Side Fluorescence (SFl) Side Scatter (SSc); Alcuni citometri a flusso utilizzano laser a semiconduttori (633 nm) e coloranti fluorescenti per acidi nucleici che separano i leucociti utilizzando i segnali di: Forward Scatter (FSc) Side Fluorescence (SFl) Side Scatter (SSc);

Leucocytes Lymphocytes Monocytes/Macrophages Granulocytes: Neutrophils Eosinophils Basophils Leucocytes comprise three major types. The lymphocyte makes up about 20% of the total wbcs, in the normal adult. A higher proportion is seen in children. Monocytes comprise about 5-10% of the total Granulocytes form the largest group, with about 60-70% of the total. Of these the majority are neutrophils, with small populations of eosinophils and basophils.

I leucociti vengono anche differenziati sulla base della maggiore o minore quantità di mieloperosidasi (MPO) contenuta nei granuli citoplasmatici. Il sangue viene diluito, le emazie lisate e i leucociti colorati per la (MPO). Granulociti ed eosinofili = massima quantità di MPO Monociti = quantità medio -piccola di MPO Linfociti e Blasti = piccola quantità di MPO I GB: entrano in una cella di flusso in cui per ciascuna cellula vengono determinate le proprietà bi-dimensionali di dispersione e di assorbimento della luce. Le cellule colorate assorbono più luce e la disperdono con un angolo diverso rispetto alle cellule non colorate. Il risultato finale e' un citogramma leucocitario bi-dimensionale.

LINEE GUIDA F. LEUCOCITARIA La refertazione delle popolazioni leucocitarie deve essere espressa in numero assoluto. La quantificazione percentuale, che da sola può fornire informazioni equivoche, può talvolta integrare efficacemente il numero assoluto; pertanto, la doppia refertazione mantiene un valore informativo.

Lymphocytes 1.5 - 3.5 x 109/l Count varies with age Adaptive immune response T cells – cell mediated immunity B cells – antibody mediated immunity NK cells Lymphocytes can be split into different functional groups by their antigenic makeup The major types are B and T cells and these cannot be differentiated from the blood film alone. The majority of lymphocytes are small cells with little cytoplasm and well condensed nucleus. A small proportion are larger with more abundant cytoplasm and may show a few azure granules. A small but significant group are the Natural Killer cells, which have a cytotoxic action, these may be the large lymphs seen in the blood film

Linfocitosi Linfociti ≥4500 cellule/l negli adulti Linfociti ≥9000 cellule/l nei bambini fino ai 10 anni Infezioni virali, tubercolosi ,brucellosi e tifo Infezione da Bordetella pertussis Linfomi LINFOCITOSI : Count varies with age Viral infection Other infections(Syphillis, toxoplasmosis, mycoplasma) Drug sensitivity (delantin) Miscellaneous (Autoimmune, hyperthyroidism, Addison’s, graft rejection)

Linfocitopenia Linfociti1500 cellule/l negli adulti Linfociti 3000 cellule/l nei bambini Immunodeficit congeniti ed acquisiti Stress da aumentata increzione di glucocorticoidi Aplasia midollare Alcune forme di LLA Immunosoppressione da farmaci LINFOPENIA: Decreased production (Inherited immunodef.AIDS) Increased destruction (Steroids/ Cushing’s Radiation, chemo Intestinal loss Malignancies Misc. TB, CVD, sarcoidosis)

Monocytes 0.2 – 0.8 x 109/l Antigen presentation Cytokine production Phagocytosis Monocytes only pass transiently through the peripheral blood and most of their lifespan occurs as macrophages in the tissues. They are large cells with a bean or horseshoe shaped nucleus and a greyish cytoplasm. They have a major role in the immune response, where they process and present antigens to the T lymphocytes. They are also major producers of cytokines. They are motile cells which are able to phagocytose foreign material.

Monocitosi Monociti ≥ 800 cellule/l Tubercolosi Endocarditi batteriche acute Brucellosi Malaria Morbo di Chron Sarcoidosi LES ed artrite reumatoide Linfoma di Hodgkin MONOCITOSI: Infections (TB, SBE, syphillis, salmonella, listeria, leprosy, brucellosis) Hodgkins Collagen Vascular Disease Gastrointestinal disorders (Ulcerative colitis, regional anuritis)

Monocitopenia Monociti 150 cel/l Stress da glucocorticoidi Aplasia midollare Leucemia a cellule capellute Farmaci immunosoppressivi MONOCITOPENIA: Steroids Hairy Cell Leukemia (HCL)

Neutrophils 2.5 - 7.5 x 109/l Large reserve pool 3-5 lobes Granular cytoplasm Transient in blood Major phagocytic role Bacterial killing Neutrophils form the major cell type in the peripheral blood, although they only spend about 12-24 hours before migrating to the tissues. They are highly motile cells, which rapidly migrate to the site of infection, in response to factors released by bacteria and activated cells already present at the site. They readily phagocytose and destroy bacteris etc, utilising enzymes and hydrogen peroxide

Neutrofilia Neutrofilia ≥7500cellule/L Infezioni batteriche – germi piogeni (ma anche fungine, parassitarie ed alcune virali) Flogosi immuni e non immuni (ustioni ,emorragie,tumori metastatici, infarto,embolia polmonare) Stress, Adrenalina e Glucocorticoidi Sindromi mieloproliferative ( Leucemia mieloide cronica,mielodisplasia e policitemia vera) Farmaci (glucocorticoidi)

