Hematopoietic System- 1 Peripheral Blood Linda F. Cunningham, MD January 2, 2003

Hematopoietic System- 1Hematopoietic System- 1Peripheral BloodPeripheral Blood

Linda F. Cunningham, MDLinda F. Cunningham, MD

January 2, 2003January 2, 2003

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• ASCP 1.3 Normal Peripheral Blood ASCP 1.3 Normal Peripheral Blood SmearSmear

Hematopoietic SystemHematopoietic SystemClinical ProblemsClinical Problems

• AnemiaAnemia

• Cytosis and Cytopenia Cytosis and Cytopenia – LeukocytosisLeukocytosis

– ThrombocytopeniaThrombocytopenia

• LymphadenopathyLymphadenopathy

Hematopoietic SystemHematopoietic System

• BloodBlood– PlasmaPlasma– Cells and ProteinsCells and Proteins

• Bone MarrowBone Marrow

• Lymphoid OrgansLymphoid Organs– Lymph Nodes, Spleen, and Thymus Lymph Nodes, Spleen, and Thymus

BloodBlood

• Connective TissueConnective Tissue

• Mesodermal OriginMesodermal Origin

• CompositionComposition

– CellsCells

– Aqueous Plasma MatrixAqueous Plasma Matrix

• ProteinsProteins

• Organic and inorganic saltsOrganic and inorganic salts

Integrated HistologyIntegrated HistologyBloodBlood

• Histology of red blood cells (RBCs, Histology of red blood cells (RBCs, erythrocytes), white blood cells (WBCs, erythrocytes), white blood cells (WBCs, leukocytes), and plateletsleukocytes), and platelets

• Composition, biochemistry, and Composition, biochemistry, and physiology of plasma proteins, inorganic physiology of plasma proteins, inorganic salts, and organic compoundssalts, and organic compounds– Fibrinogen for clotting (coagulation)Fibrinogen for clotting (coagulation)

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Medical HistologyMedical HistologyLaboratory TestsLaboratory Tests

Collect blood (venipuncture) in tube Collect blood (venipuncture) in tube without anticoagulant without anticoagulant – clotting occurs, using clotting proteins and clotting occurs, using clotting proteins and

plateletsplatelets– aqueous plasma becomes aqueous plasma becomes serumserum– serumserum is plasma without fibrinogen is plasma without fibrinogen– serum is sample used to analyze proteins, serum is sample used to analyze proteins,

glucose, etcglucose, etc

Medical HistologyMedical HistologyLaboratory TestsLaboratory Tests

Collect blood in tube with an anticoagulant Collect blood in tube with an anticoagulant and centrifugeand centrifuge

• Two (2) major layers formTwo (2) major layers form– Bottom Bottom solid cellularsolid cellular layer layer

• Largest layer, red cells, bottom red layerLargest layer, red cells, bottom red layer

• White cell, “buffy coat” small white/gray layerWhite cell, “buffy coat” small white/gray layer

• Top layer, invisible thin platelet layerTop layer, invisible thin platelet layer

– Top Top aqueous plasmaaqueous plasma layer layer

Medical HistologyMedical Histology

HematocritHematocrit

• Volume occupied by red blood cells in the Volume occupied by red blood cells in the anti-coagulated centrifuged bloodanti-coagulated centrifuged blood

• Expressed as % of total volume of bloodExpressed as % of total volume of blood

• Adult male reference range, Adult male reference range, 39 – 49%39 – 49%

• Adult female reference range, Adult female reference range, 35 – 45%35 – 45%

ASCP 1.08

Blood CellsBlood Cells

• Erythrocytes (RBCs)Erythrocytes (RBCs)

• Leukocytes (WBCs)Leukocytes (WBCs)– GranulocytesGranulocytes

• NeutrophilsNeutrophils

• EosinophilsEosinophils

• BasophilsBasophils

– MonocytesMonocytes– LymphocytesLymphocytes

• PlateletsPlatelets

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Integrated HistologyIntegrated HistologyErythrocytesErythrocytes

• RBC size and shapeRBC size and shape

• RBC membrane structure and proteinsRBC membrane structure and proteins

• RBC cytoplasmic componentsRBC cytoplasmic components

• RBC maturation and destruction in the RBC maturation and destruction in the circulationcirculation

• RBC expected lifespan in the circulationRBC expected lifespan in the circulation

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• Histology 2-6.3 a, b, c: Mature red cell Histology 2-6.3 a, b, c: Mature red cell peripheral blood smear, disc. 7.5-7.8 micron, peripheral blood smear, disc. 7.5-7.8 micron, eosinophilic cytoplasmic hemoglobin staining; eosinophilic cytoplasmic hemoglobin staining; scanning EM; red cell cytoskeleton maintains scanning EM; red cell cytoskeleton maintains shape shape

