诊断学英文课件：Testing of bone marrow cytology

1、Testing of bone marrow cytology,Application of bone marrow examination,?,1) Diagnosis for hematopoietic system diseases: leukemia, myeloma, aplastic anemia, Gaucher disease, Niemann-Pick disease, etc 2)Auxiliary diagnosis: metabolic diseases, IDA, hypersplenism. 3)Others：parasite infectious diseases

2、(malaria, black fever); lupus cell, stem cell cultivation.,Function of bone marrow examination,Indications for Bone Marrow Examination,Contraindication: Hemophilia,Bone marrow aspiration removes a small amount of bone marrow fluid and cells through a needle put into a bone. The bone marrow fluid and

3、 cells are checked for problems with any of the blood cells made in the bone marrow.,The aspiration are done by a trained medical practitioner (nurse or doctor). Posterior iliac crest (any age),Examination of bone marrow,1. Naked-eye observation: bone marrow particles 2. Microscopic examination,Low

4、power lens examination Oil immersion lens examination,1）Low power lens examination Quality of bone marrow smear The extent of bone marrow hyperplasia Megakaryocyte Unusual cells,(1)Quality of bone marrow smear: bone marrow particles marrow film Wright stain,(2) The extent of bone marrow hyperplasia

5、Judge the proliferative degree of bone marrow : 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 Degree Karyocyte : Mature Erythrocyte D

6、iseases Extreme hyperplasia 1:1 leukemia Hyperplasia 1:10 leukemia; anemia Active proliferation 1:20 normal myelogram; anemia Hypoplasia 1:50 aplastic anemia(chronic) Extreme hypoplasia 1:200 aplastic anemia(acute),The extent of bone marrow hyperplasia is posited using the 5 grades classification,(3

7、) Megakaryocytes Four principal aspects about megakaryocytes should be inspected: 1) amount 2) degree of maturity 3) function of generating platelets 4) shape of megakaryocytes and platelets,(4)check whether abnormal cells exist: Abnormal histiocyte Metastatic carcinoma cell Gaucher cell Niemann-Pic

8、k cell Pay attention to observing at the end or the edge of the film Once abnormal cells were found, they should be inspected under oil immersion.,2) oil immersion assay Choose the boundaries of the films coating body and tail. count the karyocytes differentially: G:E is defined as the ratio of the

9、percentage of granulocyte series to erythrocyte series. Reference value: (2-4):1 (2) observe the morphology of cells: Including the shape of mature erythrocyte and whether special abnormal cell and parasitic agents abound.,Bone marrow cytomorphologic examination,Peripheral blood cells,Evolution of b

10、lood cells, 1000 x,With the development and maturity of blood cells: 1) Cell volume: from big to small，except megakaryocytes 2) Cytoplasm: volume increased，but it changes little in lymphocytes. Color turns from deep blue to light blue, even carmoisine. And in the erythrocyte series finally turn into

11、 reddish yellow. Granules in cytoplasm changes as follow: no granulesazurophil granulesspecific granules(neutral, acidophilic and basophilic strippling),3) nucleus: Size: from big to small；From regular to irregular, even with sublobes. But megakaryocytes is from small to big. And that of erythrocyte

12、 series becomes small and regular and eventually disappears. Chromatin: from fine and tight to rough and dense, color changes from light to deep Nucleole: clear blurred disappeared Nuclear membrane: unclearclear,4) ratio of cell nucleus and cell-substance From big to small, i.e. from big nucleus wit

13、h little cytoplasm to small nucleus with abundant cytoplasm (but the opposite occurs in the megakaryocyte),Erythrocyte series Granulocyte series Lymphocyte series Monocyte series Megakaryocyte series Plasmacyte series Other common cells,Normal morphologic characteristics of blood cell,1. Erythrocyte

14、 series,Normal red cells are produced in the bone marrow from erythroid precursors or erythroblasts. The earliest morphologically recognisable red cell precursor is derived from an erythroid progenitor cell which in turn is derived from a multipotent haemopoietic progenitor cell.,(1)normoblast,Somet

15、imes on the edge of cell there are hemisphere or tumor-like ecphymas. Sometimes there is olistherozone surrounding the nucleus with light color.,(2)basophilic erythroblast,There are still tumor-like ecphymas and olistherozone surrounding the nucleus.,(3) Polychomatic normoblast,(4) Ortho-chromatic n

16、ormoblast,(5) Reticulocyte,The reticulocyte is an incompletely mature red blood cell after the orthochromatic normoblast loses its nucleus.,(6) erythrocyte,2. Granulocyte series,Myeloblast promyelocyte myelocyte,neutrophilic myelocyte,eosinophilic myelocyte,basophilic myelocyte,Metamyelocyte,Stab Gr

17、anulocyte,segmented granulocyte,neutrophilic segmented granulocyte,eosinophilic segmented granulocyte,basophilic segmented granulocyte,(1)myeloblast Shape: round or elliptical Diameter: 11-18m nucleus constitutes over 2/3 of cell volume Nucleus shape: round or elliptical Chromatin: fine-sand-like, l

18、ight prunosus, smooth like gossamer Nucleole: 2-5, clear, light blue or colorless Cytoplasm: little, clear sky blue around the nucleus Granules: no or a few,(2)promyelocyte Shape: round or elliptical Diameter: 12-22m Nucleus: big, round or oval Chromatin: thick granular or net-like, distributing une

19、venly Nucleole: clear or disappeared Cytoplasm: comparatively much, light blue or blue Sometimes there is olistherozone near the nucleus Granules: non-specific azurophilic granules, distributed unevenly, with different size, shape and quantity,(3)myelocytes,Neutrophilic myelocyte,The nucleus constit

