检验医学英语

Chapter 2 Bone marrow Examination Bone marrow (BM) is spongy tissue found inside some of your larger bones. Bone marrow has a fluid portion and a more solid portion. A bone marrow aspiration is usually done at the same time as a biopsy. In an aspiration, a sample of the liquid portion of your marrow is withdrawn. A bone marrow biopsy and aspiration are often called a bone marrow examination. 2.1. Clinical application of bone marrow examination A bone marrow exam offers detailed information about the condition of your blood cells. Sometimes, collecting a blood sample through a vein in your arm provides enough information about your health. But if those results are abnormal or don't offer enough details, you may need further evaluation with an examination of your bone marrow. Because your bone marrow is essentially a blood cell factory, it's normally rich in young cells. Examining bone marrow gives a much more detailed picture of the types, amount and condition of these newly forming blood cells. The marrow can be studied to determine the cause of anemia, the presence of leukemia or other malignancy, or the presence of some “storage diseases” in which abnormal metabolic products are stored in certain bone marrow cells. A. Indications of bone marrow examination The basic indication for performing a bone marrow evaluation is to answer questions that a routine hematology examination of a blood sample does not answer. One need not take the additional effort to take a bone marrow aspirate and biopsy, if, for example, the blood already clearly indicated an immune mediated hemolytic anemia, a typical inflammatory response, or even a leukemia with clearly diagnostic features in EDTA blood is present. Most cases of anemia (low red blood cells) can be diagnosed by simple blood tests. When these tests are negative, examining the marrow can reveal problems with the red blood cells that are uncommon causes of anemia (sideroblastic anemia, aplastic anemia). The most common indications for bone marrow analysis are a deficiency of cells from one, two or all three cell lines. Cytopenias also suggest decreased bone marrow function so one should check for various bone marrow diseases. These would be a thrombocytopenia and/or leukopenia. Additionally, leukemia may be hidden in the marrow blast cells may be numerous in the bone marrow but few or no blast cells are seen in the blood (aleukemic leukemia). The more common indications are fever, hepatomegalia, splenomegalia, lymphadenectasis of unknown origin and juvenile cell in peripheral blood. Hypercalcemia may be caused by lymphosarcoma, which may be located in the bone marrow. Plasma cell myeloma may be suggested by hyperproteinemia or by lytic lesions in the spine. B. Contraindications of bone marrow examination. The only absolute contraindication is hemophilia and other congenital hemorrhagic disorders. 2.2. Aspiration of bone marrow and preparation of marrow smears: A. Site of aspiration The samples are preferably obtained from the posterior iliac crest (spina iliaca posterior superior)(Figure 2.2-1). In this location, a bone marrow aspiration is technically easy, less hazardous and less painful than a sternal aspiration. The anterior iliac crest can also be used as the site for bone marrow aspiration in order to avoid a repositioning of patient. B. Preparation of marrow smears The first portion of material aspirated, about 0.2 ml of marrow, is used for the preparation of several thin smears.The preparation and staining of these smears are same to peripheral blood smears. 2.3. The development rules of hematocyte It has been demonstrated that the blood cells are derived from the multipotential stem cell. The various blast cells which will be further differentiated have similar morphologic characteristic, a large cell with a nucleus and nucleoli. These structures are regularly changed as cells differentiate. The transformation from an immature cell to a mature cell always involves changes in the cytoplasm, nucleus and cell size. A. Cell size Cell size changes from large to small except megakaryocyte. B. Nucleus a. Size changes from large to small; nucleus disappears in matured red blood cell. b. Shape changes from round to segment in granulocytic series. c. Chromatin changes from fine, sparse to coarse, condensed. d. Membrane changes from unconspicuous to conspicuous. e. Nucleoli changes from predominant to vanish. C. Cytoplasm a. Amount changes from less to more. b. Color changes from dark blue to light blue in granulocyte, and to red in red cell. (Basophilic is proportional to the cytoplasmic content of ribonucleic acid-RNA as the cell matures, there is a gradual loss of cytoplasmic in general, the more basophilic the cytoplasm, the less mature the cell.) c. Granules changes from none to less and then more. In cells of myeloid series cytoplasmic differentiation is characterized