Haematology and blood transfusion Andrea Knight, PhD Definition & function nthe branch of medical science concerning blood and blood-forming tissues nstudy of etiology, diagnosis, treatment, prognosis & prevention npathophysiology nVariations from normal blood element counts nBleeding disorders - hemophilia nMalignant disorders – leukaemia, lymphoma, myeloma nHaemoglobinopathies nbone marrow and stem cell transplantation nblood transfusion & blood banking n n Organs & tissues nperipheral blood nbone marrow n nspleen nlymph nodes n(liver) n n n 17-02_BloodCllClssf_1 Scanning EM Light microscope Composition of blood nSpecialized connective tissue nBlood cells (elements) suspended in plasma nBlood volume: 5-6 litres in males and 4-5 litres in females nClinically important hematocrit n% of blood volume consisting of erythrocytes (red blood cells) nMale average 47; female average 42 nPlasma contains water, ions, 3x important proteins: albumin, globulins, fibrinogen n nSerum nBlood that is allowed to stand will clot nplasma without the clotting factors n n nDensity gradient centrifugation nSolution: Lymphoprep, Ficoll (1.077 g/ml) nlayered over with whole blood or bone marrow as 1;1 volumes nBuffy coat n nPracticals: small groups 4-6 students welcome to learn in our labs; to arrange by email: knight@med.muni.cz n 17-01_WholeBloodSep_1 nAnticoagulants: n-to prevent the coagulation (clotting) for: n renal dialysis n deep vein trombosis (DVT) n pulmonary embolism n myocardial infarction n ischemic stroke n-food supplements with blood-thinning effects: beer, papaya, cranberries n-encouraging clotting are avocado, spinach n nA key event in the blood coagulation is the conversion of fibrinogen into fibrin by the serine protease enzyme thrombin. nThrombin is produced from prothrombin, by the action of an enzyme, prothrombinase (Factor Xa along with Factor Va as a cofactor), in the final states of coagulation. nFibrin is then cross linked by factor XIII (Fibrin Stabilizing Factor) to form a blood clot. nThe principal inhibitor of thrombin in normal blood circulation is antithrombin. n n n 17-07_PartofaClot_1 Zástava krvácení nhemostáza má tři složky: n 1.reakce cév 2.reakce trombocytů 3.koagulace: srážení krve DA3C10FFU3 DA3C10FFU4 DA3C10FFU1 CBC test = complete blood count nRBC count (red blood) nWBC (white blood) nin thousands/cumm ndifferential WBC nPlatelet count in thousands/cumm 17-05_LeukcytePrcntg_1 Adult Normal Ranges in FBC nMCV: mean cell volume nPCV: packed cell volume (or haematocrit) – percentage of the blood volume that is made from RBCs nHypoxia can lead to increased amount of RBC nMCH: mean cell haemoglobin – average amount of haemoglobin per RBC nMCHC: mean cell Hb concentration nRDW: red cell distribution width n Parameter Male Female Haemoglobin g/dL 13.5 - 18.0 11.5 - 16.0 WBC x109/L 4.00 - 11.00 4.00 - 11.00 Platelets x109/L 150 - 400 150 - 400 MCV fL 78 - 100 78 - 100 PCV 0.40 - 0.52 0.37 - 0.47 RBC x1012/L 4.5 - 6.5 3.8 - 5.8 MCH pg 27.0 - 32.0 27.0 - 32.0 MCHC g/dL 31.0 - 37.0 31.0 - 37.0 RDW 11.5 - 15.0 11.5 - 15.0 Neutrophils 2.0 - 7.5 2.0 - 7.5 Lymphocytes 1.0 - 4.5 1.0 - 4.5 Monocytes 0.2 - 0.8 0.2 - 0.8 Eosinophils 0.04 - 0.40 0.04 - 0.40 Basophils < 0.1 < 0.1 http://www.pathology.leedsth.nhs.uk/pathology/ClinicalInfo/CommonTestsampInvestigations/FullBloodCo unt.aspx Variations from normal nLymphopenia : too few lymphocytes nNeutropenia: too few neutrophils nThrombocytopenia : too few platelets n nNeutrophilia: too many neutrophils nThrombocytosis: too many platelets nLeucocytosis : too many WBC Erythrocytes nAlso called red blood cells (RBC) nBiconcave discs and flexible nPlasma membrane but no nuclei or organelles nPacked with hemoglobin molecules nOxygen carrying protein n4 chains of amino acids, each with iron which is binding site for oxygen/CO2 nyoung RBC still containing ribosomes are called reticulocytes nLifespan 100-120 days 17-03_Erythrocyte_1 Parameter Male Female Haemoglobin g/L 135 - 180 115 - 160 RBC x1012/L 4.5 - 6.5 3.8 - 5.8 hb3col Hemoglobin nHeterotetramer nHbA1 α2β2 96-98% nHbA2 