Complete Blood Count (CBC, Hemogram) Doç. Dr. Jale Çoban Klinik Biyokimya Uzmanı Topics: 1- Blood 2- Blood Cells 3- Complete Blood Count (CBC) 4- CBC Parameters 5- Aim of CBC Testing BLOOD As a vital component, blood has 2 phases: - Cellular phase and - Liquid phase All metabolic substances are present homogenously in this liquid phase. Whole Blood Centrifuged with Anticoagulant BLOOD COMPONENTS • • Plasma (Vs. Serum) – Proteins – Lipids – Carbohydrates – Ions – Water • Cellular • Erythrocytes • Leukocytes • • Polymorphonuclear or granulocytes • Neutrophil • Basophil • Eosinophil • Monomorphonuclear or agranulocytes • Monocytes • Lymphocytes • Platelates The hierarchy of haematopoiesis 4 Complete Blood Count Parameters: Complete Blood Count: Analysis of blood cells Complete Blood Count: Analysis of blood cells Complete Blood Count (CBC, Hemogram) Red blood cells White blood cells Platelets Erythrocyte and indices Erythrocyte and indices • RBC –Red blood cell –Number of all erythrocytes • Hgb – Hemoglobin concentration • HCT – Hematocrit value: Erythrocyte ratio of total blood volume • MCV – Mean erythrocyte volume in total sample • MCH – Mean hemoglobin volume per RBC • MCHC – Mean hemoglobin concentration of RBC • RDW – SD – Calculated distribution width of erythrocytes, standard deviation • RDW – CV- Calculated distribution width of erythrocytes, coefficient of variation • RET – Reticulocyte count Erythrocyte = Red Blood Cells = RBC Erythrocyte = Red Blood Cells = RBC • Erythrocytes have an oval shape, biconcava, flattened, with a depression in the center. • This design is optimized for the exchange of oxygen with the environment that surrounds it which gives them flexibility to pass through the capillaries, where they release the load of oxygen. • The diameter of an erythrocyte is typically 6-8 micrometers. Erythrocyte = Red Blood Cells = RBC • The half-life of the erythrocyte normal average is 120 days • Erythrocyte does not have nucleus and mitochondria • Erythrocytes are quantitatively the most numerous of the blood. 4.5 – 6 millyon cells / uL Erythrocyte = Red Blood Cells = RBC • Red blood cells contain hemoglobin, which is responsible for transporting oxygen and carbon dioxide. It is also the pigment that gives red color to blood. Haemoglobine (Hgb) Haemoglobine (Hgb) (g/dL) • Hemoglobin is a protein in red blood cells. • The main role of hemoglobin is to transport oxygen from the lungs to tissues, and CO2 from tissues to the lungs, by unstable binding of oxygen and CO2 with bivalent iron heme. • Normal results vary, but in general are: Male: 14 - 18 g/dL Female: 12 - 16 g/dL Hemoglobin Ölçümü • Hemoglobin potasyum ferrisiyanür “Drabkin Solüsyonu” ile siyanmethemoglobine dönüştürülmekte ve hemoglobin yoğunluğu, 540 nm.deki adsorbansın spektrofotometrik olarak ölçülmesi ile hesaplanır Hematocrit (Hct) (%) • The hematocrit is the proportion of blood volume that is occupied by red blood cells. • Hct = MCV x RBC • Referance range: Man: %40 – 50 Femine: %36 – 45 Hematocrit/Packed Cell Volume HCT/PCV 6 Erythrocyte indices (MCV, MCH, MCHC) • MCV – Mean erythrocyte volume in total sample • MCH – Mean hemoglobin volume per RBC • MCHC – Mean hemoglobin concentration of RBC MCV (Mean Corpuscular Volume) 84-90 fl • The MCV is expressed in femtoliter(fl) =(10-15L) and hematology analysers either directly measure this parameter Empedans Yöntemi Kırmızı Küre Bir Kırmızı Kürenin İlerleyişi