by the appearance of granules. The granules are probably concerned with enzyme systems. When cytoplasmic granules first appear. They are few, coarse and wine red. The number gradually increases, and the granules differentiate into three types. These are eosinophilic, basophilic and neutrophilic granules. In erythroid cells, no granules are present during all stages of differentiation. D. Ratio of nucleus to cytoplasm changes from high to low. 2.4. Morphology of the marrow cells A. Erythroid series a. Proerythroblast (Pronormoblast) :Round or irregular, 15-22μm (2-3 times that of a mature red cell), the cytoplasm is deeply basophilic in appearance. No granules are present. Nucleus is oval or round in shape, exhibiting chromatin aggregation. Normally, 1 to 4 nucleoli are present. b. Basophilic normoblast(Early normoblast): Similar to the pronormoblast but slightly smaller (12-20μm), cytoplasm is more abundant and less basophilic than that of pronormoblast. Nucleus reveals deeply stained chromatin bands. Nucleoli are sometimes present. c. Polychromatic normoblast (Intermediate normoblast): It is up to twice the size of a mature red cell (10-15μm) round or irregular shape and polychromatic cytoplasm which is more abundant. Nucleus is reduced in size with deeply basophilic chromatin aggregation. d. Orthochromic normoblast (Late normoblast): It is slightly larger than the mature red cell (7-12μm), with regular round shape. The cytoplasm is hemoglobinized and abundant in amount, the nucleus is pyknotic. e. Reticulocyte: This cell stains as a mature red cell with the Wright’s dyes. It can be differentiated only supravital dye staining technique. B. Myeloid series a. Myeloblast: It is 10-20μm, round and regular. Its cytoplasm stains basophilic by Wright’s stains. No granules are present. Nucleus is round in shape, no chromatin aggregations are normally present but 2-6 nucleoli are usually seen. b. Promyelocyte: It is 12-25μm, regular in shape. Its cytoplasm is less basophilic than its precursor and is more abundant, often with purple granules. Nucleus may still be seen in the early cell. c. Myelocyte: It is 10-18μm in diameter, regular and round. Its nucleus is smaller than that of the promyelocyte and some chromatin aggregates are seen in nucleus. Nucleoli are absent. The cytoplasm is light blue to pink and may contain azurophilic and specific granules which may be eosinophilic, basophilic or neutrophilic. d. Metamyelocyte: It is 10-16μm in diameter. The nucleus is typically kidney shaped. The chromatin structure in the nucleus is more cyanotic than that of the myelocyte and is usually condensed into irregular thick and thin areas. The cytoplasm is abundant, pale or pink in color and contains specific granules. e. Stab granulocyte (Band granulocyte): It is 10-13μm in diameter. The nucleus is elongated, sausage shaped or deeply indented. It is not segmented but may be slightly constricted at 1 to 2 points. The chromatin is continuous, thick and coarse. The cytoplasm is similar to that of metamyelocyte. f. Segmented granulocyte: It is 10-13μm in diameter. The nucleus is central or eccentric, with heavy thick chromatin masses. It is divided into several lobes connected to each other by thin chromatin bridge. The cytoplasm is abundant, slightly eosinophilic or colorless, and contains specific granules. The basophilic and eosinophilic granules are large and overlie the nucleus. The neutrophilic granules are very fine. C. Monocytic series a. Monoblast : 15-25μm in diameter, round or sometimes oval. The nucleus is round and sometimes indented with 2 to 6 nucleoli. The chromatin pattern may resemble that of a myeloblast showing delicate blue or purple stippling. The cytoplasm is often relatively large in amount, may contain a few azurophilic granules, and stains pale blue or gray. The cell border is irregular with pseudopods and indentations. b. Promonocyte: 15-20μm in diameter. The nucleus is large, ovoid, and convoluted and indented. The chromatin forms a loose, open network. There may still be a nucleolus. The gray-blue cytoplasm contains fine azurophilic granules. c. monocyte: 12-20μm in diameter. The nucleus is kidney shaped, horseshoe shaped or round and often lobulated. The chromatin is distributed in a linear arrangement of delicate strands. No nucleoli are seen. The cytoplasm is abundant with gray-blue “ground-glass” appearance. It contains azurophilic dust. D. Lymphocytc series a. Lymphoblast : 10-18μm in diameter. The nucleus is round or oval and the chromatin has a stippled delicate pattern. 