Basics_of_the_CBC_and_Hemostasis_Tests_-_2023 (1).pptx

BASICS OF THE CBC AND TESTS FOR HEMOSTASIS Michael E. Woods, PhD October 5, 2023 REQUIRED READING  Provided in LEO LEARNING OBJECTIVES By the end of this session you should be able to:  Explain the concept of reference (normal) range  Explain the theory of the automated cell counter  Explain...

BASICS OF THE CBC AND TESTS FOR HEMOSTASIS Michael E. Woods, PhD October 5, 2023 REQUIRED READING  Provided in LEO LEARNING OBJECTIVES By the end of this session you should be able to:  Explain the concept of reference (normal) range  Explain the theory of the automated cell counter  Explain the components of the complete blood count (CBC) and its application in patient evaluation  Compare and contrast the reporting of leukocyte differential counts as relative percentages vs. absolute numbers, in terms of the advantages and disadvantages of each system  List and discuss the laboratory diagnostic procedures used to approach patients with bleeding disorders, including PT, aPTT, and the PFA-100.  Discuss the use of Erythrocyte Sedimentation Rate HISTORY OF THE MICROSCOPE AND VISUALIZING BLOOD CELLS Illustration of “red corpuscles” of blood by A van Leeuwenhoek (Letter 42 of Arcana Natura, 1695) Illustration of “red and white corpuscles” in blood by Beale. The legend under the illustration indicates that the picture was originally printed in September 1863. (Plate 38, page 261, The Microscope in Medicine, 1877). BLOOD CONTENTS CAN STILL BE CHARACTERIZED USING A MICROSCOPE THE CBC IS A STANDARD ELEMENT OF PATIENT EVALUATION  Total RBC Count  Total WBC Count  Peripheral Smear  Shape, size & color of RBCs  Differential WBC count  Platelet Count  Hemoglobin Concentration  Blood indices – MCH, MCV, MCHC WHEN IS A CBC ORDERED?  Routine health examination  When a person has any number of signs and symptoms that may be related to disorders that affect blood cells  Fatigue or weakness  Infection  Inflammation  Bruising or bleeding  With a disease known to affect blood cells  Chemotherapy and other drugs known to affect blood cells TODAY, WE USE TWO METHODS FOR AUTOMATED CELL COUNTING Electrical-Impedance Light-Scatter MAX WINTROBE LED THE CHARACTERIZATION OF RBC INDICES AND DEFINITIONS OF ANEMIA  “I … discovered that there were no reliable normal blood values. What was called “normal” was based on only a few counts that had been made in the nineteenth century. So I proceeded to collect normal blood values. Others elsewhere, also mindful of this deficiency, were beginning to do the same. A major problem, however, was methodology, and this was what led me to devise the hematocrit as a simple and accurate means of quantitating blood.”  Max Wintrobe Weisse AB. Conversations in medicine: The story of twentieth-century American medicine in the words of those who created it. Chapter 5: Maxwell M. Wintrobe, M.D., Ph.D. (1901–). New York: New York University Press; 1984. p. 75–92 HEMATOCRIT IS THE PERCENTAGE OF TOTAL BLOOD VOLUME OCCUPIED BY RED BLOOD CELLS (RBCs) Fred HL. Maxwell Myer Wintrobe: new history and a new appreciation. Texas Heart Institute Journal. 2007 ;34(3):328-335. PMID: 17948084; PMCID: PMC1995040. ERYTHROCYTE SEDIMENTATION RATE (ESR) IS A NON-SPECIFIC MARKER OF INFLAMMATION  Rouleaux formation is influenced by plasma fibrinogen levels  ESR increases with acute and chronic infections, some tumors and degenerative diseases.  In such situations, the ESR values are much greater than 20mm/hr.  ESR denotes only the presence of tissue damage or disease; does not correspond with severity  It may be used to follow the progression of the disease or monitor the effectiveness of treatment. Red blood cells have settled over the course of 1 hour, leaving plasma at the top of the tube. Reading: 18 MEAN CORPUSCULAR (CELL) VOLUME (MCV) IS THE AVERAGE VOLUME OF RED BLOOD CELLS  Used to characterize anemias  MCV <80 fL is called microcytic  Occurs in iron deficiency anemia  MCV 80-100 fL is called normocytic  Occurs in hemolytic anemia  MCV >100 fL is called macrocytic  Occurs in vitamin deficiency anemia  Newborn = 96 to 108 fL  This is an average, so a combination of large and small cells could give a normal value  Refer to RDW RED BLOOD CELL DISTRIBUTION WIDTH (RDW)  This is the coefficient of the RBC volume distribution.  Indicates variation in the size of RBC.  