c6細胞免疫學檢測和流式細胞術(shù)

1、細胞免疫檢測和流式細胞術(shù)第二臨床醫(yī)學院袁小澎一、簡介為什么要檢測免疫細胞珠江醫(yī)院檢驗醫(yī)學部檢測了有什么用？珠江醫(yī)院檢驗醫(yī)學部免疫細胞功能是什么珠江醫(yī)院檢驗醫(yī)學部珠江醫(yī)院 檢驗醫(yī)學部檢測的重要性對腫瘤患者的研究，免疫細胞分群的，研究，標志物，預后的關(guān)系珠江醫(yī)院 檢驗醫(yī)學部Note: This table containsSampleDate collectedtota17892011/2/917902011/2/9珠江醫(yī)院 檢驗醫(yī)學部samples prepared by enzyme digestion of solid tumor only except a

2、s noted% vaible of total% of viable cells% of viable leukocytesCommentltumorleucoCD45+EpCam+CD45-T cellsB cellsmonoother80.880.680.218.412.181.628.40.747.423.566.953.683.857.618.842.442.73.436.017.917912011/2/144.143.495.516.417922011/2/2381.481.181.260.827.53.131.

3、517932011/3/1187.491.841.26.01.194.063.2Breast Ca17942011/3/1677.672.081.961.522.938.557.116.911.114.917952011/3/1683.384.279.418.69.381.454.66.320.718.417962011/3/2120.619.660.133.6Belco prep17972011/3/2287.687.887.573.713.826.318.211.314.456.1Ascites17972011/3/2256.

4、551.372.034.529.165.524.47.636.031.9Tumor17982011/3/2972.176.569.748.818.051.236.813.422.527.3腫瘤治療策略珠江醫(yī)院 檢驗醫(yī)學部白血病免疫分群從1976年FAB分型M從2001年FAB分型MICM細胞形態(tài)學（Morphology）免疫學（Immunology）細胞遺傳學（Cytogenetics）分子生物學（Molecular biology）珠江醫(yī)院 檢驗醫(yī)學部細胞分型的方法玫瑰花試驗等-利用T淋巴上具有綿羊紅細胞受體珠江醫(yī)院 檢驗醫(yī)學部白細胞分化抗原是白細胞（還包括血小板、血管內(nèi)皮細胞等）在正常分化

5、成熟不同譜系（lineage）和不同階段以及活化過程中，出現(xiàn)或消失的細胞表面標記。它們大都是穿膜的蛋白或糖蛋白，含胞膜外區(qū)、穿膜區(qū)和胞漿區(qū)。有些白細胞分化抗原是以磷脂酰肌醇（ inositol phospholipids,ip）連接方式“錨”在細胞膜上。少數(shù)白細胞分化抗原是碳水化合物半抗原。珠江醫(yī)院 檢驗醫(yī)學部珠江醫(yī)院 檢驗醫(yī)學部單克隆抗體技術(shù)(monoclonal antibody technique) 1975年英國科學家Milstein和Kohler所發(fā)明，并獲得1984年諾貝爾醫(yī)學獎。珠江醫(yī)院檢驗醫(yī)學部生物發(fā)光現(xiàn)象珠江醫(yī)院 檢驗醫(yī)學部信號檢測-熒光信號特異熒光珠江醫(yī)院 檢驗醫(yī)學部自發(fā)熒

6、特光異熒光 激發(fā)能量損失激光能量級發(fā)射激發(fā)態(tài)珠江醫(yī)院 檢驗醫(yī)學部珠江醫(yī)院 檢驗醫(yī)學部珠江醫(yī)院 檢驗醫(yī)學部流式細胞儀特點單細胞懸液或生物顆粒分析速度快當代最先進的細胞定量分析技術(shù)多參數(shù)檢測基數(shù)大，精度高珠江醫(yī)院 檢驗醫(yī)學部The relationship between cytometry and cell structure90o偏振光照射顯示出細胞的分葉性Lobularity90o去偏振散射可區(qū)分Neutrophils and Eosinophilscomplexity-10o 中間角度7o的散射顯示了細胞的復雜性Depolarization0o(Axial Light Loss)LASER

7、 LIGHT scatter indicates細胞大小sizeFLOWCELL珠江醫(yī)院 檢驗醫(yī)學部珠江醫(yī)院 檢驗醫(yī)學部流式細胞儀的工作原理光學系統(tǒng)激光光源光收集系統(tǒng)液流系統(tǒng)流動室液流驅(qū)動系統(tǒng)電子系統(tǒng)光電轉(zhuǎn)換數(shù)據(jù)處理系統(tǒng)細胞分選系統(tǒng)珠江醫(yī)院檢驗醫(yī)學部鞘液系統(tǒng)珠江醫(yī)院 檢驗醫(yī)學部液流系統(tǒng)：流動室、液流驅(qū)動系統(tǒng)珠江醫(yī)院 檢驗醫(yī)學部期流式細胞儀檢測范圍細胞大小細胞粒度細胞表面面積核漿比例DNA含量與細胞周RNA含量蛋白質(zhì)含量特異性抗原（細胞表面/胞漿/核）細胞內(nèi)細胞因子細胞活性酶活性激素結(jié)合位點細胞受體珠江醫(yī)院 檢驗醫(yī)學部細胞功能細胞結(jié)構(gòu)靈敏度：一般能測出單個細胞上CD15; on maturing

