PATOFIZIOLOŠKA DIJAGNOSTIKA POREMEĆAJA BELE KRVNE LOZE
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- Ernest Dorsey
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1 UNIVERZITET U NOVOM SADU POLJOPRIVREDNI FAKULTET DEPARTMAN ZA A VETERINARSKU MEDICINU PREDMET: PATOLOŠKA FIZIOLOGIJA IOLOGIJA VEŽBA 4. PATOFIZIOLOŠKA DIJAGNOSTIKA POREMEĆAJA BELE KRVNE LOZE Dr vet. Marko R. Cincović
2 PLAN IZLAGANJA PREGLED LEUKOPOEZE PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE
3 PREGLED LEUKOPOEZE
4 PREGLED LEUKOPOEZE
5 PREGLED LEUKOPOEZE Mijeloblast
6 PREGLED LEUKOPOEZE Neutrofilni promijelocit
7 PREGLED LEUKOPOEZE Neutrofilni mijelocit
8 PREGLED LEUKOPOEZE Neutrofilni metamijelocit
9 PREGLED LEUKOPOEZE Neutrofil sa nesegmentiranim jedrom
10 PREGLED LEUKOPOEZE Neutrofil sa segmentiranim jedrom
11 PREGLED LEUKOPOEZE EOZINOFILNI PROMIJELOCIT
12 PREGLED LEUKOPOEZE EOZINOFILNI MIJELOCIT
13 PREGLED LEUKOPOEZE EOZINOFILNI METAMIJELOCITI
14 PREGLED LEUKOPOEZE NESEGMENTIRANI - ŠTAPASTI EOZINOFIL
15 PREGLED LEUKOPOEZE SEGMENTIRANI EOZINOFIL
16 PREGLED LEUKOPOEZE BAZOFILNI MIJELOCIT
17 PREGLED LEUKOPOEZE SEGMENTIRANI BAZOFIL
18 PREGLED LEUKOPOEZE MONOBLAST
19 PREGLED LEUKOPOEZE PROMONOCIT
20 PREGLED LEUKOPOEZE MONOCIT
21 PREGLED LEUKOPOEZE LIMFOCIT
22 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKOCITOZA LEUKOPENIJA NEUTROFILIJA NEUTROPENIJA EOZINOFILIJA EOZINOPENIJA LIMFOCITOZA LIMFOCITOPENIJA
23 TREBA ZNATI: SKRETANJE U LEVO: Ako se u perifernoj krvi javljaju mlađi oblici razvoja granulocitne loze, sa velikim brojom štapastih granulocita i metamijelocita, onda se takva pojava zove skretanje u levo. SKRETANJE U LEVO, UZ LEUKOPENIJU JE PROGNOSTIČKI LOŠ ZNAK!!!
24 TREBA ZNATI: Ako je skretanje u levo jače izraženo, tako da se u perifernoj krvi pojavljuju mijelociti u većem broju, uz pojavu promijelocita i mijeloblasta, onda se ta pojava naziva mijeloidna leukemična reakcija. Ovo stanje se razlikuje od mijeloproliferativnih bolesti na osnovu: 1. nalaza niskih vrednosti alkalne fosfataze u granulocitima 2. prisustvo filadelfijskog hromozoma 3. veliki % mladih ćelija granulocitne loze 4. eozinofilija i bazofilija u perifernoj krvi govori u prilog hronične granulocitne leukoze
25 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE Klasifikacija oboljenja bele krvne loze: LEUKEMIJE MIJELOPROLIFERATIVNE BOLESTI MIJELODISPLASTIČNI SINDROM LIMFOMI MALIGNA PROLIFERACIJA HISTIOCITNIH I DENDRITIČKIH ĆELIJA (APĆ) MULTIPLI MIJELOMI I DRUGE MONOKLONALNE GAMOPATIJE
26 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKEMIJE DEFINICIJA Leukemije su maligne bolesti krvi kod kojih postoji progresivno i nesvrsishodno razmnožavanje ćelija jedne od leukocitnih loza, koje je često praćeno povećanjem leukocita u perifernoj krvi, kao i povećanjem leukocitne mase u organizmu.