• Robbins 320:rbc membraneRobbins 320:rbc membrane

• Rodak 29: Schematic red cell membraneRodak 29: Schematic red cell membrane

Leukocytes- GranulocytesLeukocytes- Granulocytes

• Granules Composition and FunctionGranules Composition and Function– PrimaryPrimary Specific GranulesSpecific Granules

• Neutrophils, 60-70 %Neutrophils, 60-70 %• Eosinophils, 2-4%Eosinophils, 2-4%• Basophils, 0-1% (Tissue Mast Cells)Basophils, 0-1% (Tissue Mast Cells)

– Secondary Nonspecific Azurophilic GranulesSecondary Nonspecific Azurophilic Granules

• Granulocyte Circulation and MigrationGranulocyte Circulation and Migration• Tissue Inflammatory CellsTissue Inflammatory Cells

Leukocytes-GranulocytesLeukocytes-GranulocytesFunctional HistologyFunctional Histology

• GranulocytesGranulocytes– NeutrophilsNeutrophils– EosinophilsEosinophils– BasophilsBasophils

• Inflammatory response with margination, Inflammatory response with margination, emigration, tissue actionemigration, tissue action

• PhagocytesPhagocytes– Vacuoles and granulesVacuoles and granules– LysosomesLysosomes

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• Histology 2-6.7 a, b: Neutrophil, multilobed nucleus, Histology 2-6.7 a, b: Neutrophil, multilobed nucleus, pale cytoplasm with few granules; EM granules pale cytoplasm with few granules; EM granules

• 2-6.10 a, b: Eosinophils, bilobed, bright red granules; 2-6.10 a, b: Eosinophils, bilobed, bright red granules; EM granules with central dense crystalloid EM granules with central dense crystalloid

• 2-6.11 a, b, c: Basophils and Mast cells, large dark blue 2-6.11 a, b, c: Basophils and Mast cells, large dark blue granules; Mast cells in tissue; EM histamine released granules; Mast cells in tissue; EM histamine released from granules in cytoplasmfrom granules in cytoplasm

• Pathology 1-5.3 a, b, c: Tissue neutrophils; margination Pathology 1-5.3 a, b, c: Tissue neutrophils; margination and emigrationand emigration

• Histology 2-6.5: Neutrophil phagocytosis, ingest Histology 2-6.5: Neutrophil phagocytosis, ingest extracellular particles; primary granules lysosomal extracellular particles; primary granules lysosomal enzymesenzymes

MonocytesMonocytesFunctional HistologyFunctional Histology

• Agranulocytes Agranulocytes ** (granules by EM) (granules by EM)

• SizeSize

• Cytoplasmic ComponentsCytoplasmic Components– Azurophilic granules are Azurophilic granules are lysosomeslysosomes

– Phagocytes Phagocytes

• Circulation and MigrationCirculation and Migration

– Macrophages (Histiocytes)Macrophages (Histiocytes)

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• Histology 2-6.14: Monocyte, large cell, Histology 2-6.14: Monocyte, large cell, pale vacuolated cytoplasm, irregular pale vacuolated cytoplasm, irregular nucleusnucleus

• Harmening 1-32: Monocytes, variation; Harmening 1-32: Monocytes, variation; fine granules, vacuoles, gray-blue fine granules, vacuoles, gray-blue cytoplasm; pseudopodscytoplasm; pseudopods

• Robbins 377: Tissue monocyte in case of Robbins 377: Tissue monocyte in case of TB Langerhan cell TB Langerhan cell

LymphocytesLymphocytesIntegrated HistologyIntegrated Histology

• Size (small, medium, large)Size (small, medium, large)

• Site of Differentiation and MaturationSite of Differentiation and Maturation

• Heterogeneous Heterogeneous

• Major Classes in CirculationMajor Classes in Circulation– T lymphocytes (T cells) 80% of lymphocytesT lymphocytes (T cells) 80% of lymphocytes– B lymphocytes (B cells) 20% of lymphocytesB lymphocytes (B cells) 20% of lymphocytes

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• Histology 2-6.12 a, b: Small lymphocyte, Histology 2-6.12 a, b: Small lymphocyte, 6-9 micron, basophilic cytoplasm 6-9 micron, basophilic cytoplasm (ribosomes) , high N/C ratio; EM(ribosomes) , high N/C ratio; EM

• Harmening 1-36: Small mature; Harmening 1-36: Small mature; intermediate size; indented nucleus; large intermediate size; indented nucleus; large lymphocyte with granuleslymphocyte with granules

• 1-40: Monocyte and Lymphocyte1-40: Monocyte and Lymphocyte• 1.39: Lymphocyte and PMN1.39: Lymphocyte and PMN

PlateletsPlateletsIntegrated HistologyIntegrated Histology

• SizeSize

• NumberNumber

• Cytoplasmic ComponentsCytoplasmic Components– GranulesGranules– Tubular SystemTubular System

• FunctionFunction– HemostasisHemostasis

Medical HistologyMedical HistologyLaboratory EvaluationLaboratory Evaluation

Complete Blood Cell (CBC) countComplete Blood Cell (CBC) count provides information about the absolute provides information about the absolute and relative numbers of blood cells in a and relative numbers of blood cells in a specimen of blood.specimen of blood.

