Investigation of the Leukocyte Series PDF

PAGE 1 https://www.umfst.ro PA 6- Investigation of the leukocyte https://edu.umch.de series. 2024 May Assist. Prof. Irina-Bianca Kosovski,...

PAGE 1 https://www.umfst.ro PA 6- Investigation of the leukocyte https://edu.umch.de series. 2024 May Assist. Prof. Irina-Bianca Kosovski, MD, Ph Assist. Prof. Andreea-Catalina Tinca, MD, Ph Assist. Prof. Andreea Cozac Szoke, MD, Ph Introduction PAGE 2 The immune system has: 1. a nonspecific or innate cellular and humoral component 2. a specific or acquired immune cellular and humoral component. White cells use the blood for transport from the bone marrow to their major sites of activity. Most functions of white blood cells take place when they leave the circulation to enter tissues. Introduction PAGE 3 WBCs can be classified in 5 types of cells: Neutrophils Eosinophils Basophils Lymphocytes Monocytes WBCs can be investigated base on: 1. Peripheral blood smear 2. Automated complete cell blood count (CBC) Netter's Illustrated Pharmacology, Raffa, Robert B., PhD; Rawls, Scott M., PhD; Beyzarov, Elena Portyansky, PharmD. Published January 1, 2014. Pages 205-243. © 2014. Peripheral blood smear PAGE 4 Blood smear allows studying the morphology of blood elements. Leukocyte formula represents percentage expression of different types of leukocytes. Blood smear study is part of an early investigation of any patient because it provides important information on all blood cells-> may indicate either directly diagnosis or suggestions for extra investigation. Normal values of leukocyte formula: Granulocytes 50 – 75 % Eosinophils 0–4% Basophils 0–1% Lymphocytes 20 – 45 % Monocytes 3–9% Peripheral blood smear PAGE 5 Leukocyte formula Type of WBC % Never Neutrophils 60 Let Lymphocytes 30 Monkey Monocytes 6 Eat Eosinophils 3 Bananas Basophils 1 Peripheral blood smear - Neutrophils PAGE 6 Appearance and structure Neutrophils, also known as ‘polymorphonuclear leucocytes,’ measure 12–14 μm in diameter. Nuclei are segmented, with 2–5 lobes connected by thin chromatin threads. Stevens & Lowe's Human Histology, Lowe, James S., BMedSci, BMBS, DM, FRCPath; Anderson, Peter G., DVM, PhD; Anderson, Susan I., BSc, MMedSci, PhD. © 2020. Cytoplasm stains pink/purple and contains glycogen and numerous granules. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Peripheral blood smear - Neutrophils PAGE 7 Schematic diagram of precursor cells in formation of neutrophils Textbook of Histology, Gartner, Leslie P., PhD. © 2021. Peripheral blood smear - Neutrophils PAGE 8 Location Neutrophils circulate in blood for up to 10 hours and represent the majority (~ 60%) of the leucocyte blood population. They migrate into tissues in response to chemotactic agents, where they survive for a further 1–3 days. Function Neutrophils are the first cells to arrive at a zone of inflammation, comprising a prominent aspect of the innate immune system. They recruit further immune cells with chemotactic mediators. Neutrophils destroy microorganisms by: Release of hydrolytic enzymes from their granules Phagocytosis Dead neutrophils are the major constituent of pus. Peripheral blood smear - Lymphocytes PAGE 9 Appearance and structure Lymphocytes are the smallest of the leucocytes (~ 6–8 μm diameter). The term ‘lymphocytes’ includes T cells, B cells and natural killer (NK) cells. They all derive from lymphoid lineage. Each population expresses characteristic molecular surface markers. Their shape varies from spherical to pleomorphic. Lymphocyte nuclei are large, round, densely staining and may be situated off-centre (‘eccentrically’). Basophilic (blue-staining) cytoplasm is sparse, so the nucleus:cytoplasm ratio is high. However, this ratio falls on lymphocyte activation, since cytoplasmic Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, volume increases with intracellular synthetic activity. Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Peripheral blood smear - Lymphocytes PAGE 10 Location Lymphocytes usually represent ~ 20%–25% of the blood population of leucocytes. They circulate in blood and the lymphatic system when travelling between bone marrow and lymphoid tissue such as the spleen or lymph nodes. Function Lymphocytes produce antibodies (B cells) or kill foreign or infected cells (T cells and NK cells). Their lifespan is variable, according to antigenic interaction. Memory B or T cells, for example, survive for decades. Peripheral blood smear - Monocytes PAGE 11 Appearance and structure Monocytes are the largest leucocytes (≤ 25 μm diameter). They have a large, eccentric, reniform nucleus. Nucleoli are often visible, giving the nucleus a ‘moth-eaten’ appearance. Basophilic cytoplasm contains numerous lysosomes and vacuole- like spaces, producing a ‘ground-glass’ appearance. Microtubules, microfilaments, pinocytotic vesicles and filopodia/pseudopodia are present at the cell periphery. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Peripheral blood smear - Monocytes PAGE 12 Location Monocytes spend 1–3 days in blood, where they represent ~ 3%–9% of bloodstream leucocytes. They then enter the tissues, where they differentiate further and become macrophages. Macrophages survive within tissues for months to years. Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. Published January 1, 2009. Pages 71-85. © 2009. Peripheral blood smear - Monocytes PAGE 13 Function: Macrophages make up the reticuloendothelial system and are found in tissues throughout the body; They phagocytose elderly/damaged red cells, pathogens and cellular debris; They process and present antigen to lymphocytes as part of the adaptive immune response; They have a pro-inflammatory function, releasing a variety of cytokines. Peripheral blood smear - Eosinophils PAGE 14 Appearance and structure Eosinophils are larger than neutrophils (~ 12–17 μm diameter). Their nucleus is bilobed or reniform (kidney-shaped). Cytoplasm stains brick-red and contains numerous cationic granules with cytotoxic contents including: major basic protein eosinophil-derived neurotoxin peroxidase Crash Course Haematology and Immunology, cationic protein Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Peripheral blood smear - Eosinophils PAGE 15 Schematic diagram of precursor cells in formation of eosinophils Textbook of Histology, Gartner, Leslie P., PhD. © 2021. Peripheral blood smear - Eosinophils PAGE 16 Location The vast majority of eosinophils reside the tissues (mainly at epithelial barriers including lungs, skin and gastrointestinal tract). They circulate in blood for 1–2 days, where they represent ~ 1%–4% of the circulating leucocyte population. Function eosinophils play the primary role in attacking multicellular parasitic organisms; are capable of phagocytosis, particularly of antigen–antibody complexes; eosinophils (similarly to mast cells and basophils) are also important mediators of allergic reactions. Peripheral blood smear - Basophils PAGE 17 Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Appearance and structure Basophils are ~ 14–16 μm in diameter with an ‘S-shaped’ nucleus. Cytoplasm is densely packed with large blue-staining (basophilic) granules that may obscure the nucleus and cause bulging outward of the cell membrane, giving a ‘roughened’ appearance to the perimeter. These granules contain: heparin histamine chemotactic factors Stevens & Lowe's Human Histology, Lowe, James S., BMedSci, BMBS, peroxidase DM, FRCPath; Anderson, Peter G., DVM, PhD; Anderson, Susan I., BSc, MMedSci, PhD. © 2020. Peripheral blood smear - Basophils PAGE 18 Schematic diagram of precursor cells in formation of basophils Textbook of Histology, Gartner, Leslie P., PhD. © 2021. Peripheral blood smear - Basophils PAGE 19 Location Basophils are scarce (< 1% of circulating leucocytes), with a lifespan in the circulation of 1–2 days (whilst travelling to lymphoid and nonlymphoid tissues). Function Basophils are functionally very similar to mast cells, although their lineages differ and mast cells are restricted to tissues and thus do not appear in the blood. They both release a similar array of mediators in response to immunoglobulin E (IgE) cross- linking and thus are thought to participate in type 1 hypersensitivity reactions-> role in allergic response. Peripheral blood smear PAGE 20 Robbins and Cotran Atlas of Pathology, Klatt, Edward C., MD. Published January 1, 2021. Pages 63-92.e5. © 2021. Peripheral blood smear PAGE 21 Left and right shift of neutrophils 1. Left shift Describes the phenomenon where an increased proportion of immature neutrophils are released from the bone marrow into peripheral blood. Causes: inflammatory cytokines produced in response to inflammation or infection bone marrow disorders bacterial infection-> a rise in neutrophils. Initially, the demand for mature neutrophils temporarily outstrips the supply available in the bone marrow, so immature neutrophils are also released into blood. Band cells are most common, but earlier versions may be released also in response to increasingly severe inflammatory stimuli. Peripheral blood smear PAGE 22 Left and right shift of neutrophils 2. Right shift Leucocytes persist longer than normal in the circulation and acquire morphological characteristics of ‘hypermaturity’ (e.g., hypersegmented and enlarged ‘giant’ neutrophils). Causes: noninfectious inflammatory processes: malignancy bone marrow synthesis disorders: megaloblastic or iron-deficiency anaemia Peripheral blood smear PAGE 23 White count differential 9 Parameter Normal range (×10 /L) Most frequent blood abnormality Total white cell count 4.0–11 ↑ : ‘leucocytosis’ ↓ : ‘leucopenia’ Lymphocytes 1.3–4.5 ↑ : ‘lymphocytosis’ ↓ : ‘lymphopenia’ Neutrophils 2–7.5 ↑ : ‘neutrophilia’ ↓: ‘neutropenia’(‘granulocytopenia’) Eosinophils 0.04–0.4 ↑ : ‘eosinophilia’ Basophils 0–0.1 ↑ : ‘basophilia’ Monocytes 0.2–0.8 ↑ : ‘monocytosis’ Peripheral blood smear PAGE 24 Blood film abnormalities of leucocyte morphology and their diagnostic inferences Abnormality Description Diagnostic inference Hypersegmentation ≥ 5 lobes per nucleus Megaloblastic anaemia of neutrophils Left shift of Various earlier developmental stages of Severe infection neutrophils neutrophil CML Band cells are the most common, but all Pregnancy stages are possible Use of G-CSF Netter's Essential Biochemistry Hematology Ronner, Peter, Hudnall, S. David, PhD. Published Janua MD. Published January 1, ry 1, 2012. Pages P1-P32. © 2012. 2018. Pages 402- 415. © 2018. Peripheral blood smear PAGE 25 Blood film abnormalities of leucocyte morphology and their diagnostic inferences Abnormality Description Diagnostic inference Blast cells The most immature form of the blood cell Acute leukemias (AML and ALL) lineage (myeloblast or a lymphoblast could Occasional blast seen with be seen in the peripheral blood in AML leucoerythroblastic reaction and ALL, respectively) Netter's Integrated Review of Medicine AML Uy, Natalie F.; ALL Parikh, Kinjan. Published J anuary 1, 2021. Pages 639- 641. © 2021. Peripheral blood smear PAGE 26 Blood film abnormalities of leucocyte morphology and their diagnostic inferences Abnormality Description Diagnostic inference Auer rods Rod-like accretions of granular material AML which may appear in the cytoplasm of leukaemic blast cells in AML Smear cells (also known Remnants of a cell, lacking a clearly Reactive lymphocytosis as ‘smudge’ cells or identifiable cell membrane or nucleus CLL ‘basket’ cells) These are seen when abnormally fragile cells are abundant in the blood Robbins & Cotran Pathologic Basis of Disease, Kumar, Vinay, MBBS, MD, FRCPath; Goldman-Cecil Medicine Abbas, Abul K., MBBS; Bain, Barbara J. Published January 1, Aster, Jon C., MD, 2020. Pages 1020-1027.e2. © 2020. PhD. © 2021. Peripheral blood smear PAGE 27 Blood film abnormalities of leucocyte morphology and their diagnostic inferences Abnormality Description Diagnostic inference Leucoerythroblastic Presence of immature leucocytes Severe haemorrhage change including occasional Severe haemolysis blast and immature (still nucleated) Sepsis red cells Myelofibrosis (either primary or secondary to metastatic disease in bone marrow) Peripheral blood smear PAGE 28 Blood film abnormalities of leucocyte morphology and their diagnostic inferences Kumar and Clark's Clinical Medicine, Sive, Jonathan; Foggo, Vanessa. Published January 1, 2021. Pages 379- 410. © 2021.  Microscopic examination of the bone marrow PAGE 29 Netter's Essential Histology Ovalle, William K., PhD; Nahirney, Patrick C., PhD. Pages 169- 185. © 2021. Automated complete cell blood count (CBC) PAGE 30 Automated blood count is a series of tests used to assess the composition and concentration in the blood cellular components. This process is automated and uses stains, cell size and light scatter to differentiate among different cell types. It consists in performing the following tests: the WBC count the classification of leukocytes (leukocytes’ formula or differential) the erythrocyte count the hemoglobin concentration measurement the hematocrit the erythrocyte indices the platelet counts Automated complete cell blood count (CBC) PAGE 31 The test is performed from the venous or capillary whole blood collected on anticoagulant K3EDTA: Normal WBC count in men and women over 16 years WBC - White blood cells Analysis Value Unit NEUT - neutrophils (absolute number WBC 3.6-10 x103/μL and percentage) LY 20.0 - 45.0 % LY# 1.2-3.4 x103/μL MONO - monocytes (absolute MO 1.7-9.3 % number and percentage) MO# 0.1-0.6 x103/μL EO - eosinophils (absolute number GR 42.2-75.2 % and percentage) GR# 1.4-6.5 x103/μL BA 0-1 % BASO - basophils (absolute number BA# 0.01-0.08 x103/μL and percentage) EO 0-4 % LY – lymphocytes (absolute number EO# 0.03-0.04 x103/μL and percentage) Automated complete cell blood count (CBC) PAGE 32 Systematic approach to CBC interpretation for WBC Step 1: Ensure that the WBC count is accurate. Review WBC histogram and/or scatterplot and correlate with counts. The presence of nucleated RBCs may require correction of the WBC count. Step 2: Compare the patient’s WBC count with the laboratory’s established reference interval. Steps 3 and 4: Examine the differential information (relative and absolute) on variations in the distribution of WBCs. Step 5: Make note of immature cells in any cell line reported in the differential that should not appear in normal peripheral blood. Step 6: Make note of any morphologic abnormalities and correlate film findings with the numerical values Automated complete cell blood count (CBC) PAGE 33 CBC showing granulocytosis Automated complete cell blood count (CBC) PAGE 34 CBC in a case of chronic granulocytic leukemia  PAGE 35 Leucocytosis Leucocytosis = an increased total white cell count. One particular type of leucocyte, most commonly neutrophils, usually predominates, with smaller increases in the other types of leucocyte. The nature of the predominant population is mandatory for forming any diagnosis. Reactive leucocytosis: an acute rise in total white cell count is usually in response to infection, particularly if it occurs rapidly. This is termed a ‘reactive leucocytosis.’ A very high WBC count (>50,000/mm3) that is not a leukemia is known as a “leukemoid reaction” and much more pronounced than just the “left shift” with bandemia and the occasional metamyelocyte with acute inflammation.  PAGE 36 Leucocytosis Benign causes of neutrophilia: pregnancy or recent delivery vigorous exercise post-antibiotic diarrhoea vaccination cigarette smoking (possibly the most common cause of mild neutrophilia) Leucocytosis PAGE 37 Diseases associated with increases in particular leucocyte population Leucocyte population General causative mechanism Specific examples Neutrophil Bacterial infection (acute and chronic) (neutrophilia) Acute inflammation and/or tissue Infarction, surgery, necrosis rhabdomyolysis, myositis, burns, trauma, vasculitis, RA Myeloproliferative disorders CML, primary myelofibrosis Metabolic or endocrine disease Thyrotoxicosis, gout Drugs G-CSF, steroids, adrenaline Leucocytosis PAGE 38 Chronic myelogenous leukemia Robbins and Cotran Atlas of Pathology, Klatt, Edward C., MD. Published January 1, 2021. Pages 63-92.e5. © 2021. Leucocytosis PAGE 39 Diseases associated with increases in particular leucocyte population Leucocyte population General causative mechanism Specific examples Lymphocytes (lymphocytosis) Viral infection EBV, CMV, rubella Bacterial infection TB, brucellosis, syphilis, pertussis Parasitic infections Toxoplasmosis, rickettsial infections Neoplastic CLL, ALL, lymphoma Other Postsplenectomy Leucocytosis PAGE 40 Infectious mononucleosis Chronic lymphocytic leukemia Robbins and Cotran Atlas of Pathology, Klatt, Edward C., MD. Published January 1, 2021. Pages 63-92.e5. © 2021. Leucocytosis PAGE 41 Diseases associated with increases in particular leucocyte population General causative Leucocyte population Specific examples mechanism Monocytes Neoplastic Most commonly in CMML, CML and AML (monocytosis) May be seen in some lymphoma and myelodysplastic syndromes Bacterial infection TB, bacterial endocarditis, syphilis, typhoid, brucellosis Viral infection Varicella zoster Leucocytosis PAGE 42 Diseases associated with increases in particular leucocyte population Leucocyte General causative Specific examples population mechanism Eosinophils Parasitic infection Helminth (worm) infections, schistosomiasis, (eosinophilia) malaria Hypersensitivity Asthma, hay fever Drugs Drug-related eosinophilia, DRESS Skin disease Eczema, psoriasis, urticaria, pemphigus, dermatitis herpetiformis Neoplastic Hodgkin lymphoma, T cell non-Hodgkin lymphoma, CML, hypereosinophilic syndrome, solid tumours Post-infectious Convalescence phase following any infection Leucocytosis PAGE 43 Hypereosinophilic Syndrome (peripheral blood and bone merrow ) Ferri's Clinical Advisor 2021, Ferri, Fred F., MD. Published January 1, 2021. Pages 718.e1-718.e2. © 2021. Leucocytosis PAGE 44 Diseases associated with increases in particular leucocyte population Leucocyte General causative Specific examples population mechanism Basophils Myeloproliferative disorders Classically CML, although basophilia may be (basophilia) seen in PRV, PMF and ET IgE-mediated Anaphylaxis hypersensitivity reactions Infection Viral, helminths, mycobacterial Endocrine Hypothyroidism, diabetes, estrogen treatment Autoimmune UC, RA Leucopenia PAGE 45 Leucopenia = a total white blood count of less than 4 × 10 9 /L. Causes of neutropenia (