Algılama Bölgesi Oscilloscope Oscilloscope Trombositlerin Kırmızı Kürelerin Arasından Seçilmesi 20 fl 2 fl Ana Hat Trombositler Değişik Büyüklükte Kırmızı Küreler MCV (Mean Corpuscular Volume) 84-90 fl • MCV = 84-90 fl NORMOCYTİC erytrocyte • MCV = < 80 fl MİCROCYTİC erythrocyte • MCV = >90 fl MACROCYTİC erythrocyte MCH (Mean Corpuscular Hemoglobin) (27-35 pg) • The MCH is expressed in pg/RBC and is calculated by hematology analyzers according to the following equation: Hgb • MCH (pg) = ------ x 10 RBC • Hgb = 15g/dl • RBC = 5x1012/L • MCH = 15/5 x 10 = 30 pg MCH (Mean Corpuscular Hemoglobin) (27-35 pg) • MCH = 27-35 pg NORMOCROMİC RBC • MCH < 27 pg HİPOCROMİC RBC • MCH > 35 pg HİPERCROMİC RBC MCHC (Mean Cell Hemoglobin Concentration) (32-36 g/dl) • The MCH is expressed in g/dl of red blood cells and is calculated by hematology analyzers according to the following equation: • MCHC = Hgb / Hct x 100 • MCHC = (Hgb/ (RBC x MCV)) x 1000 • Hgb = 15 g/dL • Hct = %45 • MCHC = 15 /45 x 100 = 33.3g/dL RDW (%) (Red cell distribution width) eritrositlerin büyüklüklerine göre dağılım genişliği • RDW reflects the size distribution of the erythrocyte population • RDW is a measurement of anisocytosis. • RDW(%) = MCVsd /MCVmean x 100 • Reference values: %11-15 • RDW normal, MCV decrease = Thalassemia • RDW increase, MCV decrease = Fe deficiency Clinical significance • MCV, MCH, MCHC and RDW are important tools for: 1. The clasification of anemias 2. The early detection of processes that will cause anemia 3. The investigation of the underlying etiology of anemias RETICULOCYTE • Reticulocyte is the precursor of mature erytrocytes, before losing the nucleus. • Referance range % 0.5 – 1.5 • Erythocytes (RBC) Based on the measured RBC count, the MCV, and Hgb concentration, hematology analyzers calculate the following parameters: 1. Hct, also referred to as packed cell volume 2. Mean cell hemoglobin content (MCH) 3. Mean cell hemoglobine concentration (MCHC). ANEMİA decrease in normal number of red blood cells or less than the normal quantity of hemoglobin in the blood. Male: < 14 g/dL Female: < 12 g/dL Classification of Anemia Based on cell size (MCV) • Macrocytic (large) MCV >100 fl (femtoliters) • Normocytic (normal) MCV 8-99 fl • Microcytic (small) MCV<80 fl Based on hemoglobin content (MCH) • Hypochromic (pale color) • Normochromic (normal color) There are several different types of anemia, each with a specific cause and treatment, including the following: • • • • • • • • iron-deficiency anemia megaloblastic (pernicious) anemia anemia of folate deficiency hemolytic anemia sickle cell anemia thalassemia aplastic anemia chronic anemia Hemolytic Anemia • Hemolytic anemia is a disorder in which the red blood cells are destroyed faster than the bone marrow can produce them. The term for destruction of red blood cells is hemolysis. Hemolytic Anemia • Intrinsic - the destruction of the red blood cells due to a defect within the red blood cells themselves. Intrinsic hemolytic anemias are often inherited, such as sickle cell anemia and thalassemia. These conditions produce red blood cells that do not live as long as normal red blood cells. • Extrinsic - red blood cells are produced healthy but are later destroyed by becoming trapped in the spleen, destroyed by infection, or destroyed from drugs that can affect red blood cells. Hemolytic Anemia Iron-Deficiency Anemia • The most common cause of anemia