27 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKEMIJE Kod leukemijske proliferacije se zapaža izraziti poremećaj u diferentovanju i sazrevanju belih krvnih ćelija. Ovaj proces je izraženiji što je leukemija akutnija!!!
28 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKEMIJE PROLIFERACIJA LEUKEMIČNIH ĆELIJA U KOSTNOJ SRŽI POTISKIVANJE NORMALNIH MATIČNIH ĆELIJA U KOŠTANOJ SRŽI KVALITATIVNE I KVANTITATIVNE PROMENE U KRVNIM ĆELIJAMA Naročito u akutnim leukemijama ANEMIJA INFEKCIJA KRVARENJE
29 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKEMIJE Klinički stadijumi bolesti u Hroničnoj limfocitnoj leukemiji (po Rai-u) Stadijum 0 I II III IV Kliničke osobine Limfocitoza u perifernoj krvi i kostnoj srži Limfocitoza sa uvećanjem limfnih žlezda Limfocitoza sa uvećanjem jetre i/ili slezine Limfocitoza sa anemijom Hb ispod 100g/l Limfocitoza sa trombocitopenijom Rizična grupa Niska Srednja Srednja Visoka Visoka
30 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKEMIJE PODELA LEUKEMIJA PO TIPU ĆELIJA GRANULOCITNE LIMFOCITNE MONOCITNE PODELA LEUKEMIJA U ODNOSU NA BROJ LEUKOCITA U PERIF. KRVI LEUKEMIČNE SUBLEUKEMIČNE ALEUKEMIČNE PODELA LEUKEMIJA PO TOKU AKUTNE HRONIČNE
31 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LEUKEMIJE AKUTNE LEUKEMIJE L1 L2 ALL Akutna limfoblasna leukemija L3 (Burkitt's s leukemija) AML Akutna mijeloblasna leukemija M0 AML sa minimalnom diferencijacijom M1 AML bez diferencijacije M2 AML sa diferencijacijom M3 Akutna promijelocitna leukemija M4 Akutna mijelomonocitna (Naegeli's( acute leukemia) Akutna monoblasna leukemija M5 Akutna monocitna leukemija Schilling's AL, AMoL M6 Akut. Eritroidna leukemija, diguglielmo's syndrome M7 Akut. Megakariocitna leukemija
32 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE MIJELOPROLIFERATIVNE BOLESTI Klonalna proliferacija mijeloidnih progenitora. Mada od ovih progenitora nastaju Različite linije krvnih ćelija u okviru mijeloproliferativnih bolesti dolazi do poremećaja u jednoj liniji ćelija. Razlikujemo: Polycythemia vera (PV)- Predominantna proliferacija eritroidne loze Esencijalna trombocitemija (ET)- Predominantna proliferacija megakariocitne linije Myelofibrosis sa mijeloidnom metaplazijom (MF) (primarna ili idiopatska mijelofibroza)- Predominantna proliferacija megakariocitne linije, ali se nalazi i fibroza u koštanoj srži. Hronična mijelocitna leukemija (CML)- Predominantna proliferacija granulocitne, primarno neutrofilne linije. Ostale varijante: Hipereozinofilni sindrom Sistemska mastocitoza Mijeloproliferativna-mijelodisplastična bolest Juvenilna mijelomonocitna leukemija.
