Investigation methods in Hematology*

ContentsCell countsBlood smearBone marrow examinationFlow cytometryCytogeneticMolecular biology*

Cell countCareful assessment of the blood elements first step in assessment of hematologic function and diagnosis.Examination of blood smears and hematologic parameters often offer an important diagnostic information, allow differential diagnosis and indicate additional specific tests.Blood elements include red cells (erythrocytes ), white cells (leukocytes) and platelets. Evaluation of blood requires quantification of each of these cellular elements by manual or automatic methods. Automatic methods are usually more precise than manual procedures.*

Cell countThe blood sample should be obtained in proper conditions. There are factors that affect the hematologic measurements : patient activity, level of hydration, medication, sex, age, race, smoking and also the age of the sample may affect the data collected.Blood is collected by venipuncture into collection tubes with anticoagulant. The most preferred anticoagulant is EDTA ( tripotassium of trisodium salts of ethylenediaminetetraacetic acid) remove calcium - because produces complete anticoagulation with minimal morphologic and physical effects on cells.

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Red blood cells parameters

hemoglobin concentration Hbhematocrit Ht (volume of packed red cells) the volume of a blood sample occupied by red cellsred cell countmean corpuscular volume (MCV)= the average volume of the red blood cellsmean corpuscular hemoglobin (MCH) measure of the average Hb content per red cell. MCH=Hb (g/l)/ red cell count

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Red blood cells parameters

mean corpuscular hemoglobin concentration (MCHC) the average concentration of Hb in a given red cell volume. MCHC=Hb (g/l)/Htred cell width (RDW) reflects the heterogeneity of red cells size in sample reticulocyte counts provide useful information about the bone marrow capacity to synthesize and release red cells in response to a physiologic challenge ( anemia). Reticulocyte count performed manually using supravital staining with metilene blue looks as a dark blue meshwork or granules ( it is staining precipitated RNA).

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Leukocyte analysis automated WBC counts may be falsely elevated in the presence of cryoglobulins , aggregated platelets

Platelets analysisfalsely low PLT caused by the presence of platelets clumps or platelets agglutininsfalsely high PLT caused by fragments of red and white blood cellsmean platelet volume (MPV) has been correlated with some diseases. MPV has an inverse relationship with PLT number : high MPV in ITP ( low number of PLT)*

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Blood smearOptimal morphology and staining are obtained from nonanticoagulated blood from a fingerstick procedure : a drop of blood not too small, not too large, is placed in the center of the clean glass slides; a second spreader slide is placed at a 30 to 45 degree angle and moved backward to make contact with the blood drop, which will spread along the slide edge. This technique creates a film of blood 3-4 cm long.Blood smears are usually stained with May-Grunwald-Giemsa stains.*

Blood smearExamination of the blood smear : initially under an intermediate power (10 to 20 X objective) in order to asses the cellular distribution and staining; should scan over the entire blood smear to ensure the abnormal populations.After use an oil immersion lens (50 to 100x objective)Important to evaluate each cell type for quantitative and qualitative abnormalities*

Red cellsevaluated for changes in size, shape, Hb distribution, presence of cellular inclusionsnormal red cell dimeter 7,2-7,9 mvariation in red cell size = anisocytosisred cell > 9 m macrocytered cell < 6 m microcytevariation in red cell shape = poikilocytosis; examples : spherocytes , teardrop cell (dacryocyte), drepanocyte (sickle shaped), schistocyte ( fragmented cells).hypochomia = poor hemoglobinization , leading to a very thin rim of Hb or an increased area of central pallortarget cell = cell with a central spot of Hb surrounded by an area of pallor ( because abnormal distribution of Hb)red cells may contain inclusions : Howell-Jolly bodies remnants of nuclear material, Pappenheimer bodies remnants of mitochondria, infectious agents (malarial parasites)*

The association between the pathologic red cell and disease state

Cabot rings ( circular blue inclusion) postsplenectomy, hemolytic anemia, megaloblastic anemiaHowell-Jolly bodies - postsplenectomy, hemolytic anemia, megaloblastic anemiaOvalocyte ( elliptocyte) hereditary elliptocytosisHypochromic red cell iron deficiency anemia, thalassemia , sideroblastic anemiaMacrocyte - increased erythropoiesis; oval macrocyte in megaloblastic anemia, round macrocyte in liver disease*

