Lesson 2-1
Introduction to
Hematology
Overview
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Blood vessels and blood circulation
Composition of blood
Origin of blood cells
Hematological diseases
Hematology laboratory
– Test methods
– Complete blood count (CBC)
Circulatory System
• Systemic circulation
– Body’s blood supply
– Arteries – oxygenated blood supply
– Veins – deoxygenated blood supply
• Cardiopulmonary circulation
– Specific to heart and lungs
– Pulmonary artery – deoxygenated
– Pulmonary veins – oxygenated
Systemic Circulation
Cardiopulmonary Circulation
Blood Vessels
• Capillaries
• Arterioles
• Venules
Composition of Blood
• Plasma
• Cellular elements
– Red blood cells
– White blood cells
– Platelets
Blood Cells
Origin of Blood Cells
• Hemopoiesis – the formation and
development of blood cells.
• Hemopoietic stem cell – an
undifferentiated bone marrow cell. High
rate of replication followed by differentiation.
Origin of Blood Cells
Stem Cell Research
• Stem Cells
• Adult
• Embryonic
Hematological Diseases
• Causes
– Faulty or insufficient production of cell type
• Anemia, Leukemia (overproduction), Thrombocytopenia
– Defective cell function
• Iron deficiency anemia, Leukemia (immature)
• Inherited – Genetic mutations
– Hemophilia and Sickle Cell Anemia
• Secondary
– Renal patients – abnormal appearing RBCs
– Mononucleosis – atypical lymphs
– Aspirin – inhibited platelet function
Burr (Echinocyte) Cells
Burr cells (Echinocytes) often seen in End Stage
Renal Disease
Hematology Laboratory
• Methods of analysis – the Coulter Principle
– a technology for counting and sizing particles using impedance
measurements. The technology was principally developed to count blood
cells quickly by measuring the changes in electrical conductance as cells
suspended in a conductive fluid passed through a small orifice.
• Safety – Biohazard
• Quality assessment – Calibrators and
Controls
• Specimens – Whole Blood or plasma
• CBC – whole blood in Purple EDTA tube
• Coagulation tests and special hematology
tests
Lesson 2-2
Hemoglobin
Hemoglobin (Hb, Hgb)
• Major component of RBCs
• Transports oxygen to tissues
• Part of complete blood cell count
(CBC) or single test or in pairing with
hematocrit (H&H)
Hemoglobin Function
• Transport protein for O2 and CO2
• Releases O2 in tissues and picks
up CO2 in exchange
• Can also be a carrier protein for
carbon monoxide
– CO has 210X more affinity for Hgb than O2
Hemoglobin
• Structure
– Heme – 4 heme groups
• One per each Globin group
– Globin – 4 globin groups
• 2 alpha chain
• 2 beta chain
Hemoglobin Levels
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Reference ranges
Adult Male: 14.0-18.0 gm/dL
Adult Female: 12.0-16.0 gm/dL
Newborn: 10.4-22.6 gm/dL
See Table 2-1 for other ranges
Physiological factors
– Age
– Gender
– Altitude
Variations in Hb Structure
• Normal hemoglobins
– A1 : 95-98% of adult hemoglobin
– A2 : 2-3% of adult hemoglobin
– Hb F : 2% of adult hemoglobin
• Fetal hemoglobin – produced by the fetus during
gestation.
Variations in Hb Structure
• Abnormal hemoglobins
– Caused by mutations of globin chain
– Inherited
• Hemoglobin S
• Hemoglobin E
• Hemoglobin C
Principles of Hemoglobin
Measurement
• Specific gravity technique
– Historic method and only an estimate
• Chemical methods
– Photometric measurement
– Drabkin’s Reagent
• Cyanmethemoglobin (Hemiglobincyanide) end product
– Azidemethemoglobin – a different end product
Procedure
• Safety precautions – Biohazard
• Quality assessment
– Calibration
– Quality Controls
• Specimen – Whole Blood
• Analyzers
– Hematology analyzers
– Dedicated hemoglobin analyzers – usually POCT
Beckman Coulter Analyzer
Hemoglobin POCT Analyzers
• HemoCue
Hemoglobin POCT Analyzers
• STAT-Site M
Lesson 2-3
Microhematocrit
Peripheral Blood Microhematocrit
• Measures the proportion of red blood cells
to plasma
• Packed cell column
• Packed cell volume
(PCV)
• Buffy Coat
Equipment and Supplies
• Microhematocrit centrifuges
Equipment and Supplies
• Microhematocrit tubes
Reference Values
• Expressed as percentage or SI unit
• Varies by age and gender
• Local Normal Ranges
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Adult Male: 42.0-52.0%
Adult Female: 37.0-47.0%
Newborn: 31.0-68.0%
See Table 2-2 for more general results
• Check results
– General rule: Hct = Hgb × 3
Clinical Significance
Low Hematocrit Results
Elevated Hematocrit
Results
Bone Marrow Diseases
Cogenital Heart Disease
Chronic Inflammatory
disease
Dehydration
Iron, Folate, Vit B12
deficiency
Kidney tumor
Internal Bleeding
Lung Diseases
Hemolytic anemia
Polycythemia Vera
Kidney Failure
Leukemia
Lymphoma
Sickle Cell Anemia
Procedure
• Safety
– Biohazard; Standard Precautions
– Plastic, flexible, or Mylar-coated tubes
• No longer use glass tubes; sharps hazard
– Self-sealing tubes are safer
– Centrifuge safety
• Internal lid
• Outer locking lid