33 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE MIJELODISPLASTIČNI SINDROM Mijelodisplastični sindrom predstavlja proliferaciju mijeloidnih progenitora, a odlikuje se neefikasnom hematopoezom. Koštana srž je normocelularna ili hipercelularna, a posmatrana diferencijacija nije kompletna. Ćelije su displastične. Klasifikacija poremećaja (FAB klasifikacija): Refraktorna anemija Refraktorna anemija sa prstenastim sideroblastima Refraktorna anemija sa viškom blastnih ćelija (5-20% blast ćel. u srži) Refraktorna anemija sa 21-30% blastnih ćelija u koštanoj srži Transformacija u akutnu leukemiju sa preko 30% blastnih ćelija u srži Hronična mijelomonocitna leukemija
34 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE MIJELODISPLASTIČNI SINDROM KADA POMISLITI NA MIJELODISPLASTIČNI SINDROM? Veterinar mora pomisliti na mijelodisplastični sindrom u uslovima neobjašnjive anemije, neutropenije, trombocitopenije ili monocitoze, sa ili bez znakova insuficijencije koštane srži. U peničnim situacijama razmaz krvi i srži moraju biti detaljno pregledani.
35 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LIMFOMI Ne-Hodkinsovi limfomi B Cell Lymphomas Precursor B cell lymphoma Precursor B lymphoblastic lymphoma Mature B cell lymphoma Small lymphocytic lymphoma Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma, maltoma) Nodal marginal zone B cell lymphoma (monocytoid B cell lymphoma) Follicular lymphoma Mantle cell lymphoma Diffuse large B cell lymphoma Primary mediastinal large B cell lymphoma Intravascular large B cell lymphoma EBV positive senile large cell B cell lymphoma Primary effusion lymphoma Burkitt's lymphoma T/NK Cell Lymphomas Precursor T cell lymphoma Precursor T cell lymphoblastic lymphoma Blastic NK cell lymphoma Mature T/NK cell lymphoma Adult T cell leukemia/lymphoma Extranodal NK/T cell lymphoma, nasal type Enteropathy-associated associated T cell lymphoma Hepatosplenic gamma-delta T cell lymphoma Subcutaneous panniculitis-like like T cell lymphoma Mycosis fungoides Sezary syndrome Primary cutaneous anaplastic large cell lymphoma Peripheral T cell lymphoma, unspecified Angioimmunoblastic T cell lymphoma Anaplastic large cell lymphoma
36 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE LIMFOMI Hodkinsovi limfomi Nodular lymphocyte predominant Hodgkin's lymphoma Classical Hodgkin's lymphoma which includes Nodular schlerosing classical Hodgkin's lymphoma Mixed cellularity classical Hodgkin's lymphoma Lymphocyte-rich classical Hodgkin's lymphoma Lymphocyte-depleted classical Hodgkin's lymphoma
37 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE MALIGNA PROLIFERACIJA HISTIOCITNIH I DENDRITIČKIH ĆELIJA (APĆ) Langerhans cell histiocytosis (Histiocytosis X, Eosinophilic granuloma, Hand-Schiiller-Christian disease, Letterer-Siwe disease) Langerhans cell sarcoma (malignant neoplasma) Histiocytic sarcoma (malignant neoplasm) Erdheim-Chester disease Interdigitating dendritic cell sarcoma (malignant neoplasm) Follicular dendritic cell sarcoma (malignant neoplasm)
38 PATOFIZIOLOŠKI POREMEĆAJI BELE KRVNE LOZE MULTIPLI MIJELOMI I DRUGE MONOKLONALNE GAMOPATIJE Multiple myeloma Plasmacytomas Waldenstrom's macroglobulinemia Primary amyloidosis Heavy chain diseases IgG heavy chain disease IgA heavy chain disease IgM heavy chain disease Monoclonal gammopathy of undetermined significance (MGUS)
39 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 1. Pregled punktata limfne žlezde 2. Određivanje broja leukocita 3. Određivanje leukocitne formule 4. Pregled razmaza periferne krvi i koštane srži 5. Citohemijske osobine ćelija u bolestima bele loze
40 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 1. Pregled punktata limfne žlezde Napraviti razmaz uvećane limfne žlezde i posmatrati najpre na malom pa na velikom uveličanju. REZULTATI Uvećanje l.č. je bitan nalaz u benignim i malignim bolestima limfocitne loze. U toku baterijske infekcije vidi se infiltracija neutrofilnih granulocita, kada se vidi slika piogenog limfadenitisa. U TBC-u u vide se džinovske Langhansove ćelije. U malgnim bolestima može se primetiti da maligne ćelije u potpunosti osti ili delimično potiskuju zdravo tkivo limfnog čvora.