is iron deficiency. • Iron is needed to form hemoglobin. • Iron is mostly stored in the body in the hemoglobin. • About 30 percent of iron is also stored as ferritin and hemosiderin in the bone marrow, spleen, and liver. Iron Deficiency Anemia Characterized by the production of small (microcytic) erythrocytes and a diminished level of circulating hemoglobin (Iron-Deficiency Anemia) Occurs If (Iron Intake < Iron Loss) Causes of Iron-Deficiency Anemia: 1. Inadequate Iron Supply: Poor nutritional intake Malabsorption (gastric surgery, achlorhydria, celiac disease, etc.) Abnormal transferrin function (congenital atransferrinemia, autoantibodies to transferrin receptors) 2. Increased Iron Requirements Blood loss Extensive and prolonged menses Gastrointestinal disorders (hemorrhoids, peptic ulser, colonic cancer, etc.) Pulmonary disorders (hemoptysis, pulmonary hemosiderosis) Urologic disorders (hematuria) Nasal disorders (nose bleeds) Chronic blood donations Dialysis Hookworm infestation Intravascular hemolysis with hemoglobinuria Paroxysmal nocturnal hemoglobinuria Cardiac valve protheses Rapid growth (between ages 2 and 36 months, adolesance) Pregnancy and lactation Causes of Iron Deficiency Anemia • • • • • • Inadequate ingestion Inadequate absorption Defects in release from stores Inadequate utilization Increased blood loss or excretion Increased requirement Stages of Iron Deficiency • Stage 1: moderate depletion of iron stores; no dysfunction • Stage 2: Severe depletion of iron stores; no dysfunction • Stage 3: Iron deficiency • Stage 4: Iron deficiency (dysfunction and anemia) Tests for Iron Deficiency • Serum iron: poor indicator, highly variable day to day and during the day • Ferritin - most sensitive—chief storage form of iron; directly proportional to iron stored in cells Tests for Iron Deficiency • Total iron binding capacity (TIBC)—capacity of transferrin to bind iron • Transferrin—globulin that binds/transports Fe from gut wall to tissues Percent saturation of transferrin (calculate by dividing serum iron by the TIBC) TIBC increases in iron deficiency • • As stored iron falls, saturation of transferrin decreases Transferrin Saturasyonu, Total ve Serbest Demir Bağlama Kapasiteleri Nelerdir ? Serbest Demir Bağlama Kapasitesi = Serbest Transferrin Serum Demiri = Demir Bağlamış Olan Transferrin Total Demir Bağlama Kapasitesi = Serum Transferrin Aktivitesi (~Düzeyi) Transferrin Saturasyonu = Serum Demiri / Total Demir Bağlama Kapasitesi Transferrin Saturasyonu = Serum Demiri / (Serum Demiri + Serbest Demir Bağlama Kapasitesi) CBC in IRON DEFICIENCY ANEMIA • • • • • • Hb: 10.7 g/dL (female 12-16; male 13.5-17.7) MCV: 72 fL (80-100) = microcytosis MCH: 26 pg (27.5-33.2) = hypochromia RDW: 15 (11.5-13.4) = anisocytosis WBC: 4900/L (4 000-11 000) N Platelet count: 450 000/L (150 000-450 000) N PERIPHERAL SMEAR in IDA SERUM IRON PARAMETERS in IDA Iron: 5 g/dL (60-150) TIBC (transferrin level): 467 g/dL (250-435) Transferrin saturation: % 7 (15-45) Ferritin:2 ng/mL (15-200) Transferrin receptor level Iron-Deficiency Anemia Iron-deficiency anemia Iron-deficiency anemia Thalassemia • Normal hemoglobin, also called hemoglobin A. • HbA has four protein chains—two alpha globin and two beta globin. • The two major types of thalassemia, alpha and beta, are named after defects in these protein chains. Thalassemia • Thalassemia is an inherited blood disease. Inherited" means they're passed