8、 myeloid cells CD15CD11bCD11bMonocytes; macrophages;NK cells; granulocytes weaker;lymphoid subsetsCell adhesion molecule;alpha integrin; part of CD11/CD18Weak on many tumors; Hairy cell leukemia strongVariably present on B-CLL but brightest on hairy cell; mantle cell negative.CD11cLeu M5Early and ma

9、ture granulocytes and monocytes; LGL subsetMost AMLs (more often negative on M6b;M7); someALLsAminopeptidase N; cleaves peptides bound to class IIMy7; LeuM7FITC conjugates may not show positivity.CD13Strong on monocytes and macrophages; weak on granulocytesEndotoxin receptor; binding triggers activa

10、tionMo2; My4;Leu M3Leukemias with monocytic differentiationDifferent CD14 antibodies show variable reactivity with monocytic leukemiasCD14Cell adhesion (Lewis-X antigen; carbohydrate X-hapten) andphagocytosisGranulocytes; monocytes;endothelial cellsReed-Sternberg cells; AMLs with differentiation, es

11、p granulocyticMonocyte reactivity is clone dependent. All antibodies are IgM. Binding often alters light scatter propertiesCD15Leu M1NK cells; mature granulocytesFc gamma RIII (IgG FcR III); GPI anchoredCD16SameLeu 11aBeta chain of integrins Mac-1 (11b/18; CR3); LFA-1 (11a/18); (11c/18; CR4)Expressi

12、on as above(with CD11)PMN & macrophage adhesionCD18Rarely used in phenotyping leukemiasB cells at all stages of maturation; follicular dendritic cells; may be lost in plasma cells but often weakSignal transduction involved in B cell differentiation and activation; ligation increases Ca+;Nearly 100%

13、B-precursor ALL; most B cell lymphomas; partial aberrant expression on some AMLsAML M2 with t(8;21) characteristically positiveCD19B4; Leu 12Most B cell lymphomas; often negative or variable in B- precursor ALL;B cells, but not on earliest B precursorsCa+ channel; B cell activationCD20B1; Leu 16Mant

14、le and marginal zone B cells; follicular dendritic cells; someepitheliaCR2 (complement receptor); C3d/EBV receptor; signal transduction complexFollicular dendritic cell tumors ; some B cell tumors; few ALLsCD21B2;OKB7Antigen recognition; B cell activation; hypersensitivityreactionsIgE heavy chain (e

15、psilon)Antigen- experienced BcellsRare in B-cell tumorsnoneIgG heavy chain (gamma)Antigen- experienced BcellsAntigen recognition; B cell activation;humoral immunityMany B-cell tumors; not lymphoplasmacytoidnoneMost low grade B cell tumors; in cytoplasm of some B precursor )pre-B) ALLsIgM heavy chain

16、 (mu)Nave B cellsnoneB cell activationKappa light chainB-cell tumors; not B-precursor ALLAntigen recognition;B cell activationB cellsNone流式細胞的免疫分型 是白血病分型的重要指標FAB標準，形態(tài)學和免疫表型結(jié)合 Minimal residual diseases (MRD)流式細胞白血病免疫分型First, it is necessary to identify an abnormal population. The second step is to de

17、scribe its phenotype.Third, the phenotype of the abnormal cells must be interpreted in the context of the morphology, in order to diagnose and classify the leukemia or lymphomaA display of CD45 vs RALS allows identification (and, with gating, quantitation) of most normal populations in bone marrow.

18、The bright CD45+, low RALS cells are lymphocytes; the moderate CD45+ high RALS cells are granulocytes; and the cells which have higher CD45 than granulocytes but higher RALS than lymphocytes are monocytes. By convention, on any set of dual parameter displays, populations assigned the same arbitrary

19、color are the same. Thus, the monocytes can also be recognized because they have considerably higher CD33 expression than the granulocytes (Note the green population on both the CD45/RALS and CD71/CD33 displays;). Nucleated red blood cells have low RALS and are dim to negative for CD45. Although deb

20、ris, and unlysed red cells will have this property, nucleated red cells are brightly positive for CD71 (Again color helps to demonstrate this). Acute lymphoblastic leukemia, B-precursor type (common ALL)Diagnosis: Acute lymphoblastic leukemia, B-precursor type (common ALL)Antigen Profile: Positive for CD71, CD19,CD10, CD22, HLADR, CD34;dimly positive for CD33.Acute myeloid leukemia(FAB