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42 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 1. Pregled punktata limfne žlezde Zahvatanje limfnih čvorova u limfomima i leukemijama Nodularna infiltracija Nodularna i difuzna infiltracija Difuzna infiltracija Parakortikalna infiltracija Infiltracija mantl zone
43 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 1. Pregled punktata limfne žlezde Neoplastične ćelije limfoma Mali limfocit Centrocit Pleomorfne ćelije Sezary ćelije Centroblast Ćelije Burkittovog NHL Imunoblast Reed-Sternbergova ćelija Limfoplazmocit i plazmocit Varijante RS ćelija Limfoblasti Histiociti
44 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 1. Pregled punktata limfne žlezde Morfologija Reed- Sternbergovi h ćelija u Hodginovom limfomu
45 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 2. Određivanje broja leukocita POTREBAN MATERIJAL Melanžer za leukocita,turkov rastvor,neubauerov hemocitometar, Mikroskop IZVOĐENJE Uvući kap krvi u melanžer za leukocite do ozanke 1. Obrisati vrh melanžera i potom uvući Turkov rastvor do nivoa 11. Tako je postignuto razblaženje 1:10. Lagano mućkati melanžer nekoliko minuta. Na komoricu staviti pokrovno staklo,tako da bude dobro fiksiran (trebaju da se vide Newtonovi obojeni koturovi)
46 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 2. Određivanje broja leukocita IZVOĐENJE Br.leu./µL L = N/4 x 10 x 10 Br leukocita u jednom kvadratiću Razređenje enje Br. LEU u 1mm³ U Litri - red veličina je 10^9 Neubauerova komorica
47 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 3. Određivanje leukocitne formule Izvaditi krv i napraviti razmaz periferne krvi, koji će biti posmatran na uvećanju od 1000x. Diferenciranje leukocita se vrši na 100 ili 200 ćelija. Broji se kao što je dato na donjoj šemi, a rezultate upisujemo prema p datoj tabeli.
48 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 3. Određivanje leukocitne formule Vrsta leukocita BROJANJE Ukupno % Neutrofili 65 65/100 Eozinofili Bazofili Limfociti Monociti
49 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 3. Određivanje leukocitne formule Vrsta Ukupan broj leukocita u 1l krvi Procenat pojedinih vrsta leukocita (%) Neutrofili Eozinofili Bazofili Limfociti Monociti (x 10^9) Čovek Konj Svinja Ovca Koza Pas Mačka Kokoš
50 DIJAGNOSTIKA LEUKOZE GOVEDA PREMA BROJU LEUKOCITA I DIERENCIJALNOJ BELOJ LOZI!!! Maksimalno 30% goveda ispolji limfocitozu Starost goveda Do 3 godine Preko 3 godine Leukozni ključ po Götzeu i sar. (1953) Broj leukocita x10^9 Do 12,0 12,0-18,0 18,0 Preko 18 Do 10,0 10,0-18,0 18,0 Preko 18,0 % limfocita Do Preko 75 Do Preko 75 Prosuđivanje Normalna Sumnjiva Leukozna Normalna Sumnjiva Leukozna
51 DIJAGNOSTIKA LEUKOZE GOVEDA PREMA BROJU LEUKOCITA I DIFERENCIJALNOJ BELOJ LOZI Leukozni ključ za članice Evropske ekonomske zajednice (1978) Starost u godinama Broj limfocita u 10^9/l periferne krvi Normalna Sumljiva Leukozna Do 11,0 11,0-13,0 Preko 13,0 Do 10,0 10,0-12,0 Preko 12,0 Do 8,5 8,5-10,5 Preko 10,5 Do 7,5 7,5-9,5 Preko 9,5 Do 6,5 6,5-8,5 Preko 8,5 Do 6,0 6,0-8,0 Preko 8,0 Do 5,5 5,5-7,5 Preko 7,5 Sličan je i Jugoslovenski leukozni ključ po Stamatoviću iz 1979.