on from parents to children through genes. • The genetic defect results in reduced rate of synthesis of one of the globin chains that make up hemoglobin. • People who have thalassemias can have mild or severe anemia. • This condition is caused by a lower than normal number of red blood cells or not enough hemoglobin in the red blood cells. Thalassemia Iron-deficiency Thalassemia ANEMİA MCV Decreased Microcyter Anemia 1. Iron-deficiency NORMAL Normocrom Normocyter Anemia 1. Hemolytic Anemia 2.Chronic disease 2.Chronic disease 3.Thalassemia 3. Haemoragy increased Macrocyter Anemia Megaloblastik 1. B12-Folat def. 2. Drogs 3. Herediter Classification of anemias, dependent on the RDW and MCV • Microcytic isocytic (β thalassemia minor) Decreased MCV Normal RDW • Microcytic anisocytic ( Iron deficiency anemia) Decreased MCV Increased RDW • Normocytic Isocytic ( Chronic disease anemia) Normal MCV Normal RDW • Macrocytic Isocytic (Pernicious anemia) Increased MCV Increased RDW Microcytic anisocytic (Iron deficiency anemia) Microcytic isocytic (β thalassemia minor) Decreased MCV Increased RDW Decreased MCV Normal RDW MİKROSİTER ANEMİLERDE AYIRICI TANI FAKTÖR IronDeficiency Anemia TALASEMİ MİNÖR cronic diesises Anemia MCV decreased decreased decreased Normal RDW increased Normal Normal Serum Fe decreased Normal decreased TDBK increased Normal decreased Ferritin decreased Normal decreased Normal increased Megaloblastic Anemias • A form of anemia characterized by the presence of large, immature, abnormal red blood cell progenitors in the bone marrow • 95% of cases are attributable to folic acid or vitamin B12 deficiency Pernicious Anemia A macrocytic, megaloblastic anemia caused by a deficiency of vitamin B12. • Usually secondary to lack of intrinsic factor (IF) • May be caused by strict vegan diet • Also can be caused by ↓gastric acid secretion, gastric atrophy, H-pylori, gastrectomy, disorders of the small intestine (celiac disease, regional enteritis, resections), drugs that inhibit B12 absorption including neomycin, alcohol, colchicine, metformin, pancreatic disease Megaloblastic (pernicious) anemia Iron defitions Sideroblastic Anemia • Microcytic, hypochromic form • Inherited defect of heme synthesis enzyme • High serum and tissue iron levels Complete Blood Count (CBC, Hemogram) Granulopoiesis 11 12 Lymphopoiesis 13 WHİTE BLOOD CELLS • WBC – Number of all leucocytes • NEUT# and % - Neutrophil count and percent • LYMPH# and % - Lymphocyt count and percent • MONO# and % - Monocyt count and percent • EO# and % - Eosanophil • Baso# and % - Basophil count and percent Complete Blood Count Parameters LEUKOCYTES (granulocytes, lymphocytes, and monocytes) 1. Polymorph nucleated cells (multiple nucleated) 2. Mononuclear cells: (one nucleus) lymphocyt monocyt LEUKOCYTES referance range Neutrophils % 50 – 70 1.5–8 x103 Lymphocytes % 20 – 40 0.6-5 x103 Monocytes %2–9 0.1-0.8 x103 Eosinophils %2–4 0.7 x103 Basophils %<1 0.1 x103 Complete Blood Count Parameters: WBC disorders • Leukocytosis : non-neoplastic causes of elevated white cell counts (-philias”): • • • • • Granulocytosis (neutophilia) Eosinophilia Lymphocytosis Monocytosis Basophilia • Leukopenia : decreased production “-penias” • • • • Neutropenia Lymphocytopenia Thrombocytopenia Pancytopenia – Drugs, viral infections, Radiation, chemotherapy etc. A high number of WBCs is called LEUKOCYTOSİS It may be due to: • Infectious diseases • Inflammatory