胞凋亡进行定性和定量的测定。

1. 细胞形态变化：通过流式细胞仪测定细胞光散射的变化来观察细胞凋亡。在细胞凋亡早期，细胞前向角光散射的能力显著降低，90°角光散射的能力增加；在细胞凋亡晚期，前向角和90°角光散射的信号均降低。此方法特异性不强，目前使用较少。

2 细胞膜功能改变：
（1） 磷脂酰丝氨酸（phosphatidylserine PS）异位：正常情况下，PS位于细胞膜内层，细胞发生凋亡时PS从细胞膜内翻转并暴露在细胞膜外层，是细胞发生凋亡的早期事件。PS与Annexin Ⅴ（一种具有强力抗凝作用的血管蛋白）具有高度亲和力。应用流式细胞仪采用FITC- Annexin Ⅴ/PI双染法进行细胞凋亡检测，可同时描述三群不同状态细胞：FITC- Annexin Ⅴ-/PI-细胞，即正常活力细胞；FITC- Annexin Ⅴ+/PI-细胞，即凋亡细胞；FITC- Annexin Ⅴ+/PI+细胞，即已死亡细胞。此种方法操作过程简单，指标敏感，应用者越来越多。
（2） PI/Hoechst33342双染法：Hoechst33342（HO）是一种DNA的特异性荧光染料，可通过完整细胞膜，应用PI/Hoechst33342可将细胞分为三群：正常活细胞（HO强/ PI-），凋亡细胞（HO弱/ PI-），（由于凋亡细胞发生DNA降解和丢失，导致HO荧光减弱），死亡细胞（HO弱/ PI+）。此种方法再结合凋亡细胞前向角光散射能力降低的特点，能更好地鉴定凋亡细胞，但HO须紫外光激发，由于很多流式细胞仪不配有紫外激光，故此法应用受限。
（3） 吖啶橙(AO)/溴化乙啶(EB)双染法: AO是一种异染性荧光染料,可通过完整的质膜,它与核酸的结合主要是嵌入DNA双链的碱基之间，其发射峰为530nm，呈绿色荧光。EB的理化特性与PI相似，不能通过完整质膜。应用AO/EB双染法也可以将细胞分成三群：正常活细胞（AO强/ EB -），凋亡细胞（AO弱/ EB -），死亡细胞（AO弱/ EB +），原理与PI/Hoechst33342双染法相似，不同的是AO/EB双染法所的激发光是被广泛使用的氩激光（488nm），而不须紫外光，其缺点是染色过程较复杂，且AO易污染设备管道，因此使用此法者较少。
（4） 放线菌素D(7-AAD)染色法：7-AAD是一种核酸染料，它不能通过正常质膜，随着细胞凋亡、细胞死亡过程，质膜对7-AAD的通透性逐渐增加，结合细胞凋亡中DNA的有控降解，最后通过7-AAD标记DNA的强弱，将细胞分为三群：7-AAD强为死亡细胞，7-AAD弱是凋亡细胞，7-AAD-为正常活力细胞。此法具有染色快速、简便、价格便宜等优点。另外，由于此法不破坏细胞膜，故还可联合使用FITC、PE标记的膜蛋白,对特殊细胞群及亚群进行多色荧光分析(虽然AO/EB双染法也不破坏被检细胞膜,但由于AO及EB的发射光波谱分别与FITC和PE的发射光波谱相似，故AO/EB法不能与FITC和或PE联合使用)。此法是应用核酸染料测定细胞凋亡的流式细胞仪法中十分实用的方法。

3. 细胞器改变： 线粒体膜APO2.7蛋白表达：早期凋亡细胞的线粒体膜出现APO2.7蛋白表达，利用荧光标记的单克隆抗体，运用流式细胞术可以检测早期凋亡细胞。

4 .DNA含量变化：
主要是PI染色法，由于凋亡细胞DNA发生有序降解，被降解的低分子量DNA片段从变性细胞膜（经乙醇及透膜剂处理）漏出细胞外，使得凋亡细胞内的DNA含量减低，在流式细胞仪测定细胞DNA含量直方图中G1峰前可出现亚二倍体峰，即所谓凋亡峰。通过测定凋亡峰百分含量，便可知凋亡细胞比例。此法简便、快速，是目前常用的、经典的测量凋亡细胞的方法。此法存在问题：少量的正常的低DNA含量细胞、由于机械损伤产生DNA含量减低的坏死细胞、染色体丢失的分裂相细胞以及细胞碎片和微核等都可能出现在亚二倍体峰内。因此，此法的特异性较低。