52 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži
53 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži
54 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži Diferencijalna dijagnoza ALL prema obliku ćelija L1 L2 L3 Ćelije Male Uniformne Velike Nejednake Velike, Jednake Citoplazma Oskudna Umerena bazofilija Raznolike u količini i stepenu bazofilije Umereno obilna, Bazofilna, Izražene vakuole Jedro Pravlnog oblika Diskretni nukleolusi Nepravilnog oblika, Izraženi nukleolusi Pravilnog oblika, Izraženi nukleolusi
55 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži DIFERENCIJALNA DIJAGNOZA Mikromegakariocitna akutna leukemija (M7) Mikroblastična AML Hematogoni Reaktivna limfocitoza AL-L1, aspirat koštane srži Blastne ćelije tipa L1 su male, često po dimenziji slične normalnim perifernim limfocitima. Jedro je okruglo do ovalno sa jednako raspoređenim hromatinom. Nukleolusi načelno nisu vidljivi. Citoplazma je svetlo plava, sa rasutim i nevidljivim granulama. Površine ćelije je ravna ili sa finim membranskim projekcijama.
56 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži DIFERENCIJALNA DIJAGNOZA Blastična varijanta mantle Limfoma Blastična varijanta NK ćelijske leukemije Blastična varijanta NK/T ćelijske leukemije Male ćelije nehematopoeznih kancera AL-L2, L2, aspirat koštane srži Slične predhodnom osim što su 1,5-2 2 puta veće. Hromatin može biti grupisan, a nukleolus vidljiv. Membrana može biti sa raznim perforacijama.
57 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži DIFERENCIJALNA DIJAGNOZA Pleomorfni limfomi B i T ćelijske linije L2 ALL M0 i M1 AML Nehematološki tumori AL-L3, Burkitt's Leukemia/Lymphoma, aspirat koštane srži Nešto veće od AL-L1 i prilično uniformnog izgleda. Citoplazma je izrazito bazofilna, i sadrži lipidne vakuole. Moguće je primetiti nukleoluse.
58 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži AML-M0, aspirat koštane srži Dimenzija kao i neutrofili. Minimalna dierencijacija. Oskudna citolazma siva do svetlo plava. Visok odnos jedro:citoplazma.
59 AML M0 Cytochemistry The blasts are myeloperoxidase (MPO), Sudan black B, and nonspecific esterase (NSE) negative. Immunophenotype Stem cell and hematopoietic progenitor cell markers: CD34+, HLA- DR+, and TdT+ Myeloid lineage markers: CD117+, CD13+ and CD33+. Note that the cytochemistry stain for MPO is usually negative, but positive by immunophenotyping. T cell markers: CD7 is frequently positive, and is an example of what has been designated bilineage, biphenotypic, mixed lineage, or hybrid acute leukemias. Cytogenetics Although no signature abnormality has been identified, complex karyotypes and abnormalities of chromosomes 5, 7, and 11 are relatively common.
60 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži AML-M1, aspirat koštane srži Dimenzija kao i neutrofili. Minimalna dierencijacija. Oskudna citolazma siva do svetlo plava. Visok odnos jedro:citoplazma.