Calculated using MCV and RBC values.  An indicator of anisocytosis.  Helpful for the diagnosis of hematological disorders and monitoring the response to therapy.  Helpful in distinguishing iron- deficiency anemia (RDW increased), from the hemoglobinopathies (RDW normal). MEAN CORPUSCULAR HEMOGLOBIN (MCH) IS THE AVERAGE CONTENT OF HEMOGLOBIN PER RBC IN PICOGRAMS (PG)  Generally, macrocytes will have more hemoglobin and microcytes will have less hemoglobin. So the values resemble those of MCV.  Adult (all ages) = 27 to 31 pg  Newborn = 32 to 34 pg  MCV is higher than in adults MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC) IS THE AVERAGE CONCENTRATION OF OR PERCENTAGE OF HEMOGLOBIN IN EACH INDIVIDUAL RED BLOOD CELL  Hypochromic: MCHC <32 g/dL (<32%)  When MCHC is decreased and then there is a deficiency of hemoglobin (in iron deficiency anemia and thalassemia)  Normochromic: MCHC 32-36 g/dL  When the values are normal (In hemolytic anemia)  Hyperchromic: MCHC >36 g/dL  When MCHC value is increased and RBC cannot accommodate more than 36 g/dl (seen in spherocytosis, newborn and infants) CURRENT CLASSIFICATION OF ANEMIA British Journal of Haematology, Volume: 121, Issue: 2, Pages: 224-232, First published: 09 April 2003, DOI: (10.1046/j.13652141.2003.04188.x) THE DIFFERENTIAL PROVIDES THE ABSOLUTE AND RELATIVE WBC COUNT Type of cells % in peripheral Absolute smear values WBCs  The number of white blood cells, various types, and their morphology 3.8 to 10.8 thousand/mm3  WBCs are distinguished from the RBCs by  The automated machine counts all the Segmented neutrophils 50 to 70 2400 to 75000/mm3 Band form 2 to 6 100 to 650 /mm3 Lymphocytes 20 to 40 1000 to 4750 /mm3 Eosinophils 0 to 4 0 to 450/mm3 Monocytes 2 to 9 100 to 700 /mm3 Basophils 0 to 2 0 to 200 /mm3 the presence of the nucleus. nucleated RBCs as WBCs, so it needs to be corrected if nucleated RBCs are present in the blood.  Differential white cell count gives the proportion of the different type of white cells like neutrophils, lymphocytes, eosinophils, monocytes, and basophil.  Peripheral Blood Smear: Visual examination of the stained slides gives the exact picture THE CBC DIFFERENTIAL CAN REVEAL UNDERLYING CONDITIONS AFFECTING NEUTROPHILS  Left shift is present in the CBC when >10–12% bands are seen or when the total PMN count (segs plus bands) is >80%  Left Shift:  Bacterial infection, toxemia, hemorrhage, myeloproliferative disorders  Right Shift:  Liver disease, megaloblastic anemia, iron deficiency anemia, glucocorticoid use, stress reaction WHITE BLOOD CELL COUNT DIFFERENTIAL—LYMPHOCYTES Type of cells % in peripheral Absolute smear values Segmented neutrophils 50 to 70 2400 to 75000/mm3 Band form 2 to 6 100 to 650 /mm3  Decrease in lymphocytes = lymphocytopenia  Most often due to AIDS, and recently COVID-19, or undernutrition  Drugs, or autoimmune disorders.  Patients have recurrent viral, fungal, or parasitic infections.  Increase in lymphocytes = lymphocytosis  Relative lymphocytosis occurs when there is a Lymphocytes 20 to 40 1000 to 4750 /mm3 Eosinophils 0 to 4 0 to 450/mm3 Monocytes 2 to 9 100 to 700 /mm3 Basophils 0 to 2 0 to 200 /mm3 higher proportion (>40%) of lymphocytes among the white blood cells (WBCs); however, the ALC is normal (<4000/µL)  Absolute lymphocytosis in children and young adults most often reflects an infection, particularly a viral infection (e.g., infectious mononucleosis) or an infection with pertussis  In older adults with an absolute lymphocytosis, a chronic lymphoproliferative disorder should be considered TESTS OF HEMOSTASIS COMMON LABORATORY TESTS OF HEMOSTASIS Tests for platelet defects  Quantitative defects = platelet numbers are too low  Measured by counting; a component of the CBC  Qualitative defects = platelets aren’t functioning properly  Measured with platelet function assays, such as the PFA-100, light transmission aggregometry  Bleeding test is a simple examination of primary hemostasis Tests for coagulation defects PROTHROMBIN TIME ASSESSES THE EXTRINSIC AND FINAL COMMON COAGULATION PATHWAYS  Measures the extrinsic (VIIa) and common (X, V, II, and fibrinogen) pathways  Used to monitor warfarin therapy  Impossible to know the normal value across laboratories due to different testing kits and reagents Tissue factor Phospholipids Calcium VIIa Xa + Va on phospholipid IIa  