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Hematopathology Learning outcomes: After this session you should be able to: 1.Have a basic understanding of hemopoiesis and blood formation. 2.Basic principle of erythropoiesis. 3.General hematological investigations particularly to anemic patient. 4.Definition, classification, and clinical prese...

Hematopathology Learning outcomes: After this session you should be able to: 1.Have a basic understanding of hemopoiesis and blood formation. 2.Basic principle of erythropoiesis. 3.General hematological investigations particularly to anemic patient. 4.Definition, classification, and clinical presentations of anemias. 5.Iron metabolism and iron deficiency anemia. 6.Differential diagnosis of hypochromic anemia. 7.B12, FA metabolism and megaloblastic anemia. Blood formation mature Blood is a suspension of non-dividing end stage cells in plasma. Haemopoiesis: is process of proliferation, differentiation and mature blood cell release. It includes erythropoiesis, leucopoiesis and thrombopoiesis processes. Sites of hemopoiesis: Fetus 0-2 m (yolk sac) 2-7 m (liver, spleen) 5-9 m (Bone marrow) Infant Bone marrow (all bones) Adult Bone marrow (axial skeleton and proximal end of long bones). Structure of BM: we have 2types ofcells in bonemarrow in hemorgy • Bone marrow consist of hemopoietic and stromal cells. Hempoietic tissue including stem, progenitor, precursor and mature cells. C.in siiEetI song • Stem cells can give rise all cell lines, able to self-renewal (proliferation) and differentiation, present in few and constant number and require certain growth factors and suitable microenvironment to work. grid fencedcangives many 19th fromsame stemcells • Progenitor cells present in early and late types, and consider intermediate stage of multi-lineage or uni-lineage cells with increase differentiation over the proliferation. astantberecognized byonlymorphology If • Precursor cells specific for single line and morphologically recognizable in the bone marrow. refognize minister Stem cells Progenitor cells Precursors cells Mature cells Structure of BM: Red marrow is the active marrow and considered as the site of hemopoiesis, while yellow marrow is the inactive marrow consist mainly of fat cells. Fat marrow (yellow marrow) is able to reverse into red marrow (active marrow) in many diseases when function of active marrow is required. Percentage of active hemopoiesis will differ according to the age and gradually reduce with progression of the age in compare with percent of fat tissue: • In adult (fat: haemopoietic tissues) 50%:50% red • In child (fat: haemopoietic tissues) yellow 25%:75% • In elderly (fat: haemopoietic tissues)sea 75%:25%na infancy AY FYI by age Extramedullary hemopoiesis means process of hemopoiesis occuring outside the BM (mainly in liver and spleen) , it occurs in certain conditions as in sever chronic hemolytic anemia and BM fibrosis. Fear Hemoro an physiology but in the Pal 82 Pr so that Erythropoiesis It mean formation of RBC and require 7 days for complete new RBC formation. This process requires stem, progenitor and precursor cells for complete formation of mature RBC. Important cells in the BM for this process are precursor cells which including: lossofnudie Reduction in size recognitable •Proerythoblast. •Early normoblasts. •Intermediate normoblasts. •Late normoblasts. •Reticulocytes. d size The cell gradually loss their nucleus until formation of reticulocyte as last precursor cell without nucleus. E hemoglobin in now •Reticulocytes: non nucleated red cells with diffusely basophilic cytoplasm due to remaining ribosomal RNA, still able to synthesis the Hb, slightly larger than mature RBC, it remains in BM about 2 days then release to circulation and remains 1-2 days to complete their maturation in the spleen. Normal range in peripheral blood is 0.5-2.5 %. •Mature