disease (such as rheumatoid arthritis or allergy) • Leukemia • Severe emotional or physical stress A low number of WBCs is called LEUKOPENİA It may be due to: • Bone marrow failure (for example, due to infection, tumor, or abnormal scarring) • Collagen-vascular diseases • Disease of the liver or spleen • Radiation therapy Complete Blood Count Parameters: Neutrophils(%40-74) Most frequent cell in peripheral blood. They have phagocytic functions. They play roles in acute and bacterial infections. Lymphocyte (%19-48) Most important cell in immun system. Second most frequent cell in peripheral blood.(adult) Most frequent cell before 3 years of age. Plays role in viral and chronic infections. T cells helps to activate or inhibit the other cells and act cytotoxic. B cells are secreting antibodies. MONOCYTES (%2-9) • After leaving the bone marrow, monocytes remain in the bloodstream about 7 days, then move into tissues, where they become macrophages. • Monocytes phagocytize bacteria and larger particles • they contain great quantities of lipase and can thus degrade bacteria with lipidic capsule, such as tuberculosis • Monocytes have a dominant role in immunity because they present the ingested antigen on their surface so that it can be recognized by lymphocytes. Complete Blood Count Parameters: Monosit(%2-9) The biggest cell in peripheral blood. Precursor of macrophages. has phagocytic activity. Eosinophil (%0-6) Especially important in allergic diseases and paracytic infections. Complete Blood Count Parameters: Basophils (%0-1.5) Especially important in storage of biologic amines (Histamin etc.) Immunglobulins are attached to this cells. Basophils (%0-1.5) • Basophils appear in many specific kinds of inflammatory reactions, particularly those that cause allergic symptoms. • When activated, basophils degranulate to release histamine, proteoglycans (e.g. heparin and chondroitin), and proteolytic enzymes (e.g. elastase and lysophospholipase). • Each of these substances contributes to inflammation. Complete Blood Count Parameters: Platelets (150-450.000/uL) • They have no nucleus. • They play role in clotting (adhesion, aggregation and secretion) 17 Thrombocytopenia • Decreased Production: – Diffuse marrow disease: • aplasia, • leukemia, – Impaired thrombocyte production: • alcohol, • drugs, • HIV – Ineffective production: • megaloblastic anemia. • Decreased Survival: – Immune • Idiopathic thrombocytopenia, • SLE, • Iso-immune, • drugs, • HIV – Non-Immune • DIC, • Thrombocytic thrombocytopenic purpura • Sequestration – Hypersplenism. • Dilutional – Massive transfusion. Definitions: Erythrocyte Leucocyte Platelets Neutrophil Lymphocyte Monocyte Eosinophil Basophil Increase Polycytemia(*) Erythrocytosis Leucocytosis Thrombocytosis Neutrophilia Lymphocytosis Monocytosis Eosinophilia Basophilia Decrease Anemia Leucopenia Thrombopenia Neutropenia Lymphopenia - Sedimentation Rate (Erythrocyte Sedimentation Rate or ESR) • sedimentation rate is the rate at which red blood cells precipitate in a period of 1 hour • It is a common hematology test which is a non-specific measure of inflammation. What is the normal sedimentation rate? • The normal sedimentation rate (Westergren method) for males is < 15 millimeters per hour, females is < 20 millimeters per hour. • The sedimentation rate can be slightly more elevated in the elderly. C-reaktive protein (CRP) • C-reactive protein is an acute phase