Hematology Lecture Notes PDF - New Era University, 2023-2024

New Era University HEMATOLOGY 1 College of Medical...

New Era University HEMATOLOGY 1 College of Medical Technology LECTURE | Prof. Olivia Victoria Bondoc S.Y. 2023 – 2024 By: Andrea Ysabel Cortuna OVERVIEW OF CLINICAL HEMATOLOGY LESSON 1: HISTORY Since before 1900, physicians and medical laboratory professionals counted RBCs in HISTORY measured volumes to detect anemia or polycythemia. Athanasius Kircher in 1657, described “worms” Anemia in the blood o loss of oxygen-carrying capacity Anton van Leeuwenhoek in 1674 gave an account o often reflected in a reduced RBC count or of red blood cells decreased RBC hemoglobin Late 1800s – Giulio Bizzozero described platelets concentration as “petites plaques” Polycythemia 1902 – development of the Wright stain by James o increased RBC count reflecting increased Homer Wright circulating RBC mass, a condition that o For visualizing blood film examination leads to hyperviscosity through the microscope Diluted blood was transferred to a glass counting Wright’s Romanowsky-type stain chamber called a hemacytometer. (polychromatic, a mixture of acidic and basic The microscopist observed and counted RBCs in dyes), and refinements thereof, remain the selected areas of the hemacytometer, applied a foundation of blood cell identification. mathematical formula based on the dilution and At present, RBC, WBC, and platelet appearance on the area of the hemacytometer counted. are analyzed through automation or visually Report RBC count in cells per microliter (mL, using 500x to 1000x light microscopy mcL, also called cubic millimeter, mm3), examination of cells fixed to a glass microscope milliliter (mL, also called cubic centimeter, or slide and stained with Wright or Wright-Giemsa cc), or liter (L) stain. The scientific term for cell appearance is morphology o Encompasses cell color, size, shape, cytoplasmic inclusions, and nuclear condensation LESSON 2: RED BLOOD CELLS RED BLOOD CELLS (RBC) Anucleate, biconcave, discoid cells filled with a Visual RBC counting was developed before 1900 reddish protein (hemoglobin), which transports and was the only way to count RBCs oxygen and carbon dioxide In 1958, automated particle counters became Appear salmon pink and measure 7 to 8 mm in available in the clinical laboratory. diameter with a zone of pallor that occupies 1/3 The 1st electronic counter was patented in 1953 of their center (biconcavity) by Joseph and Wallace Coulter of Chicago, Illinois. The Coulter principle of direct current electrical impedance is still used to count RBCs in many automated blood cell analyzers. Visual counting skills remain useful where automated counters are unavailable. 1  TRANS: HGB, HCT, AND RBC INDICES o Mean cell hemoglobin concentration (MCHC) RBCs also are assayed for hemoglobin (HGB) MCV is recorded in femtoliters (fL), and reflects concentration and hematocrit (HCT). RBC diameter on a Wright-stained blood film. Hemoglobin measurement relies on a weak MCHC, expressed in grams per deciliter (g/dL), solution of potassium cyanide and potassium reflects RBC staining intensity and amount of ferricyanide – Drabkin reagent central pallor. An aliquot of whole blood is mixed with a MCH, in pictograms (pg) expresses the mass of measured volume of Drabkin reagent hemoglobin per cell and parallels the MCHC. Hgb is converted to stable cyanmethemoglobin RBC distribution width (RDW), expresses the (hemiglobincyanide) degree of variation in RBC volume Absorbance or color intensity of the solution is o Extreme RBC volume variability is measured in a spectrophotometer at 540 nm visible on the Wright-stained blood film wavelength as variation in diameter and is called Modifications of cyanmethemoglobin method are anisocytosis. used in most automated applications o In addition to aiding in the diagnosis of o Some automated blood cell analyzers anemia, RBC indices provide stable replace it with a formulation of the ionic measurements for internal quality control surfactant (detergent) sodium lauryl of automated blood cell analyzers. sulfate. Hematocrit is the ratio of the volume of packed RETICULOCYTES RBCs to the volume of whole blood. o Manually determined by transferring Polychromatic (polychromatophilic) erythrocytes are blood to a plastic tube with a uniform newly released from the RBC production site: the bore, centrifuging, measuring the column bone marrow of RBCs, and dividing by the total length o In Wright-stained blood film, 0.5% to 