α2δ2 2% nHbF α2γ2 this dominates until 6 weeks of age n nAfterwards, Hb A dominates through life n Erythropoesis erytropoesis Methemoglobin nMetHb is the derivative of Hb, in which the iron of the heme group is oxidized from Fe2+ to Fe3+ nMetHb is no longer completely capable of reversibly binding O2 (brown) nMetHb forms continuously (present in RBC 1-2% c HB) nmust be reduced actively by normal red cell metabolism or by ascorbic acid ncyanosis & fatigue 10%, coma & fatal 50-70% nnitrates in food and water, medication-local anesthetics, G6PD deficiency n Leukocytes nA) Granulocytes nGranules, lobed nuclei nAll phagocytic nNeutrophil: Nuclei of 2-6 lobes nEosinophil: Nuclei bi lobed nBasophil: Dark purple granules nD) lymphocyte nLarge nucleus nT, B lineage nNK nE) monocyte diff. into MØ n 17-04_Leukocytes_1 Hematopoiesis nFormation of blood cells nOccurs mostly in red bone marrow nAll cells arise from same pluripotent hematopoietic stem cells nMSCs form fat cells, osteoblasts, chondrocytes, fibroblasts and muscle cells 20_08Figure_2-L Not shown are mast cells, osteoclasts, dendritic cells Bone marrow nRed marrow (medulla ossium rubra) nConsists mainly of haematopoietic tissue nSite of haematopoiesis (red and white blood cells, platelets) nYellow marrow (medulla ossium flava) nMade up of fat cells nWith age more red BM is converted to yellow BM nBM stroma nCreates a microenvironment nFibroblasts, MØ, adipocytes nOsteoblasts, osteoclasts nEndothelial cells nMesenchymal Stem cells (MSC) nPluripotent stem cells that can differentiate in vitro and in vivo into a number of cell types incl. osteoblasts, chondrocytes, myocytes, adipocytes n n n Examination of bone marrow nBM sample obtained via biopsy or aspiration nUsed to newly diagnose & confirm suspected pathology nTo examine haematopoiesis nParallel to analysis of PB drawn from a vein by a phlebotomist nInvasive procedure, not a routine n n n n n 17-08_RedBoneMarrow_1 Bone marrow harvest for transplantation nBM is collected (pelvis under general anesthesia) and infused back: nAutologous Tx - same patient nAllogeneic Tx nMatched sibling nMatched Unrelated Donor (MUD) nDonor – recipient compatibility (MHC/HLA alleles) nDonor registers around the world n Cell storage for transplantation nCells frozen in 5-10% DMSO/human serum nDMSO, Dimethyl sulfoxid nPrevents the formation of ice crystals during the freezing process nStored at liquid nitrogen (-196 °C) for months/years nDecreasing the temperature as 1°C per minute over night at -80°C in the Mr Frostie containing isopropyl alcohol The Anthony Nolan Trust nhttp://www.anthonynolan.org/ nstory of Anthony Nolan (1971-1979) nborn with a rare Wiskott-Aldrich syndrome nonly cure was Tx but no donor available nShirley Nolan (1942-2002) and her legacy to start a donor register nCurrently over 500 000 donors fully typed nImportant charity – please log-in & donate nResearch Institute & project Allostem nmajor EU grant involved 13 countries including CZ (Prof. Bartunkova, Prague) nEssential contribution to EBMT n n MHC proteins nMajor Histocompatibility Complex, chr. 6 nHLA, human leukocyte antigens nTransplant antigens to prevent graft rejection nHLA I. class (HLA-A, B, C) nExpressed on all nucleated cells nHLA II. class (HLA-DP, DQ, DR) nExpressed on cells of IS nMHC III. class ncomplement nProf. S Marsh at ANRI, President of the European Federation for Immunogenetics nAllele frequencies vary in different populations and ethnic groups Haematopoietic stem cell transplantation nProgenitor stem cell transplantation derived from: nBM nperipheral blood ncord blood nAutologous Tx nRequires extraction/apheresis of stem cells (HSC) nStored in the liq nitrogen nPatient undergoes high-dose chemo ± radiotherapy nEstablished as the second-line treatment for lymphoma (not for AML) nAllogeneic Tx nHLA matching nRecipient’s immunosuppression nfull ablative vs Reduced intensity conditioning (RIC) nRIC pioneered by Prof Stephen Mackinnon at University College London nNumerous clinical trials ongoing n Post HSCT nCytokine storm nGraft-versus-host