RBC: non nucleated cells of 8µm in diameter, biconcave, flexible, able to pass through microcirculation with minimal diameter 3.5 µm. Life span 120 days, generate energy as ATP and generate reducing power. •Normoblast (precursor of RBC) normally present in the BM and not present in peripheral blood and when appear called nucleated RBC (NRBC). Control of erythropoiesis: Mainstimulus is PHYEE.fm differ sina.ie iiit mainlyinkidnefd n • Functional feedback: achieved by Erythropoietin (Epo). • Certain hormones, like growth hormone and androgen enhance erythropoiesis, while estrogen suppresses erythropoiesis. • Nutritional factors like FA, B12, iron, B6, B2, vitamin C and E. Erythropoietin (Epo): It is a glycoprotein produced mainly by the kidney in the adult, stimulates red cell progenitors and precursors cells for RBC formation from the BM. Secretion of Epo is triggered by reduced oxygen tension of blood: • Reduced Hb (anemia). • Hypoxia (cardiac and lung disease). • Damage to renal circulation. Hypoxia Epo of great clinical significant in many disorders using recombinant Epo like end stage renal disease and anemia of chronic disease. Initial hematological investigations ith commeg.BY Complete blood count; include Hb level, WBC count, platelets count, RBC indices and WBC differential count. Blood film stained usually with Leishman stain, to examin red cell morphology, leucocyte morphology, leucocyte differential count and platelet morphology. inflammation is good for but 1 specific no ESR (erythrocyte sedimentation rate): red cell sedimentation depends mainly on plasma proteins as fibrinogen and other acute phase proteins that increase the red cells rouleaux and extend of their sinking. oneof firefight É nÉsé protein Erythrocyte sedimentation rate (ESR) is a blood test. It measures how quickly erythrocytes, or red blood cells, separate from a blood sample that has been treated so the blood will not clot. During this test, a small amount of your blood will be put in an upright tube. casesofAnemia n every have to hatsthe knot Reticulocytes response it activeor not count (Retic count) to assest 1 to •Reticulocyte: are young, immature non nucleated red cells qq.e.my Bondffgrow containing remnant RNA. m • Increase retic count occur in hemolytic anemias, hemolysis and recovery state from anemia. •Decrease retic count occur in bone marrow failure. Retic count: is the estimation of number of reticulocytes among mature peripheral RBC detected by a special stain, normal range 0.5-2.5 %. Blde methelin Blood film 6ede9 PMdsasIw it we retic ftp.i Bone marrow examination: There are two a six if types of marrow procedures: 1.Bone marrow aspirate: done from iliac crest or sternum, in which a specimen is aspirated using a wide bore needle from the active marrow, smeared, stained and then examined for any abnormalities. 2.Bone marrow biopsy: here a core of bone marrow tissue is taken, processed and stained as in histopathological specimens. Main indications of BM examination: 1.Marrow infiltration with leukemia, lymphoma, secondary carcinomas and myelofibrosis. 2.Cytopenias of unexplained causes: leucopenia, thrombocytopenia, anemia. Bone marrow examination Bone marrow aspiration Bone marrow biopsy fit Definition of terms in RBC disorders CBC • Normocytic: normal size (normal MCV). Bloodfilm • Normochromic: normal Hb amount (normal MCH), RBC showing central pallor less than 1/3 of cell diameter. __ • Hypochromic: decrease in Hb amount (decrease in MCH), central pallor more than 1/3 of cell diameter. RDI • Anisocytosis: variation in RBC size as microcytic (decrease in MCV), macrocytic (increase in MCV). • Poikilocytosis: variation in RBC shape as sherocytes, target, tear drop, sickle, fragmented, oval, rod cells, etc.. • Polychromasia, rouleaux formation and agglutination of RBC. m É Poikilocytes riskier innate Red cell indices • Hemoglobin (normal range