Medical HistologyMedical HistologyComplete Blood Cell Count (CBC) Complete Blood Cell Count (CBC)

• Red blood cell countRed blood cell count, size, variation in , size, variation in size, and hemoglobin contentsize, and hemoglobin content

• White blood cell countWhite blood cell count and differential and differential into granulocytic cells, lymphocytes, into granulocytic cells, lymphocytes, monocytes, and abnormal cellsmonocytes, and abnormal cells

• Platelet countPlatelet count

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• Histology 2-6.16 a, b, c, d: Platelets, 1.5-Histology 2-6.16 a, b, c, d: Platelets, 1.5-3.5 micron; cell membrane for adhesion, 3.5 micron; cell membrane for adhesion, microtubules for aggregation; granules microtubules for aggregation; granules (alpha, dense, lysosomes)(alpha, dense, lysosomes)

• 6.17 a, b: Platelet aggregation after 6.17 a, b: Platelet aggregation after adhering to damaged endothelium adhering to damaged endothelium

Medical HistologyMedical Histology Cell Counts Cell Counts

• Cytosis: increasedCytosis: increasedLeukocytosis, neutrophilia, Leukocytosis, neutrophilia, lymphocytosis, erythrocytosislymphocytosis, erythrocytosis

• Cytopenia: decreasedCytopenia: decreasedPancytopenia = all Pancytopenia = all Leukopenia = Low WBC countLeukopenia = Low WBC countNeutropenia = Low neutrophil countNeutropenia = Low neutrophil countLymphocytopeniaLymphocytopenia

LeukocytosisLeukocytosis

• Leukocytosis is a condition in which the Leukocytosis is a condition in which the leukocyte count is higher than the leukocyte count is higher than the reference rangereference range

• Usually affects a specific type of Usually affects a specific type of leukocyte- neutrophil, basophil, leukocyte- neutrophil, basophil, eosinophil, lymphocyteeosinophil, lymphocyte

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• LeukocytosisLeukocytosis

• NeutrophilicNeutrophilic

LeukocytosisLeukocytosis

• ReactiveReactive– Infections, inflammation, necrosisInfections, inflammation, necrosis– Others Others

• MalignantMalignant– Leukemia (uncontrolled proliferation of Leukemia (uncontrolled proliferation of

leukocytes, causing an accumulation of leukocytes, causing an accumulation of leukemic cells in bone marrow and blood)leukemic cells in bone marrow and blood)

– OthersOthers

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• Reactive = Benign = Self-LimitingReactive = Benign = Self-Limiting– ASCP 1-04 and 1-07 NeutrophiliaASCP 1-04 and 1-07 Neutrophilia– ASCP 3-43 MonocytosisASCP 3-43 Monocytosis– ASCP 3-26, 3-27, 3-33 LymphocytosisASCP 3-26, 3-27, 3-33 Lymphocytosis– Essential #138 IM Essential #138 IM

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• ASCP 400X NeutrophilsASCP 400X Neutrophils– 1.43 Normal1.43 Normal– 3.1 Reactive3.1 Reactive– 6.1 Chronic Myelocytic Leukemia6.1 Chronic Myelocytic Leukemia

• ASCP MonocytosisASCP Monocytosis– 3.433.43

• ASCP LymphocytosisASCP Lymphocytosis– 1.121.12

Medical Histology Medical Histology Differential WBC CountDifferential WBC Count

PatientPatient• WBC=100, 000WBC=100, 000

• Neutrophils 86%Neutrophils 86%– 86,00086,000

• Lymphocytes 4%Lymphocytes 4%– 40004000

Reference RangeReference Range• 4,000 -10,0004,000 -10,000

• 50 – 60%50 – 60%– 1800 – 70001800 – 7000

• 30 – 40%30 – 40%– 1500 - 40001500 - 4000

Medical Histology Medical Histology Differential WBC CountDifferential WBC Count


• Neutrophilic leukocytosis (Neutrophilia)Neutrophilic leukocytosis (Neutrophilia)

• Absolute lymphocyte count within Absolute lymphocyte count within reference rangereference range

What is the most likely cause?What is the most likely cause?

