Introduction to anemia Gülay Ç İ LER ERDA Ğ,MD

Hematopoiesis Hematopoiesis Begins in the embryo during the third week of gestation Begins in the embryo during the third week of gestation Developmental hematopoiesis occurs in three stages Developmental hematopoiesis occurs in three stages Mesoblastic Mesoblastic In the yolk sac first produces erythrocytes and leukocytes In the yolk sac first produces erythrocytes and leukocytes Hepatic Hepatic By the 12th week of gestation liver and the spleen are the predominant sites By the 12th week of gestation liver and the spleen are the predominant sites Myeloid Myeloid By the 30 weeks gestation, the bone marrow assumes its ultimate role as the major site of production of the formed elements of the blood By the 30 weeks gestation, the bone marrow assumes its ultimate role as the major site of production of the formed elements of the blood

AgeSite of hematopoiesis Embryoyolk sac then liver 3 rd to 7 th month Spleen and liver 4 th and 5 th months marrow cavity - esp. granulocytes and platelets 7 th monthmarrow cavity - erythrocytes Birth mostly bone marrow; spleen and liver when needed Birth to maturity number of active sites in bone marrow decreases but retain ability for hematopoiesis Adult bone marrow of skull, ribs, sternum, vertebral column, pelvis, proximal ends of femurs

The predominant cellular element in the blood is eryhtrocyte: RBC (red blood cell) The predominant cellular element in the blood is eryhtrocyte: RBC (red blood cell) Under the stimulus of erhythropoietin and other humoral factors Under the stimulus of erhythropoietin and other humoral factors erythroid burst forming units (from the pluripotent stem cells) and colony forming units give rise to identifiable erythroblasts erythroid burst forming units (from the pluripotent stem cells) and colony forming units give rise to identifiable erythroblasts Begin to synthesize Hb Begin to synthesize Hb Nucleated RBC Nucleated RBC Nucleus extruded Young erythrocyte Nucleus extruded Young erythrocyte

Hb Hb Binds to oxygen in the lungs and transports it to the tissues Binds to oxygen in the lungs and transports it to the tissues Tetramer consisting of two pairs of unlike polypeptide chains each attached to a HEME ( molecule of protoporphyrin ferrous iron) Tetramer consisting of two pairs of unlike polypeptide chains each attached to a HEME ( molecule of protoporphyrin ferrous iron) The primary Hb The primary Hb in the postnatal life is HbA (2 α 2 β ) in the postnatal life is HbA (2 α 2 β ) Initial months of gestation HbF (2 α 2 γ ) Initial months of gestation HbF (2 α 2 γ )

At the time of birth γ and β chain synthesis is approximately equal and 60%-80% of the total Hb is HbF At the time of birth γ and β chain synthesis is approximately equal and 60%-80% of the total Hb is HbF γ glb synthesis almost ceases during the initial months of life γ glb synthesis almost ceases during the initial months of life By 6 months of age the percentage of HbF approximates that of adults(<2%) By 6 months of age the percentage of HbF approximates that of adults(<2%) HbF is resistant to both alkali and dilute acid. It binds less avidly than does HbA to 2,3 diphosphoglycerate HbF is resistant to both alkali and dilute acid. It binds less avidly than does HbA to 2,3 diphosphoglycerate an organic phosphate in the erythrocyte important in modulating oxygen uptake and release by Hb an organic phosphate in the erythrocyte important in modulating oxygen uptake and release by Hb The affinity of HbF for oxygen is high The affinity of HbF for oxygen is high

Because of intrauterine hypoxia and the high affinity of HbF for oxygen(resulting in a shift to the left of oxygen-Hb dissociation curve) Because of intrauterine hypoxia and the high affinity of HbF for oxygen(resulting in a shift to the left of oxygen-Hb dissociation curve) Erythropoietin secretion is enhanced during fetal life Erythropoietin secretion is enhanced during fetal life During the final months of gestation and at birth values for Hb and Hct are higher than those for older children During the final months of gestation and at birth values for Hb and Hct are higher than those for older children