disease (GvHD) as a major complication post SCT nT cells present in the transplant recognize the host’s (recipient’s) cells as foreign nMinor histocompatibility antigens nAcute within 100 days as major challenge to transplant mortality and morbidity (grade 1-4) nChronic as moderate to severe nSkin, liver, gut and GI tract, lung nDonor T cells mediate graft -versus-tumour effect (versus leukaemia, lymphoma or myeloma) n Graft – versus - tumour effect nGvL (versus leukaemia) nMost prominent in CML patients, (also in ALL) nGvM (myeloma) n SCT and CMV nHCMV cytomegalovirus nCommon beta-herpes virus (HHV5) nPrimary infection followed by a latent infection nVigorous immune response, persistent suppression of viral replication nCMV seropositivity associated with immune senescence of virus-specific CD4+ and CD8+ T cells (Prof. Paul Moss, Graham Pawelec, Mark Wills) nMultiple strategies to evade the host immune system nImmunocompetent vs immunocompromised host nDonor+ Recipient+ nD+ R- nD- R+ nD- R- n n Blood transfusion nprocess of receiving blood intravenously nto replace a lost blood component (red blood cells, plasma, platelets or clotting factors) ndonated blood processed/separated by centrifugation ntested for infections (HIV 1, 2, HTLV 1, 2, Hep B, C , syphilis and CMV) nstored in Blood Bank ncompatibility testing between D and R ntyping of recipient's blood determines the ABO blood groups and Rh status nsample tested for any alloantibodies that may react with donor blood 20_04Figurea-L ABO blood groups nIf a blood transfusion is given to a person who has antibodies to that type of blood, then the transfused blood will be attacked and destroyed (transfusion reaction) 20_04Figureb-L ABO blood group types nEurope: nA 45% nB 16% nAB 6% nO 33% 20_04Table-T Rh blood group system nconsists of 50 defined blood-group antigents nThe commonly used terms Rh factor nRh positive (85%) nRh negative (15%) refer only to the D antigen nWe either have or don’t have it on the surface of red cells nCondition of hemolytic disease of the newborn nIncompatibility between mother and the fetus Haematological disorders 800px-Hematopoiesis_simple_svg.png Disorders of Erythrocytes nPolycythemia: too many cells nAnaemia: not enough cells n n n n Anaemia - symptoms nAmong over 400 types of anaemia nDefined as a condition that develops when: nDecrease in the total number of red blood cells (RBC) nDecrease of the amount of haemoglobin and/or its reduced ability to carry oxygen nSymptoms: nfatigue and tiredness, pain, shortness of breath, fast heartbeat, cold hands nbone deformities (found in thalassemia major) nleg ulcers (sickle cell anaemia) nenlarged spleen nin children - poor performance at school nin elderly, in patients n n Anaemia - causes nBlood loss nCommon in women, pregnancy nAcute: trauma and surgery nChronic: many types of cancers (colon, bladder carcinomas), IBD patients nDecreased production of RBC - result of BM failure & differentiation of stem cells nPure red blood cell aplasia (PRCA) nAplastic anaemia; along with exposure to chemicals, radiation, drugs, viral infection nFanconi anaemia; 22x identified genes involved in DNA repair nImpaired/ faulty production of RBC and maturation of erythroblasts nDeficiency of Vitamin B12; Pernicious anaemia nIron deficiency anaemia; deficient heme synthesis nSevere type Myelophthisis; displacement of BM by malignant tumours or fibrosis nMyelodysplastic syndrome (MDS) nIncreased destruction of RBC Increased destruction of RBC classified as hemolytic anaemias nHereditary spherocytosis ndefect in RBC cell membrane caused by mutations in gene relating to proteins that allow RBC to change shape; then destroyed by the spleen nAntibody mediated nprimarily by IgG attacking the RBCs, leaving their Fc exposed to monocytes and MΦ, destroyed as spherocytes in the spleen nWarm autoimmune hemolytic anaemia (WAIHA) at 37°C nCold antibody induced anaemia (at 28-31°C) n n n n Disorders of Erythrocytes - Hemoglobinopathies nare inherited single-gene disorders ncharacterized by decreased and/or unstable