Tissue factor (TF), phospholipid, and patient plasma incubated for 5 minutes  CaCl added to 25–30 mM to recalcify citrated plasma  Measure time for clot formation Detector Clot INTERNATIONAL NORMALIZED RATIO (INR) IS USED TO MONITOR PATIENTS ON WARFARIN THERAPY  Used to standardize results across analytical systems  INR =  International Sensitivity Index (ISI) provided by manufacturer Prothrombin Time Roshal, Mikhail, MD, PhD, Transfusion Medicine and Hemostasis: Clinical and Laboratory Aspects, Chapter 124, 799-803 Copyright © 2013 Copyright © 2013, 2009 Elsevier Inc. All rights reserved ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT) ASSESSES THE INTRINSIC AND FINAL COMMON COAGULATION PATHWAYS  Measures activity of intrinsic and common pathways of coagulation  Induced by surface (contact) Kaolin Contact system Phospholipids Calcium IXa + VIIIa activation of factor XII  Partial = Phospholipid is present, but no Tissue Factor Xa + Va on phospholipid  Mix equal parts of a negatively charged surface (Kaolin, silica and ellagic acid) and phospholipid (cephalin) mixture and patient plasma for >5 minutes  CaCl added to 30 mM to recalcify citrated plasma  Measure time for clot formation IIa Detector Clot MIXING STUDIES HELP DETERMINE WHETHER A CLOTTING DEFECT IS DUE TO A LACK OF FACTOR OR FACTOR INHIBITION Prolongation of the coagulation time of the screening test  1:1 mixture of patient plasma and normal plasma to distinguish between clotting factor deficiency and an inhibitor Mixing: Patient Plasma + Normal Plasma  Has largely been replaced by factor assays and a lupus anticoagulant screen  If mixture fails to correct Pt or aPTT within 3–4 seconds, this is strongly suggestive of:  A coagulation factor inhibitor (e.g., acquired FVIII antibody)  Antiphospholipid antibody (e.g., lupus anticoagulant) Correction Factor Deficiency No Correction Lupus Anticoagulan t Specific Factor Inhibitor INTERPRETATION OF PT AND PTT IN PATIENTS WITH A BLEEDING OR CLOTTING SYNDROME PT RESULT PTT RESULT EXAMPLES OF CONDITIONS THAT MAY BE PRESENT Prolonged Normal Liver disease, decreased vitamin K, decreased or defective factor VII, chronic low-grade disseminated intravascular coagulation (DIC), anticoagulation drug (warfarin) therapy Normal Prolonged Decreased or defective factor VIII, IX, XI, or XII, von Willebrand disease (severe type), presence of lupus anticoagulant, autoantibody against a specific factor (e.g., factor VIII) Prolonged Prolonged Decreased or defective factor I, II, V or X, severe liver disease, acute DIC, warfarin overdose Normal Normal or slightly prolonged May indicate normal hemostasis; however, PT and PTT can be normal in conditions such as mild deficiencies in coagulation factor(s) and mild form of von Willebrand disease. Further testing may be required to diagnose these conditions. FACTOR-SPECIFIC TESTING IS NECESSARY TO PINPOINT FACTOR DEFICIENCIES  One-stage assays use lyophilized, immunoadsorbed human plasma deficient in the coagulation factor under study  Mixed 1:1 with patient plasma  PT or aPTT is dependent on the amount of factor present in the patient plasma  More accurate with serial dilutions, which dilutes out any inhibitors present SCREENING TESTS FOR PLATELET DISORDERS Platelet count Bleeding time  Number of platelets in 1 μL  Time for a small puncture  Used to exclude a  Normal range is 1–9 minutes of blood quantitative platelet defect  Normal range is 150,000 to 450,000 per μL  Thrombocytosis = >450,000/μL  Thrombocytopenia = <150,000/μL wound to stop bleeding (1–13 minutes in children)  Slightly longer in females  Has largely been replaced by other tests  Does not specifically indicate a platelet defect HIGH-SHEAR PLATELET FUNCTION ANALYZERS— PFA-100  Measures platelet adhesion, activation and aggregation leading to rapid occlusion of the aperture and cessation of blood flow.  Membrane is coated with either collagen/epinephrine or collagen/ADP  Aspirin will prolong the CT for C/EPI but not C/ADP  Results are interpreted as normal or abnormal (elevated)  Substitute for bleeding time FIBRIN DEGRADATION PRODUCTS ARE USEFUL FOR DETECTING CHANGES IN FIBRINOLYTIC MECHANISMS  Digestion of cross-linked fibrin by plasmin gives rise to fragments, including D dimers.  Fibrinolysis panel, including D dimers, may be used to help diagnose thrombosis (excessive blood clotting).