5. DNA断裂点标记：细胞凋亡时发生DNA断裂,利用末端转移酶(TdT)可以将dUTP标记到断裂点上,称作原位缺口末端标记（TUNEL）技术。此法有直接标记和间接标记两种，前者的标记物是FITC-dUTP，后者的标记物是生物素(biotin)标记的dUTP,需要再用FITC标记的亲和素(avidin)与生物素标记的dUTP结合，使标记反应倍增，故间接标记法灵敏度高，但操作较复杂。TUNEL还可以配合其它单抗同时进行细胞表型分析，或与DNA含量同时分析。TUNEL法因其灵敏度高而被广泛采用。

6 细胞凋亡相关基因产物检测：细胞凋亡是一个多种基因参与的复杂过程,目前已知与bcl-2基因、c-myc基因、P53基因等有关，这些基因都有相关产物，目前针对这些相关产物已有单克隆抗体生产，应用这些单抗，通过流式细胞仪可检测造血细胞凋亡相关基因蛋白表达水平，相互关系。
流式细胞仪测定细胞凋亡方法、层次众多，且具有快速、准确特点，应用十分广泛。在实际应用中应注意采用多种方法结合使用，使结果更加可靠准确。

细胞分选

流式细胞仪能够分选某一亚群细胞,分选纯度＞95%。目前细胞分选主要用于研究，临床应用较少。血液学应用最多的是造血干细胞的研究，最近随着造血理论的深入研究关于造血干细胞究竟是否都是CD34+细胞出现一些争论,实验研究证明, CD34-造血干细胞较CD34+造血干细胞更具造血潜能,这些实验研究所用的CD34- 和CD34+细胞就是通过细胞分选获得的。小鼠造血干细胞分选一般按lin-c-Kit+CD34+/ lin-c-Kit+CD34-分选,人造血干细胞分选一般按lin-CD34+/ lin-CD34-分选。

为避免某些遗传性血液病如海洋性贫血、异常血红蛋白病的纯合子出生，产前诊断非常重要，这些疾病的主要靶细胞是红细胞，而孕妇血循环中存在着胎儿有核红细胞，只是数量非常少，利用流式细胞仪可从孕妇血液中分选出胎儿有核红细胞（分选条件：CD45-GPA+）进行基因分析，作出产前诊断。
利用流式细胞仪分选免疫担当细胞进行细胞免疫学研究也是目前的热门课题。总之，流式细胞仪能够分选出你想得到的任何一亚群细胞，只要你想得到的某一亚群细胞有合适的单克隆抗体标记，流式细胞仪的分选功能将得到越来越多的科学研究和临床应用。

other

流式细胞仪可能在两方面对骨髓增生异常综合症（MDS）有用，一是测定CD34阳性细胞数，以监测病情，二是测定核蛋白增殖因子（PCNA）,有报告PCNA再在生障碍性贫血、骨髓增生异常综合症、白血病三种疾病中表达有明显差异，可辅助鉴别诊断。

此外流式细胞仪也可检耐药蛋白,如肺耐药相关蛋白（LRP）、多药耐药蛋白（MRP）、 P170等。
流式细胞仪也可检测细胞因子，细胞内细胞因子如白介素系列（IL-1—IL-14）,肿瘤坏死因子(TNF),干扰素(IFN)等,只要用适当的打孔剂即可用抗上述细胞因子单克隆抗体标记后用流式细胞仪测定。

China Disposable Pap Smear Kit,Pap Smear Test Kit manufacturers, welcome Disposable Cytological Kit,Gynecological Pap Smear Test Kit purchasers from worldwide to visit our site.

Disposable Pap Smear Kit

Disposable Pap Smear Kit,Pap Smear Test Kit,Disposable Cytological Kit,Gynecological Pap Smear Test Kit

Luck Medical Consumables Co.,LIMITED , https://www.luckmedical.com