forward scatter (fsc) side fluorescence (sfl) side scatter (ssc);
DESCRIPTION
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: - PowerPoint PPT PresentationTRANSCRIPT
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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);
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Leucocytes
• Lymphocytes
• Monocytes/Macrophages
• Granulocytes:
Neutrophils
Eosinophils
Basophils
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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 MPOMonociti = quantità medio -piccola di MPOLinfociti 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.
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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.
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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
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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
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Linfocitopenia
• Linfociti1500 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
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Monocytes
0.2 – 0.8 x 109/l
• Antigen presentation• Cytokine production• Phagocytosis
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Monocitosi
• Monociti ≥ 800 cellule/l
• Tubercolosi• Endocarditi batteriche acute• Brucellosi• Malaria• Morbo di Chron• Sarcoidosi• LES ed artrite reumatoide• Linfoma di Hodgkin
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Monocitopenia
• Monociti 150 cel/l
• Stress da glucocorticoidi• Aplasia midollare• Leucemia a cellule capellute• Farmaci immunosoppressivi
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Neutrophils
• 2.5 - 7.5 x 109/l• Large reserve pool
• 3-5 lobes• Granular cytoplasm• Transient in blood• Major phagocytic role• Bacterial killing
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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)
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Neutropenia
• Neutropenia grave:neutrofili500 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)
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HypersegmentationHypersegmentation
• Shift to the right• More mature cells
kept on• Infective and
inflammatory sites• Severe burns• TB, post chemo,
pregnancy
““Shift to the left”Shift to the left”
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Eosinophils
• 0.2 - 0.8 x 109/l• Bilobed nucleus
Red granules
Weakly phagocytic• Anti-parasitic action• Modulation of
hypersensitivity and allergic reactions
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Eosinofilia
• Eosinofili ≥800 cellule/l
• Allergie• Infestazioni da metazoi• Farmaci• Malattie autoimmuni sistemiche• Neoplasie• Sindromi ipereosinofile
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Eosinopenia
• Eosinofili 70 cellule/l
• Aumentata increzione di glucocorticoidi
• Infezioni batteriche acute
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Basophils
• 0.1 - 0.2 x 109/l• Bilobed nucleus• Large black granules
• Inflammatory response
• Hypersensitivity /allergy
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Basofilia
• Basofili ≥ 20 cellule/ l
• Ipersensibilità immediata• Colite ulcerosa• Leucemia mieloide cronica (basofila)• Policitemia vera
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BasopeniaBasopenia
• Stress
• Infection
• Steroids
• Cushing’s syndrome
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Platelets
• Fragments of cytoplasm from BM megakaryocyte
• Major role in coagulation
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Platelets 2
• Lifespan in peripheral blood 7-10 days
• Range 150-400x 109/l
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METODI USATI PER IL CONTEGGIO AUTOMATICO DELLE PIASTRINE
CITOFLUORIMETRICO
IMPEDENZOMETRICO
– ELETTRO OTTICO
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Conta Ottica PLTo Conta ad impedenza PLTi
Conta piastrinica
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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)
Metodo impedenziomet
rico
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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
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PDW
• Indica l'anisocitosi piastrinica.Aumenta in corso di:• An. Megaloblastica• An. Aplastica• AREB• LMC• In presenza di crioglobuline e paraproteine
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MPC
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
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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
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Quantitative abnormalities 2
• Thrombocytosis - Increased count
• > 450 x 109/l
• Reactive eg infection inflammation
• Chronic bleeding
• Haematological malignancy
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INTERFERENZA NELLA CONTA PIASTRINICA
•Frammenti eritrocitari
•Frammenti di globuli bianchi
•Parassiti, batteri
•Ombre eritrocitarie
•Piastrine giganti
•Aggregati piastrinici
•Effetto da anticoagulante
•Frammenti eritrocitari
•Frammenti di globuli bianchi
•Parassiti, batteri
•Ombre eritrocitarie
•Piastrine giganti
•Aggregati piastrinici
•Effetto da anticoagulante
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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
Gli AUTOANTICORPI EDTA – DIPENDENTI interagiscono con antigeni criptici della glicoproteina II b, smascherati dalla chelazione del calcio da parte dell’EDTA
•IgG EDTA- e temperatura – dipendenti•Crioagglutinine EDTA – dipendenti•IgG EDTA- dipendenti e temperatura – indipendenti
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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