1 or 2 nucleoli are present, which are usually well outlined. The cytoplasm is no granular, stained deep blue. b. Prolymphocytc : 10-20μm in diameter. The nucleus is oval but slightly indented and may contain nucleoli or nuclear remnants. The chromatin appears coarser than that of the lymphoblast. Cytoplasm stains basophilic and scantly azurophilic granules are occasionally present. c. Large lymphocyte: 12-18μm in diameter. The dense, round, oval or slightly indented nucleus is centrally or eccentrically located. Its chromatin is dense and clumped. Cytoplasm is abundant and appears hyaline blue with the Wright’s stain. Small numbers of minute azurophilic granules may be present. d. Small Lymphocyte : 6-8μm in diameter. Approximately that of a mature red cell nucleus is same as that of large lymphocyte. Scanty cytoplasm is seen which usually forms a narrow rim and often contains few azurophilic granules. e. Plasmacyte: Up to twice the size of a mature red cell (8-9μm ) oval shape. The nucleus is eccentrically placed. The condensed chromatin forms clumps that may be concentrated in the periphery of the nucleus showing typical “cartwheel’’ pattern. No nucleoli are seen. The cytoplasm is dark blue and there is usually a clear zone close to the nuclear membrane. The cytoplasm is no granular but may contain vacuoles. E. Megakaryocytic series a. Megakaryoblast : 20-30μm diameter. Nucleus is large, indented and irregular in shape with a fine reticulum chromatin network. Occasionally 1 or 2 small indistinct nucleoli are present. Cytoplasm appears moderately basophilic and the periphery shows pseudopodia like structure. No granules are present. b. Promegakaryocytc: 30-50μm in diameter. It is much larger than the megakaryoblast. The nucleus is large, indented and poly lobulated, rarely multinucleated. The chromatin appears coarse and may show some clumping. Nucleoli may be still present. Cytoplasm is abundant and appears basophilic with early azurophilic granules. c. Megakaryocyte: 40-100μm in diameter. It is the largest cell found in the normal bone marrow. The nucleus is lobulated and irregular in shape. The chromatin is heavy clump, and nucleoli are not seen.The cytoplasm is abundant, appearing slightly basophilic or polychromatic with numerous azurophilic granules. The cytoplasm of the platelet-producing (active) megakaryocyte produces pseudopod-like projection and contains aggregates of azurophilic granules surrounded by pale halos. This structure gives rise to platelets at the periphery of the megakaryocytes. The cytoplasm of non-platelet-producing (inactive) megakaryocyte is free from azurophilic granules and aggregates. d. Platelet: 2-3μm in diameter, non nucleated each platelet consists of a central group of azurophilic granules, granulomere, and a surrounding light blue hyalomere. 2.5. The procedure of bone marrow cytological examination A. Procedure of bone marrow cytological examination a. Observe the quality of the stains smear under the low power and evaluate whether it is fit for study. Thin and well-stained areas were selected for cell count. Observe the ratio between erythrocytes and nucleated cells and determine the degree of hyperplasia. b. Count the number of megakaryocytes in the whole film. c. Examine the whole smear particularly its tail portion for special cells such as tumor cells or other large pathologic cells. d. A minimum of 200 nucleated cells should be counted under the oil immersion and the relative positive proportions of various cells are enumerated. B. Determination of Bone marrow proliferaative degree According to the ratio between erythrocytes and nucleated cells, five grades can be enumerated (table 2.5-1). Table2.5-1 Bone marrow proliferative degree Grade erythrocyte nucleated nucleated cell% Extreme hyperplasia 1:1 >50 Obvious hyperplasia 10:1 >10 Normal hyperplasia 20:1 1-10 Hypoplasia 50:1 0.5-1 Extreme hypoplasia 200:1 <0.5 2.6.Clinical significance of bone marrow examination A. significance of bone marrow proliferative degree a. Extreme hyperplasia: leukemia, especially chronic granulocytic leukemia. b. Obvious hyperplasia: leukemia, hyperplastic anemia, idiopathic thrombocyto- penic purpura, et al. c. Normal hyperplasia : normal bone marrow, some anemia. d. Hypoplasia: chronic aplastic anemia, granulocytopenia. e. Extreme hypoplasia: acute aplastic anemia B. Ratio or myeloid to Erythroid (M: E) Normally about 2-4: 1, the M: E Ratio is increased in acute and chronic infection, leukemoid reactions (e.g, chronic inflammation, metastatic tumor), acute and chronic myeloid leukemia, myelodysplastic disorders and pure red cell aplasia. Decreased in agranulocytosis, anemias with erythroid hyperplasia (megaloblastic, iron-deficiency, thalassemia, hemorrhage, hemolysis, sideroblastic), and erythocytosis (excessive RBC production). Normal in aplastic anemia (though marrow hypocellular), myelofibrosis(marrow hypocellular), multiple myeloma, lymphoma, anemia of chronic disease. 