for male 13.5– 17.5 g/dl, female 11.5-15.5 g/dl). • Packed cell volume (PCV) also called Hematocrit (Male 40-52 %, Female 36-48 %). • RBC count (Male 4.5-6.5 x 1012/L, Female 3.9-5.6 x 1012/L). a I • • • • Am MCV: mean cell volume MCH: mean cell Hb MCHC: mean cell Hb concentration RDW: red cells distribution width five to leds.ie (80-95 fl). (27-34 pg). (32-36 g/dl). (12-15 %). Example of complete blood counts and blood film Red blood cells indices Platelets count: ×109/L. 332 Hb: 14.1 gm/dl. Total WBC count: 9.7 × 109/L PCV (Hematocrit): 43.2 % Differential count (%) I RBC: MCV: MCH: MCHC: RDW: 88 fl 780 595 28 pg 24 34 32 gm/dl. 31 32 It Retic count: % shown Not 4.8 × 10 /L 12 L 18 % Neutrophils: Lymphocytes: Monocyte: Eosinophils: Basophiles: 190 400 62 35 3 0 0 _noxini.EE ESR: 8 mm/hr. Blood film morphology RBC: Normocytic normochromic red cells. WBC: Look normal in total, differential count and morphology Platelets: Look normal in count and Red blood cells disorders (Anemias and Polycythaemia) Normal Hb is: • Male: 13.5-17.5 gm/dl • Female:11.5-15.5 gm/dl (PCV 40-52 %) (PCV 36-48%) Anemia: decrease in Hb concentration below normal range for age and sex. tPolycythemia: increase in the Hb concentration above the upper limit of normal for age and sex In male In female Hb <13 g/dL or PCV < 40% Hb <11.5 g/dL or PCV < 36 % I. II. III. Classification of Anemia Etiological classification. Morphological classification. Classification based on reticulocyte response. Anemia Etiological Classification: imbalance between Impaired RBC production: 1.Iron deficiency anemia (IDA). 2.Megaloblastic anemia (MBA). 3.Aplastic anemia (AA), pure RC aplasia (PRCA). 4.Anemia of chronic disease (ACD). 5.Myelophthesic anemia. severe anaemia on son 6.Sideroblastic anemia. Increase in distruction or loss : 1.Bleeding (anemia of acute blood loss). 2.Hemolysis (HA)Intrinsic and extrinsic defects. production f.IE onie and loss IInsis Morphological classification ofFBC Hypochromic Microcytic anaemia: Iron deficiency anaemia Thalassemia Sideroblastic anaemia Lead poisoning Normochromic normocytic anaemia: Anaemia of chronic disorder Hemolytic anaemia Stem cells defects Macrocytic normochromic anaemia Megaloblastic anaemia Myelodysplastic syndromes nonnegff Liver disease Drugs Alcoholism use classificent nemia Classification of anemia ( Retic count low or normal ) anemia ( Retic count increase ) Normal BM defect Like: 1.Iron deficieny anemia 2.Megaloblastic anemia 3.Aplastic anemia 4.Anemia of chronic dis. 5.Anemia associated with BM infilteration. Peripheral blood loss Like: 1.Bleeding 2.Hemolysis (hemolytic anemia) ghygynemia manifests differently in People iii s isiii.iiiiinic General clinical features of anemia: 4 presenceofotherdiseases •Symptoms: easy fatigability, palpitation, dyspnea, headache, tinnitus, anorexia, nausea. •Signs; pallor, tachycardia, jaundice and splenomegaly (in hemolytic anemia), delay growth and sexual maturation in chronic anemia. 1 General signs General initial laboratory investigations of anemia: (1)CBC, blood film and retic count should be done for all cases presented as anemia. (2)ESR in recommended cases. (3)BM examination in certain conditions (4) special investigations done according to suspected cause of anemia, like serum iron indices, Hb electrophoresis, coomb's test, etc. presen common most Iron Deficiency Anemia Iron metabolism Iron is essential for many metabolic processes in the body. Total body iron 3-5 gm, more in male than female. Iron is absorbed in the duodenum and less in jejunum with mean 1mg/day and may increase in demand (not > 3-4 mg/day). Dietary sources include meat especially liver , egg yolk, some green vegetables and dairy products. Iron metabolism Generally iron in the body present in main 3 pools are: (1) Functional pool: (65-70%) •Hemoglobin: New RBC formation required 30 mg iron/day mainly derived from breakdown of old RBC. Each unit of blood (450ml) contains 200 mg iron. •Myoglobin, mitochondria and iron containing enzymes. (2)Storage pool: (20-25%) present in form of ferritin and hemosiderin that present in RES : BM, liver and spleen. (3)Transporting pool: (0.1%) plasma iron carried by iron transporting protein (Transferrin). 