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    3 Practical

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    The document discusses different anticoagulants used for hematology tubes including EDTA, sodium citrate, and heparin. It explains their mechanisms of action and common uses. The document also provides information on automated blood cell counting methods like electrical impedance and flow cytometry, describing how they work and some applications. Key points on immunophenotyping of ALL and common hematological tests like ESR and sickle cell solubility testing are summarized as well.

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    3 Practical

    Uploaded by

    T N
    56 views47 pages
    The document discusses different anticoagulants used for hematology tubes including EDTA, sodium citrate, and heparin. It explains their mechanisms of action and common uses. The document also provides information on automated blood cell counting methods like electrical impedance and flow cytometry, describing how they work and some applications. Key points on immunophenotyping of ALL and common hematological tests like ESR and sickle cell solubility testing are summarized as well.

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    The document discusses different anticoagulants used for hematology tubes including EDTA, sodium citrate, and heparin. It explains their mechanisms of action and common uses. The document also provides information on automated blood cell counting methods like electrical impedance and flow cytometry, describing how they work and some applications. Key points on immunophenotyping of ALL and common hematological tests like ESR and sickle cell solubility testing are summarized as well.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    56 views47 pages

    3 Practical

    Uploaded by

    T N
    The document discusses different anticoagulants used for hematology tubes including EDTA, sodium citrate, and heparin. It explains their mechanisms of action and common uses. The document also provides information on automated blood cell counting methods like electrical impedance and flow cytometry, describing how they work and some applications. Key points on immunophenotyping of ALL and common hematological tests like ESR and sickle cell solubility testing are summarized as well.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 47

    Practical hematology

    Tubes and anticoagulants


     EDTA (Ethylene Diamine Tetra-Acetate)
     Mechanism: forming Ca salts to remove Ca.
     Uses: CBC, PCR, blood films and HbA1c.

     Sodium citrate:
     Anticoagulant: 3.2%
     Mechanism: Calcium chelation.
     Use: Coagulation studies and platelet function.
    Black:
     3.8% of sodium citrate
     Action: Remove calcium.
     Uses: Erythrocyte Sedimentation Rate (ESR).
    Heparin:
     Sodium Heparin or Lithium Heparin
     Mechanism: inactivation of thrombin and thromboplastin.
     Uses: Use for osmatic fragility test and blood gases

    Note: avoid using hemolyzed, clotted or lipemic sample!!!!


    Changes in Blood at Room Temperature
    Blood Cell Enumeration-Automated Methods
    A. Electrical impedance (coulter): counting & sizing.
    • Cells pass through an aperture with an electrical current flowing through
    simultaneously. Cells do not conduct current (nonconductive) but rather they
    change electrical resistance, which is then counted as voltage pulses.
    • The number of pulses generated is proportional to the number of cells present;
    amplitude of the pulse generated is proportional to the size of the cell.
    • Sample is diluted in isotonic conductive solution that preserves cell shape and
    characteristics.
    • Dilutions used are dependent on instrument/methodology used.
    • Platelets are counted with RBCs.
    • Sample for counting WBCs is mixed with reagent to lyse RBCs.
    • A commercially available reagent, which both lyses RBCs and converts
    hemoglobin to cyanmethemoglobin, can be used to determine hemoglobin and
    WBCs in one dilution.
    • Hydrodynamic focusing is utilized to reduce cell coincidence (chance of one
    cell being counted more than once).
    2. Flow cytometry: Optical light scattering. Applications:
    • Cell counting, sizing, sorting & WBCs differential. • Immunophenotyping: Differentiating cells on basis
    of surface & cytoplasmic markers. Can determine
    • Based on both physical and immunologic features. lineage & maturity of cells in hematologic
    • Using laminar flow technology (Hydrodynamic focusing), by using malignancies. Useful for Dx, follow-up, &
    sheath fluid (normal saline). prognosis.
    • Evaluate the size and volume using the forward scatter (FSC) of the • Dx & monitoring of immunodeficiencies
    laser beam. • Dx of paroxysmal nocturnal hemoglobinuria
    • Enumeration of stem cells
    • Evaluate the internal structures (complexity) using the side scatter (SSC) • Quantitation of fetal hemoglobin
    of the laser beam.
    • Specimens analyzed by flow cytometry: Leukocytes, erythrocytes, lymph
    nodes, peripheral whole blood, bone marrow, tumors
    • Light source (tungsten halogen lamp or laser light).
    • Cells are labeled with one to several fluorescent compounds (excitation
    & light emission).
    • Energy is detected by a photomultiplier tube and converted into
    computerized data, which upon analysis provides information regarding
    number, size, and cellular composition of the population assayed.
    • The flow cytometer is composed of three main systems: (fluidics, optics
    and electronics).
    Immunophenotyping of ALL
    A. CD markers of B cell lineage:
    • CD10 (CALLA), CD19, CD20, CD34, and TdT (terminal deoxynucleotidyl transferase)
    positive.
    B. CD markers of T cell lineage:
    • CD2, CD3, CD5, CD7 and TdT.
    • Pan B cell marker: CD19.
    • Pan T cell marker: CD7.
    • T helper cell marker: CD4.
    • Cytotoxic T cell marker: CD8.