protein that is produced by the liver during an inflammatory reaction Clinical significance • An increased plasma concentration of CRP is an important indicator of: - Acute or chronic inflammation such as may accompany bacterial infection - Autoimmune or immune complex disease - Tissue necrosis and malignancy ? ? ? Iron-deficiency anemia Aim of Complete Blood Count Testing: 1- Bone marrow dysfunctions (Malignancies, therapeutic drugs, toxic substances etc.) 2- Anemia and related red blood cell diseases 3- Polystemia and related red blood cell diseases 4- White blood cells and related diseases (Leukemia, infections, drugs, immunological diseases) 5- Platelets and related diseases (GIS bleeding, thrombosis, purpura, ITP, massive transfusion, HSM ) ? ? Anemia (Bleeding) Polisitemia ? POLİSİTEMİ VERA 1 YILDAN BERİ GİDEREKL ARTAN BAŞAĞRISI TANIMLIYOR. MM Thalasemi taşıyıcı Analiz Yöntemleri (1) • Empedans yöntemi: Hücreler dar bir aralıktan geçerlerken doğru akımda meydana getirdikleri empedans (elektrik direnci) değişimi ile sayı ve büyüklükleri belirlenir. Voltaj değişiklikleri hücre büyüklüğü ile doğru orantılıdır. Analiz Yöntemleri (2) • Optik sistem: Hücrelerin üzerine gelen lazer ışını değişik yönlere yansır. Dar açı ile meydana gelen yansımalar hücre büyüklüğü, geniş açı ile ortaya çıkanlar ise hücre yapısının (granularite ve çekirdek yapısı) belirlenmesinde kullanılırlar. Optik Sistem Hücre Büyüklüğüne Göre Sinyal Oluşumu Hücreleri Büyüklüklerine Göre Tanımlanma Eşikleri Lökosit Formülü Çalışma Basamakları • Kan örneğini özel antikoagülanlı tüpden aspire edilir • Bir kısmındaki kan lizise uğratılır Hgb ve BK analizi yapılır. • Kalan kısım ise hemolize uğratılmadan kırmızı küre ve trombosit analizi için kullanılır. Analiz Öncesi Dönem Kanın alınmasındaki hastanın fizyolojik durumundan alınan kan örneğinin laboratuvara ulaşıp OTKSC’de analiz edilinceye kadar geçen dönemi içine almaktadır. •Fizyolojik (yaş, yükseklik, gebelik, sigara) •Örnek alınması (bekleme süresi, antikoagülanların etkileri ve endojen interferans) •EDTA’ya bağlı psödotrombositopeni •Trombosit satellitizmi EDTA’ya Bağlı Trombosit Kümelenmesi (Yalancı Trombositopeni) Trombosit Satellitizmi (Yalancı Trombositopeni) Ölçüm Hatalı Yüksek Beyaz Küre Kriyoproteinler Heparin Paraproteinler Çekirdekli Eritrositler* Trombosit Kümeleşmesi Kırmızı Kriyoproteinler Küre Dev trombositler Hgb Htk Karboksihemoglobin>%10 Kriyoproteinler İn vivo hemoliz Heparin BKS> 50x10e9/L Hiperbilirubinemi Lipemi Paraproteinemi Kriyoproteinler Dev trombositler BKS> 50x10e9/L Hiperglisemi (>600mg/dl) Hatalı Düşük Pıhtılaşma Ezilmiş hücreler (basket hücreleri) Üremi İmmun baskılayıcı ilaçlar Otoagglutinasyon Pıhtılaşma İn vitro hemoliz Pıhtılaşma Sulfhemoglobinemi (?) Otoagglutinasyon In vitro hemoliz Mikrositer kırmızı Ölçüm (MCV) (MCHC) Trombosit Sayımı Hatalı Yüksek Otoagglütinasyon BKS> 50x10e9/L Hiperglisemi Kırmızı küre deformabilitesinde azalma Otoagglütinasyon Pıhtılaşma In vitro hemoliz Hatalı yüksek hemoglobin ölçümü Hatalı düşük hematokrit ölçümü Kriyoproteinler Hemoliz Mikrositer kırmızı küre Kırmızı kürelerde inklüzyon cisimcikleri Beyaz kürelerde fragmentasyon Hatalı Düşük Kriyoproteinler Dev trombositler İn vitro hemoliz Mikrositer kırmızı küre Şişmiş kırmızı küre BKS> 50x10e9/L Hatalı düşük hemoglobin ölçümü Hatalı yüksek hematokrit ölçümü Pıhtılaşma Dev trombositler Heparin Trombosit kümeleşmesi “clumping” Trombosit satellitizmi