haemoglobin nThalassemia nusually results in underproduction of normal globin proteins often through mutations in regulatory genes nBeta; subtypes major (both beta globin genes missing) and intermedia nAlpha; subtypes Hb H and hydropsis fetalis nMinor; either alpha or beta globin gene missing nSickle cell disease nEstimated that 7% of world's population (~420 million) are carriers nInheritance of two abnormal Β-globin gene (chr 11) nThe gene defect is a SNP (single nucleotide polymorphism) where GAG changes to GTG and results in glutamic acid being substituted by valine (E6V) n n n 17-10_NrmlSicklCompr_1 G6PD Deficiency nGlucose-6-phosphate dehydrogenase deficiency nenzyme involved in the pentose phosphate pathway nimportant in red blood cell metabolism nPerhaps most common, world-wide congenital abnormality n> 300 variants identified nX-linked inheritance nCommon G6PD deficient variants are associated with an acute intermittent hemolysis and anemia nvast majority never symptomatic! nMediterranean and others: may hemolyze with fava beans Anaemia - diagnosis and treatment nDiagnosis nBlood test, physical exam, symptoms and medical history nComplete blood count (CBC) to determine the number, size and volume of Hb nBlood iron level to indicate of body’s iron stores nVitamin B12 levels nDetection of rare types, RBC fragility, reticulocyte count, bilirubin nTreatment nIron and folic acid supplements nBlood transfusion n Disorders of Platelets nThrombocytopenia nnormal platelet count ranges from 150,000 - 450,000 per μL nplatelet count below 50,000 per μL noccasional bruising, nosebleeds, bleeding gums n!! internal bleeding nmany causes: decreased production or increased destruction (SLE, HIV) nVitamin B12 or folic acid deficiency nLeukaemia, MDS nDecreased production of trombopoietin by the liver in liver failure nBacterial, viral infections, sepsis nHereditary: Fanconi anemia nTreatment depending on the cause nCorticosteroids nPlatelet transfusion n Disease of the bone marrow nCongenital defects nAplastic anemia nMalignancies nLeukaemia nLymphoma nMultiple myeloma Congenital defects nDyskeratosis congenita (DKC) nis a rare progressive congenital disorder resembling premature aging nEssen. bone marrow failure syndrome nDKC typically develop between ages 5-15 years nis a result of one or more mutations in the long arm of the chr X in the gene DKC1 nHeiss NS, Knight SW, Vulliamy TJ, et al." May 1998, Nat. Genet. 19 (1): 32–38 n Haematological Malignancies - Objectives nDefine the disease: acute vs chronic leukemia… nClassify leukemia nUnderstand the pathogenesis nGenetic alterations including translocations, mutations (leukaemogenesis) nUnderstand the pathophysiology nAble to list down the laboratory investigations required for diagnosis nUnderstand the basic management of leukemia patients n 17-06a_T-BLymphocyte_1 Stress ligands shedding trogocytosis 17-06b_T-BLymphocyte_1 Leukaemia I. nDefinition: nheterogenous group of malignant disorders which is characterised by uncontrolled clonal and accumulation of blasts cells in the bone marrow and body tissues nExcessive production of WBC nOften non fully differentiated cells called “blasts” nWBC have abnormal function nResistant to apoptosis nExcessive proliferation nTumour microenvironment in the bone marrow nDisruption of normal haematopoesis in bone marrow n n Leukaemia II. nClassification nAcute nAcute lymphoblastic leukemia (T-ALL & B-ALL) nAcute myeloid leukemia nChronic nChronic myeloid leukemia nChronic lymphocytic leukemia Leukaemogenesis nDevelops as a result of a genetic alteration within single cell in the bone marrow nHereditary factors (Fanconi, Down sy) nRadiation, chemicals, drugs nVirus related nOncogenes, tumour suppressor genes nRetrovirus mediated (HTLV-1, EBV) nAge related n Leukaemia and chromosomal translocations Translocation.gif Mechanisms of Translocation nIonising radiation can caused breakage of the phosphodiester backbone of both strands of DNA nDouble-strand breaks are very efficiently repaired nPotential loss of genetic material nDouble-strand