000 i.EE Iron store Ferritin > is water-soluble iron –protein complex > it contain up to 20% iron of its weight . > it is not visible by light microscopy. Hemosiderin can'tbing8 in > is partially degraded and aggregated ferritin. > is insoluble in water protein-iron complex can be seen > it contain up to 37% of its weight iron > is most commonly seen as golden-brown granular material in macrophages and is readily demonstrated to be iron by a Prussian-blue stain. i 0 •Hepcidin: is a polypeptide produced by liver and considered the major regulator of iron balance . •It inhibits iron release from macrophages, intestinal epithelial cells and hepcide others be •Decreased production in response to iron deficiency, hypoxia, and ineffective erythropoiesis need •Increased production in inflammation. Ye 19 four iron's plea more •Iron loss is mainly by shedding cells of skin and mucosal lining of GIT, plus and sweating z •Increase requirement occur in pregnancy, lactation, prematurity, menses and periods of growth. dietton ferrous 6 66 56 8 6 i.is mitotho 1 it i iiiii c.in are an e Fe 01075m Ex 56 iii 52 inpregnancy Iron deficiency anemia Iron deficiency anemia is the most common type of anemia and is more common in developing countries. Causes of iron deficiency: 1.Nutritional: common cause of iron deficiency in developing and underdeveloped countries, especially if inadequate intake is coupled with increased demand. 2.Inadequate absorption: from many causes of malabsorption e.g. Celiac disease.jejuna 3.Chronic blood loss: Common cause in adults, and most likely from the gastrointestinal tract, or in females bleeding from genital tract. 4. Increase in demand: prematurity and infancy (3-6 months), pregnancy, lactation, menstruation and adolescence period. Clinical features: • Clinical features related to underlying pathology. • General signs and symptoms of anemia. • Mucosal changes in severe IDA as mouth soreness, painless glossitis, angular stomatitis and nail changes as brittle, ridged, and spooning (koilonychias) of nails. • Pica (craving to eat unusual substances like clay and ice). • Mental development disturbance, irritability, poor cognition may occur in children. • In sever long standing IDA, patient may develop dysphagia (Plummer Vinson syndrome) which is a triad of dysphagia (pharyngeal web), glossitis and IDA, it has risk of CA, more in c It women, of unknown etiology. Pallor Nail changes Pale conjunctiva Oral changes AbIron Stages of iron deficiency anemia: 8 transferien Initially depletion occurs in iron store then the BM will be affected and finally the anemia become obvious. only 1.Depletion of iron stores: no iron in store (low serum ferritin) and no anemia. 2.Iron-deficient erythropoiesis: Low serum iron and high TIBC and still no significant anemia yet. stillCBI is Normal 3.Iron deficiency anemia: Low Hb, MCV and MCH. Parameter first Laboratory findings in IDA: 1. Hematological findings: •Reduce in Hb, according to the degree of anemia. •Reduce PCV, MCV, MCH, and MCHC. •Retic count is low. •WBC usually normal, and platelets counts may increase. e film: hypochromic microcytic, anisocytosis. •Blood •Decrease or absence in marrow iron store which can be detected by Perl's stain/Prussian blue stain which is diagnostic, but the BM examination is not an indication in suspected case of IDA. If 2. Biochemical findings: •Serum iron reduced. •Total iron binding capacity increased. •Transferrin saturation reduced <15%. •Serum Ferritin reduced which reflects storage iron. 3. Investigations for suspected