61 AML M1 Cytochemistry: The blasts are positive for myeloperoxidase (MPO) and Sudan black B, but negative for non-specific esterase (NSE). Immunophenotype: Stem cell and early hematopoietic progenitor markers: CD34+, HLA-DR+, Tdt+ Myeloid lineage markers: CD117+, CD13+, CD33+, MPO+
62 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži AML-M2, aspirat koštane srži AML-M2 sa eozinofilijom AML-M2 sa bazofilijom
63 AML M2 Cytochemistry: The blasts are positive for myeloperoxidase (MPO), Sudan black B, and chloroacetate esterase (reflecting the abortive maturation effort). The nonspecific esterase stain is negative in the agranular blasts. In acute basophilic leukemia, the diagnostic finding is metachromatic positivity with toluidine blue. Immunophenotype: Stem cell and early hematopoietic progenitor cell markers: CD34+, HLA-DR+, Tdt±. Myeloid lineage markers: CD117+, CD13+, CD33+ Lymphoid lineage markers: CD19, a B cell marker, is usually weakly positive, except in the acute basophilic subtype, and CD56± Cytogenetics: A signature cytogenetic abnormality, t(8;21) is seen in 30% or more of cases, and is seen more in the younger adults with this subtype. No signature cytogenetic abnormality has been identified in the acute basophilic subtype.
64 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži M3 AML with "angel wings" AML-M3, aspirat koštane srži
65 AML M3 Cytochemistry: The cells are strongly myeloperoxidase, Sudan black B and chloroacetate esterase positive. Immunophenotype: Stem cell and hematopoietic progenitor cell markers: CD34-, HLA-DR- Myeloid lineage markers: CD13+ and CD33+; CD117- and CD15-. Cytogenetics: The signature and diagnostic cytogenetic abnormality is the t(15;17), and has the same diagnostic significance as the t(9;22) in chronic myelocytic leukemia. It is a balanced translocation from the long arm of chromosome 17, the locus of the retinoic acid receptor-α gene, to a site on the long arm of chromosome 15 which is the locus for the promyelocytic leukemia (PML) gene.
66 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži Atipični, mladi eozinofil AML-M4, aspirat koštane srži
67 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži Mijeloperoksidaza bojenje Nespecifična esetraza bojenje AML-M4, aspirat koštane srži
68 AML M4 Cytochemistry: The clonal cells stain positive for the myeloid markers, myeloperoxidase (MPO) (d)and chloroacetate esterase, and the monocytic marker, non-specific esterase (NSE) (e) which is not inhibited by flouride. The cells with features of myeloblasts show more MPO positivity while those with features of monocytes show more NSE positivity- some cells stain with both. In the M4Eo subtype, the eosinophils stain like normal neutrophils (e.g. MPO+, Sudan black B+, NSE+) and not normal eosinophils. Immunophenotype: Stem cell and hematopoietic progenitor cell markers- CD34 positivity variable in the M4 subtype, but usually strongly positive in M4Eo subtype Myeloid lineage markers- CD13+, CD33± Monocyte lineage markers- CD14+, CD116±, CD11c±, CD4± Cytogenetics: The signature cytogenetic abnormality found in the M4Eo subtype is inv(16).
69 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži AML-M5, aspirat koštane srži
70 AML M5 Cytochemistry: In the M5a variant, the non-specific esterase (NSE) is positive (c), and myeloperoxidase (MPO) and Sudan black B are negative. In addition to a positive MSE stain, the Sudan black B may be positive in M5b variant. Immunophenotype: Stem cell and hematopoietic progenitor cell markers- CD34-, HLA-DR - Monocytic lineage markers- CD14±, CD116±, CD11c±, CD64±, CD68±, CD4±, and lysozyme± Myeloid lineage markers- CD33±, CD13±, CD117± Note: In general the M5b subtype shows more positivity with more mature monocyte markers. Cytogenetics: Cytogenetic abnormalities are found in most cases, but a signature abnormality has not been identified. Abnormalities involving the 11q23 are not infrequent, including those cases that occur following exposure to the topisomerase II inhibitors (VP-16 and VM-26).