2.5% of the column of RBCs plus plasma exceed the 7- to 8-mm average diameter and Hematocrit is also called packed cell volume stain slightly blue-gray. (PCV) o Are closely observed because they indicate The light-colored layer between the RBCs and the ability of the bone marrow to increase plasma is known as the buffy coat RBC production in anemia caused by blood o WBCs and platelets loss or excessive RBC destruction Methylene blue dyes (nucleic acid stains or vital stains) are used to differentiate and count these young RBCs. Vital (or “supravital”) stains are dyes absorbed by live cells. Young RBCs contain ribonucleic acid (RNA) and are called reticulocytes when the RNA is visualized using vital stains. All fully automated blood cell analyzers provide: o Relative reticulocyte percentage o Absolute reticulocyte count o Immature reticulocyte fraction WHITE BLOOD CELLS (WBC) WBCs, or leukocytes, are a loosely related The medical laboratory professional may use the category of cell types dedicated to protecting three numerical results, RBC count, HGB, and their host from infection and injury. HCT, to compute the RBC indices: WBCs are transported in the blood from their o Mean cell volume (MCV) source (bone marrow or lymphoid tissue) to their o Mean cell hemoglobin (MCH) tissue or body cavity destination. 2  TRANS: WBCs are so named because they are nearly Basophils (BASOs) colorless in an unstained cell suspension. o Cells with dark purple, irregular WBCs may be counted visually using a cytoplasmic granules that obscure the microscope and hemacytometer. nucleus. The technique is the same as RBC counting, but o Basophil granules contain histamines and the typical dilution is 1:20, and the diluent is a various other proteins. dilute acid solution. o An elevated basophil count is called Visual WBC counting has been largely replaced basophilia – hematologic disease by automated blood cell analyzers. Lymphocytes (LYMPHs) Medical laboratory professionals who analyze o Lymphocytes comprise a complex body fluids such as cerebrospinal fluid or pleural system of cells that provide for host fluid may employ visual WBC counting. immunity. A decreased WBC count is called leukopenia, and o They recognize foreign antigens and an increased WBC count is called leukocytosis. mount humoral (antibodies) and cell- mediated antagonistic responses. o On a Wright-stained blood film, most TYPES OF WHITE BLOOD CELLS lymphocytes are nearly round, are slightly larger than RBCs, and have Neutrophils (NEUTs, segmented neutrophils round nuclei and a thin rim of [SEGs], polymorphonuclear neutrophils nongranular cytoplasm. [PMNs]). o An increase in the lymphocyte count is o These are phagocytic cells whose major called lymphocytosis – viral infections purpose is to engulf and destroy o An abnormally low lymphocyte count is microorganisms and foreign material. called lymphopenia or lymphocytopenia – drug therapy or immunodeficiency Monocytes (MONOs) o It is an immature macrophage passing through the blood from its point of origin (bone marrow) to a targeted tissue location. o Macrophages are the most abundant cell type in the body. o Macrophages occupy every body cavity o The term segmented refers to their – some motile and some are multilobed nuclei. immobilized. o The cytoplasm of neutrophils contains o Benign monocytosis may be found in pink- or lavender-staining granules filled certain infections or in inflammation. with bactericidal substances. Leukemia o An increase in neutrophils is called o It is an uncontrolled proliferation of a neutrophilia – bacterial infection clone of malignant WBCs. o A decrease is called neutropenia – o It may be chronic or acute. medications or viral infections o It may involve any of the cell lines and are categorized by their respective Bands (band neutrophils, BANDs) immunophenotypes and genetic o Slightly less mature neutrophils with a aberrations nonsegmented nucleus in a U or S shape. o Some leukemias are more common in a o An increase in bands also signals specific age group bacterial infection – left shift ▪ Chronic lymphocytic leukemia Eosinophils (EOs) is more prevalent in people >65 o Cells with round, bright orange-red years old cytoplasmic granules filled with proteins ▪ Acute lymphoblastic leukemia is involved in immune system regulation. the most common form of o An elevated eosinophil count is called childhood leukemia eosinophilia – allergy or parasitic infection 3  TRANS: o Medical laboratory scientists participate platelet