The association between the pathologic red cell and disease state

Pappenheimer bodies sideroblastic anemia, postsplenectomyRouleaux paraproteinemiaSchistocyte microangiopathic hemolytic anemia ( DIC, TTP, severe burns)Drepanocyte sickle cell disordersSpherocytes hereditary spherocytosis, immuno-hemolytic anemiaTarget cell liver disease, postsplenectomy, thalassemiaTeardrop cell myelofibrosis

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PlateletsPLT number may be estimated from the blood film. Normal PLT counts should have several platelets ( 5-15) per oil immersion field or 1 platetet for 10 to 20 red blood cells Diameter 1-2 Leukocytesare analyzed lastneutrophils, eosinophils, basophils, lymphocytes, monocytes normally seen on the blood smearimmature myeloid cells : myelocytes, metamyelocytes, promyelocytes , blasts abnormalshould be counted at least 100 elementsshould screen for morphologic abnormalities of the cytoplasm and nucleus : toxic granulation ( azurophilic granules) increased in infection or after growth factor therapy; hypogranularity of neutrophils in myelodysplastic disorder*

Bone marrow examinationImportance : diagnosis and management of hematologic diseasesExamination of BM include 2 specimens : cytologic preparation of BM cells obtained by aspiration of BM and smear of the cells; the second needle biopsy of the bone and associated marrow, allowing optimal evaluation of marrow cellularity, fibrosis, infections, infiltrative diseases *

Bone marrow examinationIndications :evaluation of primary bone marrow tumorsstaging for bone marrow involvement by metastatic tumorsassessment of infectious diseasesfever of unknown originevaluation of metabolic storage diseases*

Bone marrow examinationMethods :younger children may have marrow examination from the anterior medial tibial area ( at birth, hematopoietic marrow is found in all bones, by early childhood fat cells replace the bone marrow hematopoietic cells in the extremities).in adults aspiration from sternum at the second intercostal space or anterior/posterior iliac crest aria. For biopsy - posterior iliac crest areaThe skin, subcutaneous tissue and periosteum in the area of biopsy are anesthetized with a local anesthetic ( lidocaine).From aspirated material we make smears; if additional material is needed for flow cytometry , cytogenetics, culture it is needed additional aspiration, collected into tubes with anticoagulant.The BM biopsy is made with a larger needle; touch preparations of the bone biopsy should be made, particularly if no aspirate was obtained.The BM aspirate or touch preparations are stained with Wright or May-Grunwald-Giemsa stains.*

Bone marrow examinationThe aspirate smear allows cytologic examination of BM cells . Should be evaluated minimum 500 nucleated cells under oil immersion magnification. Erythroid cells represents 10-40% of marrow cells; erythroid cells should be examined for abnormalities in morphology and iron content. The myeloid cells are usually predominant within BM and mature forms are more numerous. If immature forms are predominant reveals a pathologic process. Megakaryocytes represent < 1% of BM cells. Some other non-hematopoietic cells may be seen within the BM : macrophages, mast cells, stromal cells, fat cells , representing < 1% from total marrow cellularity.*

Bone marrow examinationBone marrow core biopsies and the clot are fixed in formalin; they are stained with hematoxylin and eosin or Giemsa stain for morphologic examination.The BM biopsy section provides the best representation of BM and its anatomic relationships normal localization of immature myeloid cells adjacent to bony trabeculae; useful for evaluation of infiltrative processes ( carcinoma, lymphoma), fibrosis. When aspiration of BM is not possible the biopsy remains the only method for diagnosis.