71 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži Mijeloblast i eritroblast Atipični eritroblast Ćelije sa vakuolama AML-M6, aspirat koštane srži
72 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži PAS + bojenje AML-M6, aspirat koštane srži Bojenje Fe, prusko plavo
73 AML M6 Cytochemistry: The myeloperoxidase (MPO), Sudan black B, and nonspecific esterase (NSE) stains are negative in the proerythroblasts. The myeloblastic elements may be MPO, Sudan black B or NSE positive depending on the lineage of the myeloblastic leukemia component. The periodic acid-schiff (PAS) stain frequently shows large PAS positive cytoplasmic granules in the erythroblastic elements (g-h). Immunophenotype: Stem cell and hematopoietic progenitor cell markers- CD34-, HLA-DR- Erythroid lineage markers- The erythroid cells ae positive for glycoforin A Myeloid lineage markers- The myeloblasts are variably positive for CD13, CD33, and CD117. Monocytic lineage markers- Depending on the number of monolytic elements present, CD14, CD11b and 11a, CD4, and CD64 are variably positive. Cytogenetics: No signature cytogenetic abnormality has been identified in this subtype. Complex abnormalities, usually involving chromosomes 5 and 7 are most common, similar to those seen in the myelodysplastic syndromes.
74 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži Veliki nezreo megakarioblast AML-M7, aspirat koštane srži
75 AML M7 Cytochemistry: May show positivity with acid phosphatase, PAS and NSE stains. Myeloperoxidase and Sudan black B stains are negative. Immunophenotype: Stem cell and hematopoietic progenitor markers- CD34- and HLA-DR- ; a unique feature is that the leukocyte common antigen (CD45) is negative Megakaryocyte lineage markers- CD41 (glycoprotein IIb/IIIa) and/or CD61 (glycoprotein IIIA) usually positive. CD36 (glycoprotein IIIb) is positive. Myeloid lineage markers- CD13 and CD33 may be positive Cytogenetics: No signature cytogenetic abnormality has been identified. In infants under one year of age the t(1;22) often is present.
76 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 4. Pregled razmaza periferne krvi i koštane srži Kvalitativne nenormalnosti krvnih loza u mijelodisplastičnom sindromu Ćelijska loza Eritron Granulociti Megakariociti Monociti Blasti Periferna krv Dimorfija, makrocitoza, anizopoikilocitoza, bazofilno punktirani eritroblasti, retki dakriociti, displastični er-blasti Pseude-pelger neutrof., hipersegmentisani neutrof., Ring- neutrof. sa prstenastim jedrom, Döhleova tela, agranulisanost (kompletna ili segmentirana) Džinovski trombociti, fragmenti megakariocita, sivi (gray) trombociti, mikrotrombociti Česti promonociti, vakuolisani monociti, multilobarni monociti, dvojedarne dendritičke ćelije Koštana srž Eritroidna hiperplazija, ring (prstenasti) sideroblasti, displazni eritroblasti Promijelociti sa retkim ili odsutnim azorpfilnim granulama, hipo ili agranulirani mijelociti i neutrofili, džinovski metamijelociti i mikro i makro-neutrofili Mikro-megakariociti, hipo ili asegmentisani megakariociti, polinuklearni megakariociti Češći promonociti, dvo ili tro-jedarne dendritičke ćelije, sea-blue histiociti. Emperipoleza limfocita u retikuloendotelskim ćelijama Najčešće mali mononuklearni tipa I (1-3 3 azurofilne granule), tipa II (do 15 azurofilnih granula) i tipa III (izrazito granulisani blasti sa ili bez Auerovih štapića)
77 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE Kriterijumi za odvajanje hronične granulocitne leukemije (HGL) od o atipične HGL i hronične mijelomonocitne leukemije (CMML) HGL Vrhovi neutrofila i mijelocita+metamijelocita u leukocitnoj formuli Monociti čine manje od 3% leukocitne formule Znaci dishematopoeze ne postoje ili su minimalni Bazofilija Atipična HGL Promijelociti, mijelociti i metamijelociti čine više od 15% leukocita u perifernoj krvi Monociti čine više od 3% svih leukocita u krvi Postoji disgranulopoeza na nivou mijelocita, metamijelocita i zrelih neutrofila CMML 4. Pregled razmaza periferne krvi i koštane srži Promijelociti, mijelociti i metamijelociti čine zajedno manje od 15% leukocita periferne krvi (tipično je manje od 5%) Monociti čine više od 1x10^9/l leukocita Disgranulopoeza uobičajena ali ne i obavezna
78 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 5. Citohemijske i imunološke osobine ćelija u leukemiji Određivanje broja Leu i diferencijacije bele krvne slike, uz detaljno uzimanje anamneze i fizikalni pregled mogu ukazati na leukemički status. Ipak, konačna dijagnoza se postavlja imuno-citohemijskim metodama pomoću određivanja ćelijskih markera, primenom tehnike rozeta, imunofluorescentna tehnika, tehnika monoklonskih At i dr.