underlying causes. Normochromic Normocytic (normal blood film) Hypochromic microcytic (blood film in IDA) viiiMiran Iron stain on marrow Iron is represented by blue granules f i Differential diagnosis of hypochromic microcytic anemias include four main types of anemia are: •Iron deficiency anemia. •Thalassemia, result from globin chain defect. •Anemia of chronic disease. •Sideroblastic anemia, result from defect in protoporphyrin ring. a in Anaemia of chronic disease • • • • • • • • we said whentheres ii iiii iiq a.nu One of the most common types of anaemia that associated with different diseases like infections, chronic inflammatory diseases, chronic renal failure and malignant diseases. Pathogenesis is related to decreased release of iron from macrophages (high hepcidin), reduced RBC life span, inadequate erythropoietin response to anaemia. Mild non-progressive anaemia (rarely Hb < 9 gm/dl), and the severity related to illness severity. Normochromic normocytic or mildly hypochromic red cells. Low serum iron and low TIBC. usuayinirndificiency is low Normal or high serum ferritin. Whenserumiron buthere high be will High C-reactive protein and high ESR. Type but itsnot irons red High serum Hepcidin level. No the willbe to tianya.fi available soTIBS ifeng.EE Megaloblastic anemia • Anemia due to impaired DNA synthesis, result mainly from B12 and e Folate deficiency. • All haemopoietic cell lines (erythropiesis, granulopoiesis, megakaryopoiesis) can be affected resulting in severe anemia and even pancytopenia. reduce productionof An celllines WBCRBCplatelets • The hallmark is enlargement of BM erythroid precursors (megaloblast) giving rise to abnormal large RBC in blood (macrocytes), in addition to giant granulocytic precursors in BM yielding hypersegmented neutrophils in peripheral blood. we • It is a serious disease that can cause neurological and psychiatric abnormalities which are preventable and reversible in the early stage of disease. maturation of divide can't to Grow but able erythroblast relativeto cytoplasm nucleus use of delayed B12 (cobalamin) • Forms of B12:Methylcobalamin is the main form in human body, Cyanocobalamin, form use in investigations, Hydroxycobalamin, form use in treatment. • Human is unable to synthesis B12 so requires intake. • It is present only in food of animal origin specially meat, liver and fish and less in dairy products. • It is a heat stable vitamin. • Vegetables and fruits free of B12.mind • Daily requirment is about 1- 2 µg. • Body store about 2-3 mg. (2-4 years) Functions of vitamin B12 B12 in involved in two important Biochemical reactions: 1. Conversion of Homocystiene to Methionine, and this is essential for maintaining the active form of folate inside the cells to be available for DNA synthesis. 2. Conversion of Methylmalonyl-Co A into succinyl-Co A, and this requires Deoxyadenosyl-cobalamin form, and involved in the synthesis of myelin in the CNS. thatswhy the neurological complication of I associated megaloblasticAnemiais only deficiency deficiency Not withBI folic Absorption of B12 in the ileum pass through some steps: • In stomach: B12 bind to R-binder which is produced by saliva and gastric juice. to protect Brfrom brakingTown • In duodenum: Digestion of this complex occurs by pancreatic enzymes, then the B12 will bind to Intrinsic factor (IF) which is a secreted from stomach parietal cells. • In terminal ileum: IF bind to specific receptor (Cubilin) for absorption and B12 appear in portal circulation after 6 hrs attached to transcobalamin-II (TC II). Go take BR to Bone marrowfor heckh thesis Causes of vitamin B12 deficiency: (1) Malabsorption (main cause) like: oGastric causes, as pernicious anemia, gastrectomy. oIntestinal causes, as ileal resection, blind loop syndrome, (2) Dietary deficiency: as in vegans (3) B12 abnormal metabolism as TC II and IF deficiency. Folic acid • Human is unable to synthesis FA, most foods contain FA, specially liver, green vegetables, fruits and nuts, it is destroyed by heating. • Absorption takes place in upper half of small intestine (deudenum and jejunum) • Daily requirment about 100-150 µg. morhishfranp.in • Body store 10-12 mg.