ends recognised as “foreign” DNA and destroyed nIf Double-strand breaks occur in two different chromosomes then possibility for incorrect repair taking place Frequent translocations B-ALL t(1;19) 5% B-ALL (in children) t(12;21) 22% T-ALL t(5;14) 20% T-ALL 1p32 deletion 25% AML t(15;17) 13% AML t(8;21) 7% CML t(9;22) 99% Techniques used in Molecular Diagnosis nMorphology nFlow Cytometry nPCR nFISH n 57 AML morphology aml m1 L01_004a B09s009 B09s010a B09s012 L01s021 M03s012 L01s031 L01s036 m0 m1 m2 m3 m4 m4eo m5 m6 m7 From lecture by Dr NJ Dodd BS967-7-SP: Session 6 courses.essex.ac.uk/bs/bs967/restricted/NJD%20Leukaemia.ppt M0 Undifferentiated blasts M1 AML without maturation M2 AML with maturation M3 Acute promyelocytic leukemia M4 Acute myelomonocytic leukemia M5 Acute monocytic leukemia M6 Acute erythroblastic leukemia M7 Acute megakaryoblastic leukemia FAB classification of AML Fluorescence in situ hybridization (FISH) nUse of fluorescently-labeled DNA probes to hybridize onto metaphase spread of chromosomes nAllows for the location of the probe on the chromosome to be identified n n FISH PML-RARA 3800465f2.jpg Fluorescence in situ hybridization investigation of cutaneous lesions in acute promyelocytic leukemia Wrede et al. Modern Pathology (2005) 18, 1569–1576. t(15;17) Acute Leukaemogenesis nOncogene can be activated by : n· chromosomal translocation n· point mutations n· inactivation n nIn general, several genes have to be altered to effect neoplastic transformation n n n Pathophysiology nAcute leukaemia cause morbidity and mortality through: nDeficiency in blood cell number and function nInvasion of vital organs nSystemic disturbances by metabolic imbalance Acute Lymphoblastic Leukaemia nCancer of the blood affecting the white blood cell LYMPHOCYTES nCommonest in the age 2-10 years nPeak at 3-4 years. nIncidence decreases with age, and a secondary rise after 40 years. nIn children - most common malignant disease n85% of childhood leukaemia n n Acute Myeloid Leukaemia nArise from the malignant transformation of a myeloid precursor nRare in childhood (10%-15%) nThe incidence increases with age n80% in adults n Molecular biology of CML n90% of patients have Philadelphia Chromosome (Ph) nt(9;22) balanced translocation ndisruption of the ABL (Chr 9) and BCR (Chr 22) genes nformation of two hybrid genes n5’BCR/3’ABL n5’ABL/3’BCR nOnly the BCR/ABL hybrid gene is active nBCR/ABL mRNA np210 ‘fusion’ protein t(9;22) translocation in CML nci-vol-7153-72.jpg visualsonline.cancer.gov/addlb.cfm?imageid=7153 Why is p210 (BCR/ABL) protein oncogenic? nABL nprotein tyrosine kinase ninteracts with the adaptor protein CRKL nBCR n interacts with the adaptor protein Grb2 nCRKL and GRB2 activates the RAS growth stimulation signalling pathway nInactivation of pro-apoptotic protein (BAD) nUp-regulation of the anti-apoptotic protein (BCLXL) np210 is a constitutively active tyrosine kinase resulting in the permanent activation of the RAS pathway New CML Treatment nDesign compounds that specifically target the p210 protein np210 is CML specific nimatinib (Gleevec, Glivec, STI571) nspecifically inhibits the ABL kinase nimatinib inhibits the growth of CML cells in culture nProgression-free survival at 24 months is 87% nProf John Goldman Hammersmith Hospital London nProf John Barret NIH Washington nProf Francois Mahon Bordeaux nProf Ráčil, Prof Mayer FN Brno n n n Provan and Gribben Fig 7.4.jpg Molecular Haematology Provan & Gribben Chronic lymphocytic leukaemia nMost common leukaemia in the Western countries nlymphocytosis of > 5000 cells/μl for n> 3 months nFlow cytometry of peripheral blood (phenotype CD19, CD5, CD23) nBone marrow biopsy nStaging according to Rai (I-IV) nMutated IgVH nDel11q (ATM) nDel17p nDel13q (RB1) n+12 nTP53 nProf Michael Doubek, IHOK, FN Brno n Multiple Myeloma nB cell maligancy of plasma cells CD38+CD138+ in the bone marrow nPre-malignant stage: nMGUS – monoclonal gammopathy of undetermined significance nProgression of 1% per annum nBone marrow biopsy nTherapy (IMIDS) nProf. Roman Hájek FN Ostrava n Questions n