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Testing of bone marrow cytology(2)-.The content,method and clinical significance of the bone marrow cytomorphologic examination -The test of bone marrow slides is carried out as follows:1.low power scan(1)make sure if the material-drawing and the film preparation of bone marrow aspiration is satisfied:The specimen should be consisted of 3 parts:the head,the body and the tail.-(2)judge the extent of bone marrow hyperplasia:The extent of bone marrow hyperplasia is reflected by the amount of karyocytes in bone marrow generally.Observe the ratio of karyocyte to mature erythrocyte under low power.-The extent of bone marrow hyperplasia is posited using the 5 grades classification.-(3)observe megakaryocytes:Four principal aspects about megakaryocytes should be inspected:1)amount2)degree of maturity3)function of generating platelets4)shape of megakaryocytes and platelets -(4)check whether abnormal cells exist:Abnormal histiocyte Metastatic carcinoma cellGaucher cellNiemann-Pick cellOnce abnormal cells were found,they should be inspected under oil immersion.-2.oil immersion assayChoose the boundaries of the films coating body and tail.(1)count the karyocytes differentially:Calculate the ratio of the granulocyte to the erythrocyte(G:E).(2)observe the morphology of cells:Including the shape of mature erythrocyte and whether special abnormal cell and parasitic agents abound.-3.the clinical significance of the resultThe extent of bone marrow hyperplasia:(1)extremely active hyperplasiaReflect the hyperfunction of bone marrow hematogenesisConditions:leukemia,esp.chronic myelocytic leukemia(CML)-(2)the manifestly active hyperplasiaReflect the vitality of bone marrow hematogenesisConditions:hyperplastic anemia,leukemia,proliferating bone marrow diseases,idiopathic thrombocytopenic purpura(ITP),hypersplenia -(3)the active hyperplasiaReflect the basically normal condition of bone marrow hematogenesisConditions:normal bone marrow,hyperplastic anemia,chronic aplastic anemia with local compensatory hyperplasia -(4)the decreased hyperplasiaReflect the decreased function of bone marrow hematogenesisConditions:chronic aplastic anemia,granulocytopenia,agranulemia,myelofibrosis -(5)remarkably decreased hyperplasiaReflect the nonfunction of bone marrow hematogenesisConditions:acute aplastic anemia,bone marrow necrosis -The ratio of cells in each series at each developing phase in bone marrow(1)Granulocyte seriesAlmost 50%-60%of karyocytes are granulocytes.With cell maturity,the ratio of each developing phase increased.Myeloblast1%,promyelocyte5%Myelocytes+metamyelocytessegmented granulocytesMost of the eosinophilic granulocytes and the basophilic granulocytes are usually mature in bone marrow.eosinophilic granulocytes 5%basophilic granulocytes 1%-(2)Erythrocyte seriesNormoblasts20%Pronormoblast1%Basophilic normoblast5%Polychromatic normoblast10%Orthochromatic normoblast10%G:E is defined as the ratio of the percentage of granulocyte series to erythrocyte series.Reference value:(2-4):1 -(3)Lymphocyte seriesThe percentage of cells of lymphocyte series is approximately 20%of karyocytes.Higher in childhood,it even goes up to 40%.Mature lymphocytes:majorityLymphoblasts and prolymphocytes:rare -(4)Monocyte series4%,almost all of the cells are mature monocytes.(5)Plasmacyte seriesUsu.2%,mainly mature plasmacytes.-(6)Megakaryocyte systemIn a film preparation of 1.5cm 3.0cm,the normal value is defined as 7-35 megakaryocytes can be seen.Granular megakaryocytes and thrombocytogenous megakaryocytes:commonly seenMegakaryoblasts:seldom be seenAmong the megakaryocyte system:Promegakaryocyte5:1)G or EAML,CML,acute pyogenic infection,neutrophilic leukemoid reaction,pure fragility of erythrocytes-aplastic anemiaG:E(2:1)GagranulemiaEhyperplastic anemia,polycythemia vera,secondary erythrocytosis,etc.-ChangediseasesGranulocyte seriesGranulocytic leukemia,acute inflammation,infectious diseases,neutrophilic leukemoid reactionAplastic anemia,agranulemia,granulocytopeniaErythrocyte seriesHyperplastic anemia,megaloblastic anemia,Di Guglielmo syndromeAplastic anemiaLymphocyte seriesAbsolutely increaseALL,CLL,malignant lymphoma,infectious lymphocytosis,infectious monocytosis,other viral infection,lymphocytic leukemoid reactionRelatively increaseAplastic anemia,agranulemia,granulocytopenia -ChangediseasesMonocyte seriesHematological system diseases,infectious diseases,rheumatic diseases,malignant tumor,hepatic cirrhosis,drug reactionPlasmacyte seriesMM,plasmacytic leukemia,macroglobulinemia,heavy