(4 months) • Function of folate As a co-enzyme in purine and pyrimidine synthesis Causes of FA deficiency: (1)Inadequate dietary intake (common cause due to low storage state): old age, poverty, alcoholism, psychiatric disturbance. (2)Malabsorption like celiac disease. needed is (3)Increase utilization: Mff -Physiological as pregnancy, lactation, prematurity -Pathological as hemolytic anemia, malignancy, inflammatory disease. 1 7 (4) Antifolate drugs as anti-convulsants. (5) Liver diseases. Biochemical basis of MBA: •MBA is a type of anemia characterized by impaired DNA synthesis. •DNA synthesis requires polymerization of 4 deoxyribonucleoside triphosphates, and FA deficiency impaires thymidylate synthesis which is a crucial step in DNA synthesis as this step need methylin THF polyglutamate. •B12 required to achieve the active form of folate inside the cells. •So deficiency of any vitamin will lead to impair in DNA synthesis. if EE imonuenn essentialp fgi i dgtf Clinical features of MBA • May be asymptomatic. • Usually insidious onset with signs and symptoms of anemia. • Mild jaundice (due to intramedullary destruction of red cell deÉÉÉf precursors). theorknormid destructingBonemarrow • Epithelial tissue changes as glossitis and angular stomatitis. • NTD (neural tube defect ) in FA deficiency and may occur with B12 deficiency. Ñ D • Neurological manifestations only with B12 deficiency due to defect in methylation of myelin, it is usually symmetrical affecting lower limbs more. • Psychiatric abnormality. Pernicious anemia • It is MBA due to B12 deficiency • It is caused by autoimmune attack on gastric mucosa • characterized by gastric atrophy, with plasma cell and lymphoid infiltrate in lamina properia in the wall of stomach • reduced or absent intrinsic factor with achlorhydria. • It is more in females, mostly a disease of the elderly, more in patients with autoimmune mediated disorders like autoimmune thyroid disease. • Increased risk of gastric carcinoma 2-3%. • Not common in Iraq, common in north Europe. • Mostly it is autoimmune in nature with presence of serum antibodies like anti-IF Ab, anti-parietal cells Ab and anti-gasrtin receptor Ab. Laboratory findings of MBA: Hematological findings: imacroiy.li •Variable degree of anemia, may be severe Hb < 6 gm/dl. •MCV increased >100 fl, may be up to 135 fl. •Increase MCV, MCH, and normal MCHC. •Increase RDW. •Low retic count. •Leucocytes may be reduced, platelets may be moderately reduced (pancytopenia). •Blood film: oval macrocytes, hypersegmented neutrophils, leukoerythroblastic picture may found. c E Other investigations: • low Serum B12, low serum FA. • Therapeutic trials of both vitamins and assess the response by increase Hb and retic count after several days. • High S. LDH, indirect hyperbilirubinaemia due to intramedullary destruction of red cell precursors. . Search and exclude causes for B12 and FA. • Serum Ab measuring and gastric biopsy in suspected cases of pernicious anemia. • BM findings: Hypercellular marrow, Erythroid hyperplasia, with megaloblastic maturation. Giant metamyelocytes, Abnormal megakryocyte nuclear segmentation Normochromic, Anisocytosis poikilocytosis, macrocytosis, tear drop cells oval Hypersegmented Neutrophiles cellsare very an egghead Normoblastic & Megaloblastic Erythropoiesis Response to treatment: • • • • Initial correction of patient behavior and appetite within 1-2 days. Retic count increase with peak level at 7 days. was Hb level increase 1 gm/dl /wk . if thetreatment Peripheral neuropathy may partially improve but spinal cord damage is irreversible. good