    • Immature T cell: CD2 & TdT.


    • Immature B cell: CD10, CD19, CD34 & TdT.
    + CD38 (myeloblasts)

    (TdT)

    Hairy cell leukemia: CD123, CD24, CD11c,


    CD103, CD19.
    Automated cell count errors

    • WBC counts exceeding instrument linearity limits result in increased cell turbidity and may falsely increase
    the hemoglobin, MCH, and MCHC.
    • Glucose over 600 mg/dL (hyperosmolarity) may increase the MCV and hematocrit and decrease the
    MCHC.
    • Cold agglutinins increase the MCV, MCH, and MCHC and decrease the RBC count and hematocrit.
    • Lipemia increases the hemoglobin, MCH, and MCHC.
    • Repeat the analysis if:
    • Rule of three failure on a normocytic sample, especially MCHC >37 g/dL.
    • RBC X 3 = Hgb
    • RBC X 9=Hct
    • Hgb X 3 = Hct
    2-20 fL

    Platelet aggregation, clumping,


    microcytic RBCs
    RBCs histogram
    Hematological tests
    1. Erythrocyte Sedimentation Rate:
     ESR measures degree of settling of RBCs in plasma in an anticoagulated specimen during a specific time,
    usually 1 hour.
     High fibrinogen or protein levels increase the ESR.
     ESR is increased in chronic inflammatory conditions, including rheumatoid arthritis and pregnancy (increased
    fibrinogen), bacterial infection, malignancy, tissue damage, multiple myeloma, Waldenstrom
    macroglobulinemia, and severe anemia.
     ESR is normal to decreased in polycythemia, sickle cell anemia, spherocytosis, and other conditions with
    poikilocytosis (prevents rouleaux formation).
     Sources of error causing falsely increased results: Tilted column, hemolysis, increased room temperature.
     Sources of error causing falsely decreased results: Clotted sample, excess anticoagulant, "old" blood
    (spherocytes form), decreased room temperature, vibration of tube & air bubbles in the tube.
    2. Solubility Test for Hemoglobin S:
     Hemoglobin S is insoluble when combined with a reducing agent (sodium dithionite).
     Hgb S will crystallize and give a turbid appearance to the solution.
     The test will not differentiate homozygous from heterozygous conditions containing Hgb S.
     Follow up a positive solubility test with hemoglobin electrophoresis.
    3. Hemoglobin Electrophoresis:
     Procedure for the identification of normal and abnormal hemoglobins
     Methodology is based on net negative charges, which cause hemoglobins to migrate from the negative
    (cathode) region toward the positive (anode) region. The distance a particular hemoglobin molecule migrates
    is due to its net electrical charge.
     Two types of electrophoresis: Cellulose acetate at pH 8.6 and citrate agar at pH 6.2
     Migration of hemoglobin is dependent on net negative charge and buffer pH.
    Hematological
    calculations
    RPI = (Hct / 45) * Retic / Maturation
    Atlas
    Basophilic
    erythroblast
    Polychromatophilic
    Pronormoblast erythroblast