20、utes 1/2-2/3 of the cell volume,Eosinophilic myelocyte,Basophilic myelocyte,(4)metamyelocyte Shape: round or elliptical Diameter: 10-16m Nucleus: kidney-like Chromatin: rough and chunk-like, arranged densely Cytoplasm: abundant, salmon pink, with different specific granules(neutral, acidophilic, bas

21、ophilic),Neutral metamyelocyte,Acidophilic metamyelocyte,Basophilic metamyelocyte,(5)stab granulocyte Shape: round Diameter: 10-15m Nucleus: long and narrow, girdle-shaped, both ends are blunt and round Chromatin: rough and chunk-like Cytoplasm: specific granules(neutral, acidophilic, basophilic),Ne

22、utral stab cell,Acidophilic stab cell,Basophilic stab cell,(6) 3 types of segmented granulocyte,There are filaments linking the sublobes, or the sublobes are separated from each other completely.,Neutrophilic segmented granulocyte,Eosinophilic segmented granulocyte,Basophilic segmented granulocyte,(

23、1)lymphoblast Shape: round or elliptical Diameter: 10-18m nucleus: big; round or oval Chromatin: exquisite, granular Nucleolus: 1-2, small and clear, light blue or colorless Cytoplasm: clear sky blue Granules: no,3. Lymphocyte series,There is concentration of chromatin around the inner membrane of n

24、ucleus and around the nucleoli, which makes the nuclear membrane thick and clear.,(2)prolymphocyte Shape: round or oval Diameter: 10-16m Nucleus: round or elliptical, sometimes with light incision Chromatin: comparatively exquisite and rough Nucleole: vague or disappeared Cytoplasm: little, light bl

25、ue Granules: without or just with a few big and rare azurophil granules,(3)lymphocyte,4. Plasmacyte series,plasmablast,proplasmacyte,plasmacyte,5. Monocyte series,monoblast,promonocyte,Shape: from round to elliptical or irregular,Chromatin： Delicate loose gradually become closer rough, nucleolus fro

26、m clear to disappear,Cytoplasm: gray-blue，Granules from nothing,Nuclear : Nuclear recessed into the side, after a kidney-shaped, irregularly shaped, with twisted folds.,monocyte,6. Megakaryocyte series,(3)Granular megakaryocyte,(2)promegakaryocyte,(1)megakaryoblast,(4)Thrombocytogenous megakaryocyte

27、 =mature megakaryocyte The granules in the cytoplasm are remarkably concentrated, surrounded by platelets. The edge of the cytoplasm splits into platelets. Conglomerate platelets can be seen on the inner and lateral side of cytoplasm.,(5)megakaryocyte with naked nucleus After the cytoplasm of thromb

28、ocytogenous megakaryocyte completely splits into platelets, the only nucleus is called the naked nucleus.,Cytochemical staining of the blood cell,The cytochemical staining is a kind of assay, based on cell morphology, which uses chemical reaction principles, staining bone marrow slides according to

29、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.,Peroxidase staining (POX) Sudan black B staining(SB) Neutrophil alkaline phosphatase staining (NAP) Acid phosphatase st

30、aining(ACP) -Naphthol acetate esterase (- NAE) Periodic acid schiff stain (PAS) Iron staining,1. Peroxidase staining(POX),Result: no blue or black particles in the cytoplasm: (-) Tiny and diffusely distributed granules in the cytoplasm: weakly positive Thick and dense granules: strongly positive Sig

31、nificance: identify the category of acute leukemia Acute myeloblastic leukemia: strongly positive Acute monocytic leukemia: weakly positive or (-) Acute lymphoblastic leukemia: (-) POX is most valuable in the identification of AML and ALL.,POX,2. Sudan black B staining(SB),A kind of fat-soluble stai

32、n, 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 stronger Monocyte series: weakly (+) Lymphocyte series: (-) Significance: the same as POX.,3. Neutrophil alkaline phosp

33、hatase 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 cytoplasm The intensity of response can be divided into 5 grades: -, 1+, 2+, 3+, 4+. The result is presented as the percen

34、tage of the positive cells and the score. Reference value Adult: positive ratio of NAP: 10%-40%, the score: 40-80 points Significance: acute festered infectious diseases: remarkably, viral infections: normal of slightly CML: remarkably, usu. score 0, leukemoid reaction: extremely AML, ALL, acute mon

35、ocytic leukemia: normal or Aplastic anemia, PNH,NAP,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 ac

36、id. 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 esterase Result: p

37、ositive 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, pl

38、asmacyte, 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

39、(NSE) or monocyte esterase Result: 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.,7. S

40、taining for glycogen,Also 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 promy

41、eloblast 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,7. Staining for glycogen,Si

42、gnificance: normoblast of erythremic myelosis or erythroleukemia: strongly(+),Gaucher cell: strongly(+), adenocarcinoma cell: strongly(+),8. Iron staining,Also called Prussian blue reaction Result: (1)Extracellular ferritin: observe the ferritin inside monocyte-phagocyte series in the bone marrow pa

43、rvule(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+.,Iron stain(extracellular iron）,8. Iron staining,(2)Int

44、racellular 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 nucle

45、us for over 2/3 of the nucleonic perimeter.,1.正常铁粒幼红细胞 2.环状铁粒幼红细胞,Iron stain(intracellular iron）,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(-), perce

46、ntage 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 con

47、stitutes over 15% of the normoblast.,Grouping according to cellular immunology,Also 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) Immu

48、nofluorescence method (2) immunoenzymatic staining 1) APAAP(alkali phosphatase anti-alkali phosphatase) 2) PAP(peroxydase anti-peroxydase) 3) ABC(avidin-bioepiderm-multienzyme complex),recognition of cells in different series (2) Identify different lymphocytes,2. The application of cell immunophenotyping,(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