Hemoglobin F (Hb F), composed of 2 alpha and 2 gamma globin chains, is the normal hemoglobin of the fetus and newborn. Normally in the second trimester, gamma chain production (and Hgb F levels) decrease and beta chain production increases, resulting in increasing levels of hemoglobin A (Hb A), the major normal adult hemoglobin (2 alpha and 2 beta globin chains). Hemoglobin A2 (Hb A2) (2 alpha and 2 delta globin chains) also comprises a small amount (<3.3%) of hemoglobin normally found in adults. Hb A2 values at birth are <1%. Hemoglobin F (Hb F), composed of 2 alpha and 2 gamma globin chains, is the normal hemoglobin of the fetus and newborn. Normally in the second trimester, gamma chain production (and Hgb F levels) decrease and beta chain production increases, resulting in increasing levels of hemoglobin A (Hb A), the major normal adult hemoglobin (2 alpha and 2 beta globin chains). Hemoglobin A2 (Hb A2) (2 alpha and 2 delta globin chains) also comprises a small amount (<3.3%) of hemoglobin normally found in adults. Hb A2 values at birth are <1%. Reference Values HEMOGLOBIN A days: % 1-2 months: % 3-5 months: % > or =6 months: % HEMOGLOBIN F 1-30 days: % 1-2 months: % 3-5 months: % 6-8 months: % 9-12 months: % months: % months: % > or =24 months: %

15 Newborns less than one week old have hemoglobin of g/dl. Newborns less than one week old have hemoglobin of g/dl. By six months of age, hemoglobin runs between 11 and 14 g/dl. By six months of age, hemoglobin runs between 11 and 14 g/dl. Between 1 year and 15 years of age hemoglobin runs between g/dl. Between 1 year and 15 years of age hemoglobin runs between g/dl. Normal adult hemoglobin depends on gender: Normal adult hemoglobin depends on gender: ♀12-16 g/dl ♀12-16 g/dl ♂14-18 g/dl ♂14-18 g/dl In geriatric age group, men and women have same hemoglobin range: g/dl. In geriatric age group, men and women have same hemoglobin range: g/dl.

Anemia A reduction in RBC volume or Hb concentration below the range of values occurring in healthy person A reduction in RBC volume or Hb concentration below the range of values occurring in healthy person Although a reduction in the amount of circulating Hb decreases the oxygen carrying capacity of the blood few clinical disturbances occur until Hb levels fall below 7-8gr/dl Although a reduction in the amount of circulating Hb decreases the oxygen carrying capacity of the blood few clinical disturbances occur until Hb levels fall below 7-8gr/dl Below this level pallor becomes evident in the skin and mucous membranes Below this level pallor becomes evident in the skin and mucous membranes

Physiologic adjustments to anemia Increased cardiac output Increased cardiac output Increased oxygen extraction Increased oxygen extraction Shunting of blood flow toward vital organs and tissues Shunting of blood flow toward vital organs and tissues In addition the concentration of 2,3 DPG increases In addition the concentration of 2,3 DPG increases Shift to the right of oxygen dissociation curve, reducing the affinity of Hb for oxygen Shift to the right of oxygen dissociation curve, reducing the affinity of Hb for oxygen results in more complete transfer of oxygen to the tissues results in more complete transfer of oxygen to the tissues

Anemia can be defined as a reduction in the hemoglobin, hematocrit or red cell number Anemia can be defined as a reduction in the hemoglobin, hematocrit or red cell number The pathophysiologic effects of anemia depend upon the rate at which the anemia progresses The pathophysiologic effects of anemia depend upon the rate at which the anemia progresses

In an acute hemorhage In an acute hemorhage the arterial pressure falls, cardiac output decreases, peripheral vasculature collapses and the patient rapidly enters hypovolemic shock the arterial pressure falls, cardiac output decreases, peripheral vasculature collapses and the patient rapidly enters hypovolemic shock The sudden rapid loss of 30% of the total blood volume often results in death unless there is immediate medical intervention The sudden rapid loss of 30% of the total blood volume often results in death unless there is immediate medical intervention

In a slowly developing anemia In a slowly developing anemia cardiac output increases, blood shunted from nonvital organs and hemoglobin oxygen affinity decreases due to increased levels of 2,3 DPG. cardiac output increases, blood shunted from nonvital organs and hemoglobin oxygen affinity decreases due to increased levels of 2,3 DPG. Total blood volume remains remarkably constant. More than 50% of the red cell mass can be lost slowly with minimal effects Total blood volume remains remarkably constant. More than 50% of the red cell mass can be lost slowly with minimal effects