79 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 5. Citohemijske i imunološke osobine ćelija u leukemiji Citohemijska ispitivanja omogućuju dokazivanje prisustva specifičnih enzima i drugih materija u pojedinim ćelijama, jer bojenjem po Pappenheimu nije moguće lako diferentirati blastne ćelije i različite nezrele forme; što je značajno za dijagnostiku akutnih leukemija. Vrši se ispitivanje aktivnosti mijeloperoksidaze bojenjem sudan crnim B. Perjodnom kiselinom (Schiffova PAS reakcija) se boje ćelije u cilju dokazivanja glikogena. Dokazivanje kisele fosfataze, alkalne fosfataze, nespecifične alfa-naftol butirat esteraze, dokazanje muramidaze (lizozomske aktivnsti). Takođe e se vrši i diferenciranje ćelija na osnovu prisustva karakterističnih antigena na površini ćelija ili antigena u samoj ćeliji, pomoću monoklonskih At.
80 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 5. Citohemijske i imunološke osobine ćelija u leukemiji Vrsta leukemije M-1 M-2 M-3 M-4 M-5 M-6 Peroksidaza Sudan B +/ /+ + -/+ + PAS + + +/+ + +/ ± Hloracetat esteraza /+ + +/ ± + Nespecifična butirat- esteraza - ± ± +/ Kisela fosfataza +/- + +/+ + +/ ± Muramidaza - - ± +/++ ±/
81 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 5. Citohemijske i imunološke osobine ćelija u leukemiji Vrsta leukemije 0-ALL CALLA T-ALL B-ALL FAB PODELA L1-L2 L2 L1-L2 L2 L1-L2 L2 L3 PAS Kisela fosfataza Peroksidaza Sudan B Muramidaza - / / / / / / O-ALL = Ni B ni T akutna limfoblasna leukemija CALLA = akutna limfoblasna leukemija sa prisutnim zajedničkim antigenom T-ALL = Akutna T limfoblasna leukemija B-ALL = Akutna B limfoblasna leukemija
82 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 5. Citohemijske i imunološke osobine ćelija u leukemiji Imunofenotipska klasifikacija ALL Tip leukemije Membrana Citoplazma Jedro B-ALL Rana B-prekursor ALL CD19 CD22 TdT Common-B ALL CD19/CD10 CD22 TdT Pre-B ALL CD19 µ TdT B-ALL CD19/CD22 Ig / / T-ALL Pre T-ALL CD7 CD3 TdT T-ALL CD7/CD2 CD3 TdT
83 MARKERI PATOFIZIOLOŠKE PROCENE BELE KRVNE LOZE 5. Citohemijske i imunološke osobine ćelija u leukemiji Osnovni panel monoklonalnih antitela za dijagnostiku limfoma ANTITELO CD20 CD79a CD3 CD45RO CD30 Kappa Lambda ĆELIJA B-ćelije B-ćelije T-ćelije T-ćelije i nezrele B-ćelije RS ćelije B-ćelije B-ćelije BOJENJE Membrana Citoplazma Membrana Membrana Goldžijeva zona Citoplazma Citoplazma
84 PITANJA?
85
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