counts and uncontrolled platelet in the characterization of leukemias production using Wright-stained bone marrow o Life-threatening hematologic disorder smears, flow cytometric A low platelet count is called thrombocytopenia immunophenotyping, molecular – a common consequence of drug treatment diagnostic technology, cytogenetics, and, o It is usually accompanied by easy occasionally, cytochemical staining. bruising and uncontrolled hemorrhage. Accurate platelet counting contributes to patient safety. PLATELETS OR THROMBOCYTES Platelets, or thrombocytes, are true blood cells COMPLETE BLOOD COUNT that maintain blood vessel integrity by initiating vessel wall repairs. A complete blood count (CBC) is performed on They rapidly adhere to the surfaces of damaged automated blood cell analyzers and includes the blood vessels, form aggregates with neighboring RBC, WBC, and platelet measurements platelets, and secrete proteins and small The medical laboratory professional is molecules that trigger thrombosis, or clot responsible for the integrity of the specimen and formation. ensures that it is submitted in the appropriate Platelets are the major cells that control anticoagulant and tube and is free of clots and hemostasis – a series of cellular and plasma- hemolysis. based mechanisms that seal wounds, repair vessel The specimen must be of sufficient volume, walls, and maintain vascular patency. because “short draws” result in incorrect They are only 2 to 4 mm in diameter, round or anticoagulant-to-blood ratios. ▹ The specimen oval, anucleate, and slightly granular. must be tested or prepared for storage w/in the Uncontrolled platelet and hemostatic activation is appropriate time frame and must be accurately responsible for: registered in the work list – specimen accession o Deep vein thrombosis Most laboratories employ automated blood cell o Pulmonary emboli analyzers to generate the CBC. o Acute myocardial infarctions o When one of the results from the blood o Cerebrovascular accidents cell analyzer is abnormal, the instrument o Peripheral artery disease provides an indication of this – flag o Repeated spontaneous abortions ▪ Blood film examination should (miscarriages) be performed Platelet count uses the same technique used in counting WBCs on a hemacytometer but uses a BLOOD FILM EXAMINATION different counting area, diluent, and dilution. o Phase microscopy provides for easier Preparation of a “wedge-prep” blood film on a identification glass microscope slide, and staining it using Automated blood cell analyzers have largely Wright or Wright-Giemsa stain replaced visual platelet counting and provide Visually estimate: greater accuracy. o WBC count (with the 40x or 50x One advantage of automated blood cell analyzers objective at 400x or 500x magnification) is their ability to generate a mean platelet volume o Platelet count (with the 100x OIO at (MPV) 1000x magnification) o Presence of predominantly larger Next, systematically review, identify, and platelets generates an elevated MPV tabulate 100 WBCs to determine their percent value – regenerative bone marrow distribution – WBC differential (“diff”) response to platelet consumption Lastly, examination of the morphology of WBCs, Elevated platelet counts, called thrombocytosis – RBCs, and platelets by light microscopy for inflammation or trauma abnormalities of shape, diameter, color, or Essential thrombocythemia is a rare malignant inclusions (1000x magnification) condition characterized by extremely high 4  TRANS: o Erythroid series and myeloid series ENDOTHELIAL CELLS Medical laboratory scientists, clinical pathologists, and hematologists review Wright- Endothelial cells are the endodermal cells that stained aspirate smears for morphologic form the inner surface of the blood vessel. abnormalities, high or low bone marrow cell They are important in maintaining normal blood concentration, and inappropriate cell line flow, decelerating platelets during times of distributions. injury, and enabling WBCs to escape from the The biopsy specimen, enhanced by hematoxylin vessel to the surrounding tissue when needed. and eosin (H&E) staining, may reveal COAGULATION abnormalities in bone marrow architecture indicating leukemia, bone marrow failure, or one Most hematology laboratories include a blood of a host of additional hematologic disorders. coagulation testing department. Cytochemical stains may be used to differentiate Platelets are a key component of hemostasis, and abnormal myeloid, erythroid, and