chain disease,reactive plasmacytosis,aplastic anemia,agranulemiaMegakaryocytic seriesITP,Evans syndrome,proliferating bone marrow diseases,hypersplenia,megakaryocytic leukemiaAplastic anemia,acute leukemia,soma diseases with bone marrow infiltration or destroy,acute infection,chemical or medicine poisoning,radiation disease -.Cytochemical staining of the blood cellThe cytochemical staining is a kind of assay,based on cell morphology,which uses chemical reaction principles,staining bone marrow slides according to a certain procedure,and at last observing the cytochemistry composition and its changes as seen on the microscope.It can be used in the identification of category of blood cells.-1.Peroxidase staining(POX)Result:no blue or black particles in the cytoplasm:(-)Tiny and diffusely distributed granules in the cytoplasm:weakly positiveThick and dense granules:strongly positiveSignificance:identify the category of acute leukemiaAcute myeloblastic leukemia:strongly positiveAcute monocytic leukemia:weakly positive or(-)Acute lymphoblastic leukemia:(-)POX is most valuable in the identification of AML and ALL.-2.Sudan black B staining(SB)A kind of fat-soluble stain,can make the intracytoplasmic lipid turn to brownish-black or deep black particles.Result:almost the same as POX.Granulocyte series:navemature,the positive reaction becomes strongerMonocyte series:weakly(+)Lymphocyte series:(-)Significance:the same as POX.-3.Neutrophil alkaline phosphatase staining(NAP)Result:ALP mainly exists in the mature neutrophilic granulocyte(stab or segmented),other cells(-)Positive result:gray to brownish-black particles in the cytoplasmThe intensity of response can be divided into 5 grades:-,1+,2+,3+,4+.The result is presented as the percentage of the positive cells and the score.Reference valueAdult:positive ratio of NAP:10%-40%,the score:40-80 pointsSignificance:acute festered infectious diseases:remarkably,viral infections:normal of slightlyCML:remarkably,usu.score 0,leukemoid reaction:extremelyAML,ALL,acute monocytic leukemia:normal or Aplastic anemia,PNH -4.Acid phosphatase staining(ACP)Result:positive reaction:brownish-black particles in the cytoplasm.Significance:assist in the diagnosis of hairy cell leukemia:hairy cell(+)or strongly(+),the activity can not be inhibited by L-tartaric acid.Identify T lymphocyte and B lymphocyte:T cell(+),B cell(-).Gauchers disease(+),Niemann-Pick disease(-)monocyte(-),histiocyte(-),reticular cell(-),megakaryocyte(-).-5.Naphthol AS-D chloroacetate esterase staining(AS-D NCE)Also called specific esterase(SE),granulocyte esteraseResult:positive reaction:red sediment in the cytoplasm.SE mainly exists in granulocyte series,but microleukoblast(-)or weakly(+),from promyelocyte to mature neutrophilic granulocyte(+),promyelocyte:strongly(+),activity weakened with the maturity of the cells.Eosinophilic granulocyte,lymphocyte,plasmacyte,normoblast:(-)Monocyte(-)or weakly(+)Significance:AML:strongly(+),acute monocytic leukemia and ALL(-)Acute myeloblastic-monocytic leukemia:cells of granulocyte series(+),cells of monocyte series(-)-6.Alpha-naphthol acctate esterase staining(-NAE)Also called nonspecific esterase(NSE)or monocyte esteraseResult:gray-black or brownish-black sediment in the cytoplasm.NSE mainly exists in monocyte series.Monoblast(-)or weakly(+),promonocyte and monocyte(+),granulocyte series(-)or weakly(+),lymphocyte(-)Significance:identify the acute monocytic leukemia and AML.NSEInhibited by NaFAcute monocytic leukemiaStrongly(+)yesAcute myeloblastic leukemia(-)or weakly(+)no -7.Staining for glycogenAlso called the periodic acid-Schiffs reaction(PAS)Result:positive:the cytoplasm stains red,red substance can be granular,block-like or diffused red.The degree of positive reaction can be divided into 4 grades:strongly(+),(+),weakly(+)and(-).Myeloblast(-),from promyeloblast to segmented neutrophilic granulocyte(+),the positive strengthened with the maturity of cells.Monocyte:weakly(+),lymphocyte(-)or weakly(+)Normoblast and erythrocyte(-)megakaryocyte and platelet(+),with the maturity of cells,positive reaction strengthened -Significance:normoblast of erythremic myelosis or erythroleukemia:strongly(+)7.Staining for glycogenDiseaseCellPASShape of positive substanceAMLMicroleukoblast(-)or weakly(+)Fine,granular of diffused salmon pinkALLLymphoblast and young lymphocyte(+)Coarse granular-like or chunk-likeAcute monocyte leukemiaMonoblast(+)Widespread and homogeneous red or fine granulesGaucher cell:strongly(+),adenocarcinoma cell:strongly(+)-8.Iron stainingAlso called Prussian blue reactionResult:(1)Extracellular ferritin:observe the ferritin inside monocyte-phagocyte series in the bone marrow parvule(outside the normoblast).Positive:light bluish-green and homogeneous shapeless or pearl-like,coarse grain-like,blue or deep blue,or blue-black block-like substance.The positive reaction can be divided into 5 grades:-,1+,2+,3+,4+.