It must be remembered that anemia is not a disease rather it’s a sign of disease It must be remembered that anemia is not a disease rather it’s a sign of disease The clinical effects (lack of oxygen) include The clinical effects (lack of oxygen) include Tiredness, lassitude, weakness, pallor Tiredness, lassitude, weakness, pallor Dyspnea and anginal pain are not uncommon after exercise Dyspnea and anginal pain are not uncommon after exercise Jaundice may occur in some anemias Jaundice may occur in some anemias

22 Classification of Anemias Have a variety of ways - depending on criteria used: Have a variety of ways - depending on criteria used: Functional Functional Morphological Morphological Clinical Clinical Quantitative Quantitative

23 Functional Classification of Anemias Decreased RBC production (hypoproliferative) Decreased RBC production (hypoproliferative) Defective hemoglobin synthesis Defective hemoglobin synthesis Fe deficiency Fe deficiency B12 deficiency B12 deficiency Folate deficiency Folate deficiency Impaired bone marrow or stem cell function, as in leukemia Impaired bone marrow or stem cell function, as in leukemia Increased RBC destruction, as in sickle cell anemia or hemolytic anemia Increased RBC destruction, as in sickle cell anemia or hemolytic anemia Combination of the two (sometimes called “ineffective erythropoiesis”) Combination of the two (sometimes called “ineffective erythropoiesis”)

24 Morphological Classification of Anemias Morphological based on sizes and color of RBCs Morphological based on sizes and color of RBCs Normochromic Normocytic Normochromic Normocytic Hypochromic Microcytic Hypochromic Microcytic NormochromicMicrocytic NormochromicMicrocytic NormochromicMacrocytic NormochromicMacrocytic

25 Clinical Classification of Anemias According to their associated causes: According to their associated causes: Blood loss Blood loss Iron deficiency Iron deficiency Hemolysis Hemolysis Infection Infection Nutritional deficiency Nutritional deficiency Metastatic bone marrow replacement Metastatic bone marrow replacement

26 Quantitative Classification of Anemias Quantitatively by: Quantitatively by: Hematocrit Hematocrit Hemoglobin Hemoglobin Blood cell indices Blood cell indices Reticulocyte count Reticulocyte count

27 Hemoglobin and Hematocrit 1 of 2 Anemia usually diagnosed on either hemoglobin or hematocrit values. Anemia usually diagnosed on either hemoglobin or hematocrit values. Remember, normal ranges vary depending on age, gender, state of hydration, patient positioning and local patient population. Remember, normal ranges vary depending on age, gender, state of hydration, patient positioning and local patient population.

28 Hemoglobin and Hematocrit 2 of 2 On basis of H&H, anemia can be classified as mild, moderate, or severe. On basis of H&H, anemia can be classified as mild, moderate, or severe. On basis of duration of onset, anemia can be classified as either chronic or acute. On basis of duration of onset, anemia can be classified as either chronic or acute. Rules of Three: Rules of Three: RBC X 3 = Hemoglobin RBC X 3 = Hemoglobin Hemoglobin X 3 = Hematocrit Hemoglobin X 3 = Hematocrit Ratio of Hb and Hct will vary with cause of anemia and affect the RBC indices, particularly the MCV (Mean Corpuscular Volume). Ratio of Hb and Hct will vary with cause of anemia and affect the RBC indices, particularly the MCV (Mean Corpuscular Volume). Microscopic examination of peripheral blood smear is required for evaluation of anemia. Bone marrow aspirates and smear evaluation may also be needed. Microscopic examination of peripheral blood smear is required for evaluation of anemia. Bone marrow aspirates and smear evaluation may also be needed.