    Polychromatic cell
    (reticulocyte)

    Orthochromatophilic Mature
    erythroblast erythrocytes
    Mature platelets
    Megakaryoblast
    Promegakaryocyte Megakaryocyte
    Myeloblast Band cell
    Promyelocyte Metamyelocyte
    Myelocyte
    Mature
    monocyte
    Monoblast Promonocyte
    Macrophage
    Small
    lymphocyte

    Lymphoblast
    Prolymphocyte

    Large granular lymphocyte (LGL) or NK cell

    Plasma cell
    Hint: Compare the small lymphocyte nucleus with
    RBCs

    Microcytic cell

    Macrocytic cell
    Normocytic cell
    Polychromatic cells

    Anisocytosis (high RDW) Hypochromic cells

    Normal RBCs
    Bite cell

    Blister cell
    Acanthocytes Target cell or Tear drop cell
    codocyte

    Sickle cell
    Burr cell
    Schistocyte or
    (echinocytes)
    helmet cell

    Stomatocyte Spherocytes
    Rouleaux formation Cold agglutination
    (coining) in MM disease (IgM)
    Red cell inclusions and parasites:
    1. Basophilic stippling: dark purple color, (RNA),
    seen in thalassemia, lead poisoning.
    2. Cabot ring: oval or eight ring, nuclear membrane
    remnants, seen in: lead poisoning, megaloblastic
    anemia.
    3. Heinze bodies: denatured Hb, seen in: G6PD
    deficiency.
    4. Hemoglobin C: rod shape crystal, seen in: HbC
    disease, HbSC disease.
    5. HbH: golf ball like, seen in: HbH disease, alpha
    thalassemia.
    6. Howell-jolly bodies: nuclear remnants of DNA,
    hemplytic anemia, megaloblastic anemia,
    thalassemia, sickle cell anemia.
    7. Pappenheimer bodies: iron after ineffective
    erythropoiesis.
    IDA Beta-thalassemia minor Beta-thalassemia major Megaloblastic anemia

    Immune hemolytic anemia HDN Hereditary spherocytosis Hereditary elliptocytosis


    MAHA Sickle cell anemia Hb-C disease Hb-SC disease
    Toxic granulation
    Pelger-huet anomaly (hyposegmented neutrophils) with dhole body
    Dhole body

    Hypersegmented neutrophil Vacuolated neutrophil

    Normal neutrophil
    Reactive lymphocytes
    Acute myeloid leukemia
    Myeloblast
    Myeloblast Myeloblast

    Promyelocytes Myeloblast
    Chronic myeloid
    leukemia
    Acute lymphoblastic leukemia
    Chronic lymphocytic leukemia
    Phagocytosed
    fungi (yeast)
    Phagocytosed
    histoplasma

    Trypanosoma Trypanosoma
    brucei cruzi
    Anaplasma-Ehrlichia Borrelia Toxoplasma gondii Leishmania
    Leishmaniasis (Amastigotes)
    inside macrophage

    Microfilaria

    Babesia
    Ring stages in thick film
    Malaria
    Alder Reilly anomaly May Hegglin anomaly Chediak-higashi syndrome

    Mast cell Thrombocytosis Platelets satellitism


    Lymphocyte VS myelocyte

    Monocyte VS large lymphocyte


    Reticulocyte stained by new methylene blue Reticulocyte stained by brilliant cresyl blue

    Platelets aggregation
    (clumping)
    This has been a presentation of Alyazeed Hussein
    Thanks for your attention and kind patience

    @Alyazeed7ussein

    @elyazeed7

    Alyazeed Hussein, BSc, SUST

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