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Immunohystochemical testsPerformed in Pathology DepartmentOn solid samples : bone marrow biopsy, lymph node biopsy or any other tissueUsing monoclonal antibodiesVery important for a positive diagnosis of acute leukemias, chronic lymphoproliferative disorders, metastatic tumors with bone marrow involvement*

Cytochemical stainsuseful in diagnostic and classification of acute leukemias , differential diagnosis between myeloid and lymphoid acute leukemiamade on peripheral blood films, bone marrow aspirates or touch preparations from BM, lymph node or other tissue biopsies*

Cytochemical stainsMyeloperoxidaseprimary granules of neutrophils and secondary granules of eosinophils contain MPO. Lymphocytes and nucleated red blood cells lack the enzyme.Positive in AML Sudan Black Bthe pattern of staining is parallel with MPO positive staining for granulocytic cells and eosinophilsSpecific esteraseused to identify cells of granulocytic lineageNonspecific esteraseused to identify monocytic cells, but do not stain granulocytes and eosinophils.*

Cytochemical stainsTerminal deoxynucleotidyl transferase TdT- intranuclear enzyme found in immature lymphoid cells- useful marker to identify ALL and lymphomas Periodic Acid-Schiff (PAS)detects intracellular glycogen found in variable quantities in most hematopoietic cellsAML6 PAS intense positivePerls stainused to identify iron in nucleated red blood cells normally red cell precursors contain one or more blue granules in 20-50% of the cells. When these granules surround more than 2/3 of the nucleus of the red cell precursor, the cell is called ringed sideroblast.

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Flow cytometryFlow cytometry is the measurement of cell properties (cytometry) as cells move in single file ( flow) in a fluid column and interrupt a beam of laser light. The method allows the quantitative and qualitative analysis of many properties ( multiparameter) of cell population from body fluids.Using monoclonal antibodies.*

Flow cytometryVery important for :determination of lineage ( myeloid or lymphoid ), if morphology and cytochemistry are not enough for diagnosisdistinction between B- and T-cell acute leukemiasdetection of mixed lineage acute leukemiasdetection of monoclonality in B-cell lymphoproliferative disorders important for prognosis evaluation of minimal residual disease*

Flow cytometryPrecursor B-cell acute lymphocytic leukemia : CD34, CD19, HLA-DR, CD10T-cell acute lymphocytic leukemia : CD34, CD2, CD7 ,TdT, some are CD10 positiveAcute myeloid leukemia : CD34, CD 117, CD33, CD13, CD15, CD11b, cMPO.B-cell chronic lymphoproliferative disorders : CD5, CD23, CD19, CD20, FMC7, CD38; important to differentiate types of lymphoproliferative disorders ( mantle lymphoma, CLL , follicular lymphoma, hairy cell leukemia).Peripheral T-cell lymphoma clonality may be suspected by phenotypic abnormalities of major T-cell markers Cd4, CD8 *

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CytogeneticsChromosome structure studyCytogenetic analysis became very important for :diagnosis, classification,management of therapy, prognosis ,quantification of therapy response research of hematologic disorders*

CytogeneticsDiagnosisthe presence of a clonal chromosomal abnormality establish the presence of a clonal bone marrow disordervery important for myelodysplastic syndrome diagnosis in patients with mild cytopenias and bone marrow with minimal or no dysplasia. The presence of Philadelphia chromosome=t(9,22) establish the diagnosis of chronic myeloid leukemia Classificationthe more recent World Health Organization (WHO) classification of neoplastic diseases of hematopoietic and lymphoid tissues uses cytogenetic finding into a number of subtypesExamples : t (8,21); inv (16); t (15,17) define distinct subsets of AMLdel (5q) define a distinct subset of MDSt (9,22) (q34:q11) present in the bone marrow and peripheral blood in 95% of patients with CML*

CytogeneticsPrognosisAML, ALL, MDS with normal karyotypes have an intermediate response to treatmentAML therapy related with abnormalities of chromosome 5 and 7 poor prognosisDe novo AML better prognosisStratification of treatment cytogenetic abnormalities are used in guiding patient management, especially the choice of postremission therapy. *

Molecular biologyThe molecular biology techniques have an important role for diagnosis and enable a large-scale synthesis of a number of recombinant proteins for therapy.Techniques :Southern Blot analysisNorthern Blot analysisIn Situ HybridizationPolymerase Chain Reaction AnalysisQualitative tests RT-PCR :BCR-ABL major CMLBCR-ABL minor ALLFIP1L1-PDGFRA primary eosinophilic syndromeJAK2 policittemia vera, essential thrombocytemia, idiopathic myelofibrosisQuantitative tests for diagnosis and monitoring the treatmentAML1-ETO AML2PML-RARa (bcr1) APLPML-RARa (bcr2) APLInversion 16 AML4BCR-ABL major CMLBCR-ABL minor - ALL*

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