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• Harmening 15-2 Neutrophilia due to a Harmening 15-2 Neutrophilia due to a bacterial infection (septicemia) bacterial infection (septicemia)

Medical Histology Medical Histology Differential WBCDifferential WBC

PatientPatient

• WBC=100, 000WBC=100, 000

• Neutrophils 7%Neutrophils 7%

– 7,0007,000

• Lymphocytes 93%Lymphocytes 93%

– 93009300

Reference RangeReference Range

• 4000 -10,0004000 -10,000

• 50 – 60%50 – 60%

• 1800 – 70001800 – 7000

• 30 – 40%30 – 40%

• 1500 - 40001500 - 4000

Medical Histology Medical Histology Differential WBCDifferential WBC


• LymphocytosisLymphocytosis

• Absolute neutrophil count within Absolute neutrophil count within reference rangereference range

What is the most likely cause?What is the most likely cause?

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• ASCP 3-40 LymphocytosisASCP 3-40 Lymphocytosis

Medical HistologyMedical HistologyPeripheral BloodPeripheral Blood

• Review histology of peripheral blood and Review histology of peripheral blood and give clinical examples of specific give clinical examples of specific leukocytosisleukocytosis

• Give examples of a reactive leukocytosis, Give examples of a reactive leukocytosis, acute leukemia, and chronic leukemiaacute leukemia, and chronic leukemia– Histology is important for the recognition of Histology is important for the recognition of

maturation of leukemic cellsmaturation of leukemic cells

Medical Histology Medical Histology ThrombocytopeniaThrombocytopenia

• Decreased plateletsDecreased platelets– Interrupt blood coagulation and prevent Interrupt blood coagulation and prevent

hemostasishemostasis– Bleeding complicationsBleeding complications– The degree of thrombocytopenia determines The degree of thrombocytopenia determines

the risk for bleeding due to minor trauma, the risk for bleeding due to minor trauma, risk of spontaneous bleeding, and severe risk of spontaneous bleeding, and severe bleeding bleeding

Medical Histology Medical Histology ThrombocytopeniaThrombocytopenia

• Decreased plateletsDecreased platelets– Interrupt blood coagulation and prevent Interrupt blood coagulation and prevent

hemostasishemostasis– Bleeding complicationsBleeding complications– The degree of thrombocytopenia determines The degree of thrombocytopenia determines

the risk for bleeding due to minor trauma, the risk for bleeding due to minor trauma, risk of spontaneous bleeding, and severe risk of spontaneous bleeding, and severe bleeding bleeding

Medical Histology Medical Histology AnemiaAnemia

Decrease in the total number of circulating Decrease in the total number of circulating erythrocytes and/or a decrease in the erythrocytes and/or a decrease in the quality or quantity of hemoglobin.quality or quantity of hemoglobin.

Number and severity of the symptoms Number and severity of the symptoms depend upon the body’s ability to depend upon the body’s ability to compensate for the reduced oxygen compensate for the reduced oxygen carrying capacity of the blood.carrying capacity of the blood.


• Decreased Production ( Look in the Decreased Production ( Look in the Bone Marrow) Bone Marrow) – Hemoglobin synthesisHemoglobin synthesis

– DNA synthesis and nuclear maturationDNA synthesis and nuclear maturation

– Stem cell defectsStem cell defects

– Bone marrow infiltrationBone marrow infiltration


• Acute Blood Loss (Hemorrhage)Acute Blood Loss (Hemorrhage)

• Increased Destruction (Hemolysis)Increased Destruction (Hemolysis)– Membrane abnormalityMembrane abnormality– Cytoplasmic enzyme abnormality Cytoplasmic enzyme abnormality – Hemoglobin abnormalityHemoglobin abnormality– RBC environment (Extracellular)RBC environment (Extracellular)

Medical HistologyMedical HistologyAnemia-MorphologyAnemia-Morphology

• Erythrocyte size = -Erythrocyte size = -cyticcytic– Normocytic, macrocytic, microcyticNormocytic, macrocytic, microcytic

• Hemoglobin content= -Hemoglobin content= -chromicchromic– Normochromic, hypochromicNormochromic, hypochromic

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• Normal red blood cells = normocytic for Normal red blood cells = normocytic for comparisoncomparison

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• Rodak # 86 Iron Deficiency MicrocyticRodak # 86 Iron Deficiency Microcytic• ASCP 4-16 Iron Deficiency MicrocyticASCP 4-16 Iron Deficiency Microcytic• ASCP 4- 20 Macrocytic with LymphocyteASCP 4- 20 Macrocytic with Lymphocyte• ASCP 4.12 AnisocytosisASCP 4.12 Anisocytosis•

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Hematopoietic System- 1 Peripheral Blood Linda F. Cunningham, MD January 2, 2003