2.7.Cytochemical cell differentiation Cytochemical technics allow further differentiation of hematopoietic cells. According to the chemical characteristics of the different elements of the cells, certain cytochemical stain may expose some of them, such as enzymes, ribonucleic acid, iron granules, etc. The reaction can be visualized based on the precipitation of an added dye. The cytochemical reactions of blood cells are sometimes helpful in the diagnosis of some disorders or in the identification of the cell line to which certain cells belong. Cytochemical reactions are most useful in:①distinguishing certain types of leukemias,②distinguishing a leukemoid reaction from leukemia,③distinguishing benign from malignant lymphocytic proliferations,④identifying the ringed sideroblasts in the sideroblastic anemia. A. Myeloperoxidase stain (MPO, POX) a. principle The enzyme peroxidase is present in the granules of myeloid cells. It may act on hydrogen peroxide with liberation of oxygen, which oxidizes benzdine into a brownish compound. b. Clinical significance The peroxidase reaction is positive in cells of the neutrophilic, eosinophilic and monocytic series. It can be used to differentiate cells of these types from lymphoid or erythroid cells, which are peroxidase negative. B. Alkaline phosphatase stain (ALP) a. Principle The enzyme is only present in the cytoplasm of granulocytes. The Alkaline phosphatase stain usually using α-naphthyl phosphate as substrates is positive in the granulocytes. The positive reaction shows that blackish-brown precipitates are diffusely scattered throughout the cytoplasm of cells. b. Clinical significance High alkaline phosphatase activity of granulocytes is found in infections, aplastic anemia, leukemoid reaction, acute lymphocytic leukemia. Low activity is often found in chronic and acute granulocytic leukemia. C. Periodic Acid-Schiff stain (PAS) a. Principle Periodic acid (HIO4) is an oxidizing agent that converts hydroxyls groups of adjacent carbon atoms to aldehydes. The resulting dialdehydes are combined with Schiff’s reagent to give a red-colored product. A positive reaction is therefore seen with polysaccharides, mucopolysaccharides, and glycoproteins. b. Clinical significance Cells of neutrophilic or eosinophilic series all react with positive results, being mostly marked in the mature stage; monocytes have a faint staining reaction. Lymphocytes may contain a few small or large granules. Normoblasts are normally PAS negative. In erythroleukemia and in thalassemia, some of the erythroid precursors are positive. In iron deficiency anemia, the positive PAS reactions usually appear. In malignant lympho-proliferative diseases, the lymphocytes may have increased numbers of PAS-positive granules. D. Non-specific esterases stain (NSE) a. Principle The non-specific esterase reaction usually using α-naphthyl acetate as substrates is strongly positive in monocyte but weak or negative in granulocytes. The positive reaction shows that redish-brown precipitates are diffusely scattered throughout the cytoplasm of cells. b. Clinical significance NSE reaction can be used to differentiate the leukemic cells originated from monocytic or granulocytic series. The more specific esterases that react positively in monocytes are inhibited by the presence of sodium fluoride. E. Iron stain (siderocyte stain) a. Principle Siderocytes are red cells containing non-hemoglobin iron granules, which stain with a potassium ferrocyanide acid mixture. A sideroblast is a nucleated red cell containing iron granules. The iron granules stain blue. b. Clinical significance Marrow iron is representative of body iron store. In normal bone marrow, sideroblasts are ranged from 19% to 44%. The number of sideroblasts depends on the serum iron level. With iron deficiency, the sideroblasts disappear. Their absence is a reliable criterion of iron-deficiency anemia. The sideroblasts are increased in hemolytic anemias, hemochromatosis. In “ringed sideroblast” the stained iron particles surround the nucleus. These cells are found in lead poisoning and sideroblastic anemia. 2.8. Characteristic of common hemopathy 2.8.1 Acute leukemias Acute leukemias are diagnosed according to morphological criteria on peripheral blood and bone marrow smears. The additional use of cytochemical staining techniques greatly facilitates the differentiation and classification into the following major subgroups: -acute lymphocytic leukemias(ALL) -acute myeloid leukemias (AML), synonymous with the term acute non-lymphocytic leukemias (ANLL). Based on the widely accepted French-American-British(FAB) classification, acute leukemias are further subdivided into eight myeloid and three lymphocytic leukemias. The diagnosis criteria of acute leukemias have been established: At least 30% of all nucleated cells in the bone marrow are blasts(FAB). 2.8.1.1 Acute Myeloid Leukemia Acute myeloid leukemia (AML) is a clonal malignancy of myeloid bone marrow precursors in which poorly differentiated cells accumulate in the bone marrow and circulation. Signs and symptoms occur because of the absence of mature cel