lymphoid cells. plasma coagulation is the second component. o These stains include: The coagulation system employs a complex ▪ Myeloperoxidase sequence of plasma proteins, some enzymes, and ▪ Sudan black B some enzyme cofactors – clot formation ▪ Nonspecific and specific Another system of enzymes and cofactors digests esterase clots to restore vessel patency – Fibrinolysis ▪ Periodic acid-Schiff The coagulation section of the hematology laboratory provides a series of laboratory assays o These stains include: that assess plasma proteins and their interactions ▪ Phosphatase with hematologic cells. ▪ Alkaline phosphatase Focuses on blood specimen integrity for the In most laboratories, cytochemical staining has coagulation laboratory, because minor blood been replaced by flow cytometry specimen defects, including clots, hemolysis, immunophenotyping, molecular diagnostic, and lipemia, plasma bilirubin, and short draws, render cytogenetic techniques. the specimen useless Immunostaining methods have enabled the High-volume coagulation tests for acute care identification of cell lines by detecting lineage- facility includes: specific antigens on the surface or in the o Platelet count and MPV cytoplasm of leukemia and lymphoma cells. o Prothrombin time o E.g. CD42b (diagnostic for o Partial thromboplastin time/APTT megakaryoblastic leukemia) o Thrombin time (or thrombin clotting Flow cytometers may be quantitative, or time) qualitative. o Fibrinogen assay The former devices are automated clinical blood o D-dimer assay cell analyzers that generate the quantitative The prothrombin time and partial thromboplastin parameters of the CBC through the application of time assess each portion of the coagulation electrical impedance and laser or light beam pathway for deficiencies and are used to monitor interruption. anticoagulant therapy. Both qualitative and quantitative flow cytometers are employed to analyze cell populations by measuring the effects of individual cells on laser ADVANCED HEMATOLOGY PROCEDURES light, and by immunophenotyping for cell membrane epitopes using monoclonal antibodies The hematology laboratory provides: labeled with fluorescent dyes. o Bone marrow examinations Cytogenetics, a form of chromosome analysis, is o Flow cytometry immunophenotyping employed in bone marrow aspirate examination o Cytogenetic analysis to find gross genetic errors such as the o Molecular diagnosis assays Philadelphia chromosome (chronic myeloid Bone marrow aspirates and biopsy specimens are leukemia) collected and stained to analyze nucleated cells Cytogenetic analysis remains essential to the that are the immature precursors to blood cells. diagnosis and treatment of leukemia. 5  TRANS: Molecular diagnostic techniques enhance and even replace some of the advanced hematologic methods. o Real-time polymerase chain reaction o Microarray analysis o Fluorescence in-situ hybridization o DNA sequencing systems o Detect various chromosome translocations and gene mutations that confirm specific types of leukemia and lymphoma, establish their therapeutic profile and prognosis, and monitor the effectiveness of treatment ADDITIONAL HEMATOLOGY PROCEDURES The G6PD assay phenotypically detects an inherited RBC enzyme deficiency causing episodic hemolytic anemia. The sickle cell solubility screening assay, Hgb electrophoresis,and HPLC, are used to detect and diagnose sickle cell anemia and other inherited qualitative hgb abnormalities and thalassemias. Erythrocyte sedimentation rate (ESR) detects inflammation and roughly estimates its intensity. Review of cellular counts, distribution, and morphology in body fluids other than blood o CSF, synovial (joint) fluid, pericardial fluid, pleural fluid, and peritoneal fluid HEMATOLOGY QUALITY ASSURANCE AND QUALITY CONTROL Medical laboratory professionals employ particularly complex quality control systems in the hematology laboratory. The measurement of cells and biological systems requires elaborate calibration, validation, matrix effect examination, linearity, and reference interval determinations. An internal standard methodology known as the moving average also supports hematology laboratory applications. Medical laboratory professionals in all disciplines compare methods through clinical efficacy calculations that produce clinical sensitivity, specificity, and positive and negative predictive values for each assay. Should monitor specimen integrity and test ordering patterns Ensure the integrity and delivery of reports 6