Haematology and blood transfusion Andrea Knight, PhD knight@med.muni.cz Definition & function nthe branch of medical science concerning blood and blood-forming tissues nstudy of morphology, diagnosis, treatment, prognosis & prevention npathophysiology nVariations from normal blood element counts, function 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 nliver 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) to plasma volume nMale average 44-47; female average 39-42 nPlasma contains water, ions, 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 volume nspin at 2,200 rpm for 20min, NO BRAKE nBuffy coat n n 17-01_WholeBloodSep_1 DA3C10FFU3 DA3C10FFU4 DA3C10FFU1 17-07_PartofaClot_1 nProcess in which blood changes from liquid to an insoluble clott nconversion of fibrinogen into fibrin by the serine protease enzyme thrombin. nCoagulation factors are generally indicated by Roman numerals nCofactors: calcium, phospholipids nFibrinolysis - blood clots are broken down & resorbed nTPA tissue plasminogen activator (serine protease) - conversion of plasminogen to plasmin nHyperfibrinolysis (excessive bleeding, increased vascular permeability) nHypofibrinolysis (thrombosis, embolism) nDVT, stroke, heart attack nDecreased platelet numbers (thrombocytopenia) CBC test = complete blood count 17-05_LeukcytePrcntg_1 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, packed cell vol hematokrit 0.40 - 0.52 0.37 - 0.47 RBC x1012/L 4.5 - 6.5 3.8 - 5.8 MCH, mean cell hemoglobin 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 Cell count 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 nErythrocytosis, Polycythemia: too many RBC 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 1962 Nobel Prize for Chemistry for his X-ray diffraction analysis of the structure of hemoglobin (share with John Kendrew) Heterotetramer HbA1 α2β2 96-98% HbA2 α2δ2 2% HbF α2γ2 this dominates until 6 weeks of age postnatally, Hb A dominates through life 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 Hematopoiesis nFormation of blood cells nOccurs mostly in red bone marrow nAll cells arise from the same pluripotent hematopoietic stem cells nMSCs form fat cells, osteoblasts, chondrocytes, fibroblasts and muscle cells… 20_08Figure_2-L 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 cells nE) monocyte diff. into MØ n 17-04_Leukocytes_1 CD numbers (clusters of differentiation) www.biogps.org 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, Osteoblasts, osteoclasts, Endothelial 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 nInduced pluripotent stem cells (iPSC) ntype of pluripotent stem cells that can be generated directly from a adult somatic cell nby Oct3/4, Sox2, Klf4, and c-Myc transcription factors (Yamanaka factors, Nobel prize 2012) nThe Advanced Therapies 2023 congress n n n n T cell therapy Century Therapeutics, Inc. Examination of bone marrow nInvasive procedure nBM sample obtained via biopsy or aspiration (sternum, pelvis) nUsed to newly diagnose & confirm suspected pathology nTo examine haematopoiesis nParallel to analysis of venous PB drawn 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 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 was available nShirley Nolan (1942-2002) and her legacy to start a donor register nCurrently over 750 000+ potential donors fully typed nImportant charity – please log-in & donate nResearch Institute & project Allostem nmajor EU grant involved 13 countries including CZ n(Prof. Bartunkova, Prague) nEssential clinical and research contribution to EBMT n n Our history | Anthony Nolan MHC proteins nMajor Histocompatibility Complex, locus on chr. 6 nHighly polymorphic nEnormous MHC allelic diversity 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 n 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 nStem 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 nAllogeneic Tx nHLA matching nRecipient’s immunosuppression nCalcineurin inhibitors (cyclosporin, tacrolimus) nCorticosteroids (methylprednisolone, dexamethasone, prednisolone) nCytotoxic immunosuppressants (azathioprine, chlorambucil, cyclophosphamide, methotrexate) n nFull ablative vs Reduced intensity conditioning (RIC) nRIC pioneered by Prof Stephen Mackinnon at University College London nNumerous clinical trials ongoing 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 (-196̊ C) for months/years nDecreasing the temperature as 1°C per minute over night at -80°C in the Mr Frostie containing isopropyl alcohol 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 n 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, 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 Disorders of Erythrocytes nPolycythemia: high RBC, increased Hb and hematokrit nAnaemia: low RBC nover 400 types of anaemia ndevelops when: nDecrease in the total number of red blood cells (RBC) nBlood loss – pregnancy, Acute: trauma and surgery, Chronic: many types of cancers (colon, bladder carcinomas), IBD patients nDecreased production of RBC - result of BM failure & differentiation of stem cells nIncreased destruction of RBC n nDecrease of the amount of haemoglobin and/or its reduced ability to carry oxygen n n 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 anaemia nvast majority never symptomatic! 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 nUnderstand the pathogenesis nGenetic alterations including translocations, mutations, SNPs… nLeukaemogenesis nHereditary factors (Fanconi A, Down sy) nRadiation, chemicals, drugs nVirus related (EBV, CMV) nRetrovirus mediated (HTLV-1) nAge related nOncogenes, tumour suppressor genes nUnderstand the pathophysiology nAble to list down the laboratory investigations required for diagnosis nTherapy & clinical trials nResearch ! n 17-06a_T-BLymphocyte_1 Stress ligands Immune surveillance Tumour evasion Shedding Trogocytosis Leukaemia and chromosomal translocations Translocation.gif 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% Leukaemia I. nheterogenous group of malignant disorders which is characterised by uncontrolled clonal and accumulation of blasts 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 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 nFAB classification nM0 Undifferentiated blasts nM1 AML without maturation nM2 AML with maturation nM3 Acute promyelocytic leukemia nM4 Acute myelomonocytic leukemia nM5 Acute monocytic leukemia nM6 Acute erythroblastic leukemia nM7 Acute megakaryoblastic leukemia n aml m1 L01_004a B09s009 B09s010a B09s012 L01s021 M03s012 L01s031 From lecture by Dr NJ Dodd BS967-7-SP: Session 6 courses.essex.ac.uk/bs/bs967/restricted/NJD%20Leukaemia.ppt Molecular biology of CML nPhiladelphia 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 nBCR/ABL mRNA, np210 ‘fusion’ oncoprotein as constitutively active tyrosine kinase resulting in the permanent activation of the RAS pathway n visualsonline.cancer.gov/addlb.cfm?imageid=7153 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 Mayer FN Brno nNew generations of the TKI – dasatinib, nilotinib 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