• Frequent disinfection
Procedure
• Quality assessment
– SOP manual
– Controls
– Specimen collection
• Fingerstick (capillary)
• Venous blood – EDTA collection tube
– Well mixed – manual or mechanical
– Duplicate samples because manual test
– Centrifuge calibration – speed and timer
• Too slow or too short – falsely elevated HCT
• Too fast or too long – falsely decrease HCT
Procedure
• Specimen
– Capillary blood
– Venous blood
Procedure
Procedure
• Centrifuge 2–4 minutes
– 10,000 rpm
• Reading and reporting results
– Read top of red cell column
– Duplicate tubes should be within ± 2%
– Report average
Procedure
Lesson 2-7
Preparing and
Staining a Blood Smear
Clinical Purpose
• Examine morphology of cellular
elements in the blood
• Perform a differential count of WBCs
– Usually a part of a CBC
• Estimate platelet count
Preparing a Smear
• Safety precautions
– Standard biohazard precautions
– Use care with glass slides
• Sharps danger
• Dispose of in Sharps container
– Use care with methanol
• Avoid contact with skin or inhalation of fumes
Preparing a Smear
• Quality assessment
– Clean or pre-cleaned
• Beveled edge
– EDTA anticoagulant only
• Minimal alteration of morphology and staining characteristics
• Make smear within 2 hours
– Stain smear when dry or within 1 hour of preparation
Preparing a Smear
• Collecting the specimen
– Capillary blood preferred
• Fingerstick or infant heelstick
– Venous EDTA blood acceptable
Preparing a Smear
• Making the smear
– Blood-drop dispensers
– Two-slide method
– Well mixed specimen
• 2 minutes minimum
Preparing a Smear
Preparing a Smear
Preparing a Smear
• Features of a good smear
– Feathered edge
– No holes or ridges
Preparing a Smear
• Preserving the smear
– Stain immediately or preserve by immersing in
Methanol for up to 60 seconds then let dry
• Factors affecting smear quality
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Size of blood drop
Angle of spreader slide
Speed of moving spreader slide
Moisture/debris on slide
• See Table 2-11 on page 279
Staining a Smear
• Wright’s stain – manual or automated
– Polychromatic
– Contains methylene blue and eosin (red-orange)
• Basic dye – methylene blue – nucleus
• Acidic dye – eosin – some granules, cytoplasm and other structures
– Contains methanol fixative
Staining a Smear
• Staining procedures
– Quick stains – manual method using a kit
– Two-step method use a staining rack
– Automatic strainers
• Moving belt
• Basket
Stain Quality
• Evaluating stain quality
– RBCs pink-tan
– Color of cell nuclei – purple
– Color of cell cytoplasm – varies from pink to blue
to blue-gray
Stain Quality
• Storage in a slide box – Keeps slides:
– Separated
– Dust-free
– Protected from light
Lesson 2-8
Normal Blood
Cell Morphology
Stained Blood Smear
• Safety precautions
– Not a biohazard but a sharps danger
– Dispose of slides in a sharps container
• Quality assessment
– Blood smear must be completely dry before staining
– Stain should be free of precipitate as should dry
smear
– Smear must have a feathered edge
• Use oil immersion objective
Stained Blood Smear
• Examine feathered edge
Feathered Edge Images
Identify WBCs by Evaluating
Cellular Features
• Relative cell size
• Nuclear characteristics
• Cytoplasmic
characteristics
Identification of cells:
A. RBCs B. Lymphocyte C. Neutrophil D. Eosinophil
E. Neutrophil F. Monocyte G. Platelets H. Lymphocyte
I. Band Neutrophil J. Basophil
Red Blood Cells
• Most numerous blood cell
• Non-nucleated
• Central area of pallor
– Biconcave disk shape
• 6–8 µm in size
• Stain pink-tan
Platelets
• Smallest blood “cell”
• Cytoplasmic fragment
– Derived from megakaryocyte
– Contains granules
– Cytoplasm stains blue while
granules stain red-purple
– Normal platelets do not form
many clumps
White Blood Cells
• Largest of blood cells – varies
8-20 microns
• Five basic types
– Granulocytes
• Neutrophil – inc. due to bacterial infections
• Eosinophil – inc. with parasitic infection and allergies
• Basophil – inc. due to allergies, inflammatory disease
such as rheumatoid arthritis, some leukemias
– Lymphocytes – inc. due to viral infections,
lymphocytic leukemia, lymphoma, pertussis and
whooping cough
– Monocytes – inc. due to fungal infections,
monocytic leukemias
Leukocyte Characteristics
Neutrophil
• Most numerous WBC
• Segmented nucleus
• Neutral-staining (pink-lilac) granules
• Band cell is immature form
Eosinophil
• Red-orange granules
• Nucleus has two to three lobes
• Present in low numbers
Basophil
• Only seen occasionally
• Blue-black granules
Lymphocyte
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Smallest WBC
Round to oval nucleus
Sky-blue cytoplasm
Occasional granules
Lymphocyte
Monocyte
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Largest WBC
Horseshoe-shaped or folded nucleus
Gray-blue cytoplasm
Occasional vacuoles

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