Neutropenia Neutropenia grave:neutrofili500 cellule/l Neutropenia moderata: 500-1000 cellule/ l Neutropenia lieve:1000-2000 cellule/ l Neutropenia da farmaci (immunosoppressori e antiblastici) Aplasia midollare, leucemie e linfomi Neutropenia isoimmune neonatale (aumentata distruzione da IgG materne verso Antigeni paterni) Neutropenia autoimmune (LES) Neutropenia da aumentata marginazione (setticemia da Gram -, malaria, tifo, rickettiosi) Neutropenia da sequestro splenico (cirrosi)

Hypersegmentation Shift to the right More mature cells kept on Infective and inflammatory sites Severe burns TB, post chemo, pregnancy “Shift to the left”

Eosinophils 0.2 - 0.8 x 109/l Bilobed nucleus Red granules Weakly phagocytic Anti-parasitic action Modulation of hypersensitivity and allergic reactions

Eosinofilia Eosinofili ≥800 cellule/l Allergie Infestazioni da metazoi Farmaci Malattie autoimmuni sistemiche Neoplasie Sindromi ipereosinofile Eosinofilia: Neoplasia Allergic reaction Addison’s disease CVD parasites Eosinopenia: Acute stress Infection Steroids/Cushing’s syndrome

Eosinopenia Eosinofili 70 cellule/l Aumentata increzione di glucocorticoidi Infezioni batteriche acute

Basophils 0.1 - 0.2 x 109/l Bilobed nucleus Large black granules Inflammatory response Hypersensitivity /allergy

Basofilia Basofili ≥ 20 cellule/ l Ipersensibilità immediata Colite ulcerosa Leucemia mieloide cronica (basofila) Policitemia vera Basofilia: Hypersensitivity reactions (Allergies, asthma, eczema) Hypothyroidism Hematologic malignancy Ulcerative colitis Varicella Basopenia: Stress Infection Steroids/ Cushing’s syndrome

Basopenia Stress Infection Steroids Cushing’s syndrome

Platelets Fragments of cytoplasm from BM megakaryocyte Major role in coagulation Platelets are derived from a huge cell in the bone marrow, they have a major role in coagulation, which will be discussed in later lectures

Platelets 2 Lifespan in peripheral blood 7-10 days Range 150-400x 109/l Platelets are visible as tiny particles in the background of the blood film. When the film is examined we check to see if they are normal in appearance., but it is their numbers and function which is more generally significant

METODI USATI PER IL CONTEGGIO AUTOMATICO DELLE PIASTRINE CITOFLUORIMETRICO IMPEDENZOMETRICO ELETTRO OTTICO

Conta piastrinica Conta Ottica PLTo Conta ad impedenza PLTi

Metodo impedenziometrico Piastrine : Istogramma volumetrico PL = soglia inferiore, PU = soglia superiore, PDW = platelet distribution width (larghezza della curva tagliata al 20%), P-LCR = platelet –large cell ratio (percentuale di grandi piastrine). MPC = Mean PLT Contenent (misura della concentrazione complessiva dei componenti interni delle piastrine)

MPV Più alto negli uomini Correlazione inversa non lineare fra MPV e N.ro PLT Aumenta in corso di: Diminuisce in corso di: Diabete mellito Leucemia Acuta IMA An. Megaloblastica Arteriopatie Periferiche Malattie mieloproliferative Pre-eclampsia Splenectomia

PDW Indica l'anisocitosi piastrinica. Aumenta in corso di: An. Megaloblastica An. Aplastica AREB LMC In presenza di crioglobuline e paraproteine

MPC Morfologia, Attivazione, Screening. Di conseguenza utile per: E' misura della concentrazione complessiva dei componenti interni delle piastrine, fornisce indicazioni su: Morfologia, Attivazione, Screening. Di conseguenza utile per: monitoraggio terapie antiaggreganti monitoraggio terapie antinfiammatorie

Quantitative Abnormalities 1 Thrombocytopenia - Decreased count <100 x 109/l - Increasing risk of bleeding in major surgery <50 x 109/l - Increasing risk of bleeding for CID and surgery <10-20 x 109/l - Increasing risk of spontaneous bleeding Causes Immune BM infiltration, chemotherapy etc Inherited defects An immune thrombocytopenia can occur following viral infection or as an apparently spontaneous event. In some cases it is transient and resolves quickly, in others it may become a chronic condition. Patients receiving high dose chemotherapy, may have severely decreased platelets and often require platelet transfusion until their counts have improved.

Quantitative abnormalities 2 Thrombocytosis - Increased count > 450 x 109/l Reactive eg infection inflammation Chronic bleeding Haematological malignancy In most patients an increased platelet count is of little clinical significance and often occurs as a response to infection, inflammation or chroinic bleeds.etc. In patients with chronic haematological malignancy, the platelets may also become hyperaggregable – leading to increased risk of thrombosis or hypoaggregable – leading to increased risk of bleeding.

INTERFERENZA NELLA CONTA PIASTRINICA Frammenti eritrocitari Frammenti di globuli bianchi Parassiti, batteri Ombre eritrocitarie Piastrine giganti Aggregati piastrinici Effetto da anticoagulante

Cause più comuni di conta piastrinica erronea nel paziente ematologico AGGREGATI PIASTRINICI CAUSA FREQUENTE DI CONTA SOTTOSTIMATA Sono epressione dell’agglutinazione piastrinica in vitro da parte di autoanticorpi IgG EDTA- e temperatura – dipendenti Crioagglutinine EDTA – dipendenti IgG EDTA- dipendenti e temperatura – indipendenti Gli AUTOANTICORPI EDTA – DIPENDENTI interagiscono con antigeni criptici della glicoproteina II b, smascherati dalla chelazione del calcio da parte dell’EDTA

Qualitative defects Count may be normal/low/high May be no apparent abnormality on film May show changes eg size, appearance etc Functionality is variably affected May be inherited or acquired