29 RBC Indices RBC indices include: RBC indices include: Mean Corpuscular Volume (MCV) Mean Corpuscular Hemoglobin (MCH) Mean Corpuscular Hemoglobin Concentration (MCHC) RBC Distribution Width (RDW)

30 MCV Mean cell volume Mean cell volume MCV is average size of RBC MCV is average size of RBC MCV = Hct x 10 MCV = Hct x 10 RBC (millions) RBC (millions) If fL, normal range, RBCs considered normocytic If fL, normal range, RBCs considered normocytic If < 80 fL are microcytic If < 80 fL are microcytic If > 100 fL are macrocytic If > 100 fL are macrocytic Not reliable when have marked anisocytosis Not reliable when have marked anisocytosis

31 MCH MCH is average weight of hemoglobin per RBC. MCH is average weight of hemoglobin per RBC. MCH = Hgb x 10 MCH = Hgb x 10 RBC (millions) RBC (millions)

32 MCHC MCHC is average hemoglobin concentration per RBC MCHC is average hemoglobin concentration per RBC MCHC = Hgb x 100 MCHC = Hgb x 100 Hct (%) Hct (%) If MCHC is normal, cell described as normochromic If MCHC is normal, cell described as normochromic If MCHC is less than normal, cell described as hypochromic If MCHC is less than normal, cell described as hypochromic There are no hyperchromic RBCs There are no hyperchromic RBCs

33 RDW Most automated instruments now provide an RBC Distribution Width (RDW) Most automated instruments now provide an RBC Distribution Width (RDW) An index of RBC size variation An index of RBC size variation May be used to quantitate the amount of anisocytosis on peripheral blood smear May be used to quantitate the amount of anisocytosis on peripheral blood smear Normal range is 11.5% to 14.5% for both men and women Normal range is 11.5% to 14.5% for both men and women

34 RBC Indices and Other Tests RBC indices are automatically calculated by instruments. RBC indices are automatically calculated by instruments. Microscopic evaluation will determine if RBCs are normocytic, microcytic, or macrocytic and normochromic or hypochromic. Microscopic evaluation will determine if RBCs are normocytic, microcytic, or macrocytic and normochromic or hypochromic. Use of RBC indices in differential diagnosis can provide picture of what is occurring clinically. Use of RBC indices in differential diagnosis can provide picture of what is occurring clinically. If anemia caused be bone marrow failure, requires information about RBC production. Information obtained from reticulocyte count. Reticulocyte count measures effective RBC production. If anemia caused be bone marrow failure, requires information about RBC production. Information obtained from reticulocyte count. Reticulocyte count measures effective RBC production. As study different anemias, will learn morphology. As study different anemias, will learn morphology.

35 Treatment of Anemias Treated according to cause; Should know cause before beginning treatment. Treated according to cause; Should know cause before beginning treatment. Patient can have more than one cause of anemia. Patient can have more than one cause of anemia. Must use diagnostic tests to determine cause(s). Must use diagnostic tests to determine cause(s). Do diagnostic tests before transfusions, because transfusions obscure and confuse findings. Do diagnostic tests before transfusions, because transfusions obscure and confuse findings.

36 Hgb (In the Diagnosis of Anemia) Hbg is the main component of RBCs and carries oxygen to tissues. Hbg is the main component of RBCs and carries oxygen to tissues. Three methods to measure hemoglobin: Three methods to measure hemoglobin: Cyanmethemoglobin (recommended method) Cyanmethemoglobin (recommended method) Oxyhemoglobin Oxyhemoglobin Iron Content Iron Content

37 Hct (In the Diagnosis of Anemia) 1 of 3 Is packed RBC volume Is packed RBC volume Is ratio of RBC volume to volume of whole blood Is ratio of RBC volume to volume of whole blood Usually expressed in percentage (42%) or as decimal fraction (.42) Usually expressed in percentage (42%) or as decimal fraction (.42) Venous and arterial hematocrits closely agree Venous and arterial hematocrits closely agree Specimen of choice is EDTA (ethylenediaminetetra acetic acid), oxalate or heparin Specimen of choice is EDTA (ethylenediaminetetra acetic acid), oxalate or heparin

38 Hct (In the Diagnosis of Anemia) 2 of 3 Measurement done by centrifugation or through calculations performed on many automated measurements. Calculated hematocrit is product of MCV and RBC count. Normal ranges are 42-52% in men and 37-47% in women. Normal ranges also vary among age groups, institutions, and geographic locations.