-(2)Intracellular ferritin:the ferritin in normoblast.Sideroblast:1 to 5 blue and tiny ferritin granules surrounding the nucleus of normal normoblast(mainly orthochromatic normoblast)Crico-sideroblast:one sideroblast with over 10 thick and deep stained ferritin granules,which is surrounding the nucleus for over 2/3 of the nucleonic perimeter.8.Iron staining -Reference value(1)extracellular ferritin:1+2+,mostly 2+(2)intracellular ferritin:20%90%,the average is 65%Significance:(1)iron deficiency anemia:extracellular ferritin(-),percentage of sideroblast,ofter below 15%,even 0%.(2)non-iron deficiency anemia:including thalassemia,sideroblastic anemia,hemolytic anemia,megaloblastic anemia,aplastic anemia,displacement anemia,the extracellular ferritin,often 3+4+(3)sideroblastic anemia:sideroblast,crico-sideroblast constitutes over 15%of the normoblast.8.Iron staining -.Grouping according to cellular immunologyAlso called cell immunity mark(phenotype)assay.It makes use of monoclonal antibody and immunology techniques to test the specific antigen on the surface of cytomembrane and/or in the cytoplasm.-1.Assay methods(1)Immunofluorescence method(2)immunoenzymatic staining1)APAAP(alkali phosphatase anti-alkali phosphatase)2)PAP(peroxydase anti-peroxydase)3)ABC(avidin-bioepiderm-multienzyme complex)-2.The application of cell immunophenotyping(1)recognition of cells in different series(2)Identify different lymphocytesMembrane antigenMature T lymphocyteCD2,CD3,CD4,CD7,CD8Mature B lymphocyteCD19,CD20,CD22NK cellCD16,CD56Membrane antigenMedullary systemCD13,CD14,CD15,CD32,CD33,CD65,CD91,CD156,CD166 Lymphocyte seriesCD1,CD2,CD3,CD4,CD5,CD7,CD8,CD9,CD10,CD19,CD20,CD52,CD77Megakaryocyte and plateletCD41,CD42,CD61Erythrocyte seriesGlycophorin A or B -(3)Test the T lymphocyte subgroup(4)Identify cells at various stages of differentiation(5)Identify cells in different functional states(6)Isolation and research cells in different series and stages of differentiation(7)Immunophenotypic analysis of various kinds of leukemic cells(8)Test of the leukemic tiny residueMembrane antigenHemopoietic stem cellCD34+,CD39+,Lin-Hemopoietic stem cell transform to medullary systemCD34+,CD33+Progenitor cell of T lymphocyte seriesCD34+,TDT+,CD10+,CD7+Progenitor cell of B lymphocyte seriesCD34+,CD19+High expressionNo expressionMemory T cellCD45ROCD45RAActive T cellCD45RO,CD25,CD2,CD54,CD58CD45RA -3.The immunophenotypic characteristics of acute leukemia cell(1)The immunophenotypic characteristics of cells in ALL and AML -(2)The immunophenotypic characteristics of ALL(3)The immunophenotypic characteristics of AML -.Cytogenetic analysis1.Chromosome analysisThe abnormal clone of chromosome is defined as follows:The same chromosome is tested for structural abnormalities in 2 or more cell division phases.The same chromosome is lost in 3 or more cell division phases.-(1)Denomination of chromosomeThere are 46 chromosomes in one human somatic cell,22 pairs(44)of which are euchromosomes,1 pair(XY)of which is sex chromosome.The 46 chromosomes are divided into Group A-G according to the length and the position of centromere.According to the banding characteristics of each chromosome in differential staining technique,chromosomes are divided into regions and bands.-Human chromosome fluorescence staining -(2)Karyotyping analysis and writingKaryogram is defined as a complete set of chromosomes in metaphase of cell division which are cut out and ranked according to Danvers system after magnification by photograph.The sequence of karyotype writing:chromosome number,accessory chromosome,chromosomal abnormality,separated by comma.chromosome variationabbreviation or symbolinterchangetinversioninvisobrachial chromosomeiso or iinsertioninsdeletiondelcircular chromosomerchromosome loss-chromosome accrescence+-(3)Chromosomal aberrationIncluding numerical aberration and structural aberration.There are 2 sets of chromosomes in a normal somatic cell,so it is called a diploid(2n).Chromosomal aberrationeuploidaneuploidhaploidtriploidhypodiploidhyperdiploidtetraploid -2.Common chromosomal changes in acute leukemia -Thank you!Thank you!-
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