39 RBC Indices (In the Diagnosis of Anemia) 1 of 2 RBC indices are readily available from the automated hematology counting devices RBC indices are readily available from the automated hematology counting devices MCV is measured directly or calculated from hematocrit and RBC count; MCH and MCHC are both calculated MCV is measured directly or calculated from hematocrit and RBC count; MCH and MCHC are both calculated

40 RBC Indices (In the Diagnosis of Anemia) 2 of 2 In various anemic states, indices may be altered: In various anemic states, indices may be altered: Microcytic Anemia: Microcytic Anemia: MCV usually fL MCV usually fL MCH usually pg MCH usually pg MCHC usually 22-30% MCHC usually 22-30% Macrocytic Anemia: Macrocytic Anemia: MCV usually fL MCV usually fL

41 Peripheral Blood Smear (In the Diagnosis of Anemia) Very useful in diagnosing and classifying anemias Very useful in diagnosing and classifying anemias Look for: Look for: Neutropenia Neutropenia Thrombocytopenia Thrombocytopenia Hypochromia Hypochromia Size and shape of RBCs Size and shape of RBCs Unusual leukocytes (hypersegmentation) Unusual leukocytes (hypersegmentation) Red cell inclusions: basophilic stippling, Howell-Jolly bodies… Red cell inclusions: basophilic stippling, Howell-Jolly bodies…

42 Reticulocyte Count (In the Diagnosis of Anemia) Useful in determining response and potential of bone marrow. Useful in determining response and potential of bone marrow. Reticulocytes are non-nucleated RBCs that still contain RNA. Reticulocytes are non-nucleated RBCs that still contain RNA. Visualized by staining with supravital dyes, including new methylene blue or brilliant cresyl blue; RNA is precipitated as dye-protein complex. Visualized by staining with supravital dyes, including new methylene blue or brilliant cresyl blue; RNA is precipitated as dye-protein complex. Normal range is % of all erythrocytes. Normal range is % of all erythrocytes. If bone marrow responding to anemia, should see increases in retic count. If bone marrow responding to anemia, should see increases in retic count. Newborns have higher retic count than adults until second or third week of life. Newborns have higher retic count than adults until second or third week of life.

43 Bone Marrow (In the Diagnosis of Anemia) Bone marrow aspiration and biopsy are important diagnostic tools in the determination of anemia. Bone marrow aspiration and biopsy are important diagnostic tools in the determination of anemia.

44 Other Tests (In the Diagnosis of Anemia) Hemoglobin Electrophoresis Hemoglobin Electrophoresis Antiglobulin Testing Antiglobulin Testing Osmotic Fragility Osmotic Fragility Sugar Water Test Sugar Water Test Ham’s Test Ham’s Test RBC Enzymes RBC Enzymes B12, Fe, TIBC, Folate Levels B12, Fe, TIBC, Folate Levels

Anemia is usually classified according to Anemia is usually classified according to Etiology Etiology Pathophysiology Pathophysiology Morphology Morphology

Laboratory diagnosis Initial investigation and tentative diagnosis is made with a relatively small number of tests Initial investigation and tentative diagnosis is made with a relatively small number of tests The precise diagnosis is made with further special tests The precise diagnosis is made with further special tests Screening is usually done with CBC (complete blood count) Screening is usually done with CBC (complete blood count) The exact procedures in a CBC depends upon the instrumentation in the laboratory The exact procedures in a CBC depends upon the instrumentation in the laboratory

Most laboratories now use automated multiparameter instruments. These are Most laboratories now use automated multiparameter instruments. These are Hb(hemoglobin) Hb(hemoglobin) RBC RBC MCV MCV Htc(hematocrit) Htc(hematocrit) MCH MCH MCHC MCHC RDW RDW WBC WBC Platelet and MPV Platelet and MPV Directly measured values Calculated values

MCV: MCV: MCH: MCH: MCHC: MCHC: Htc X10 RBC (90±7 fl) Hb X100 RBC (30±3) Hb X100 Htc (33±2)

spherocytosis eliptocytosis poikilocytosis stomatocytosis acanthocytosis Fragmentation hemolysis

Sickle cell anemia; target cells and sickled cells Normal RBC Target cells Heinz body anemia Thalassemia; severe hypochromia Anisopoikilocytosis, target cells

normal macrocytes Hypochromic, microcytes Target cells schistocytes