1 ANEMIA And Its Laboratory Diagnosis By: Ahmad Sh. Silmi IUG, Hematologist

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1 1 ANEMIA And Its Laboratory Diagnosis By: Ahmad Sh. Silmi IUG, Hematologist asilmi@jugaza.edu.ps

2 2 Objectives At the end of this chapter you will be able to: Define anemia Discuss the causes and clinical significance of different categories of anemia Describe the classification of anemia Explain - Microcytic anemia - Macrocytic anemia - Normochromic normocytic anemia Discuss the laboratory findings for each category of anemia Perform basic laboratory tests for the diagnosis of anemia

3 3 Chapter Outline 3.1. Definition of anemias 3.2. Classification of anemias 3.2.1. Hematologic Response to Anemia 3.2.2. Signs of Accelerated Bone Marrow Erythropoiesis 3.2.3. Physiologic Response to Anemia 3.2.4. Methods of classification 3.2.5. Anemia Diagnosis/Cause 3.2.6. Lab Investigation of Anemia 3.3. Types of anemia. 3.3.1 microcytic hypochromic anemia 3.3.2. macrocytic normocytic anemias 3.3.3. normocytic anemias 3.3.4. normocytic anemias due to hemoglobinopathies

4 4 3.1. Introduction 3.1.1. Definition of Anemia Anemia is a decrease in the RBC count, Hgb and/or HCT values as compared to normal reference range for age and sex (Also determined by alteration in plasma volume) –‘True’ anemia: –decreased RBC mass and normal plasma volume –Pseudo or dilutional anemia: –normal RBC mass and increased plasma volume –An increase in plasma volume can occur in Pregnancy, volume overload (IVs) congestive heart failure –Low Hgb and HCT values

5 5 Definition of Anemia cont’d

6 6 3.1.1. Definition of Anemia cont’d….. Anemia must also relate to the level of hemoglobin the individual normally possesses. –If an adult male usually maintains a hemoglobin level of 16g/dl, and over a period of days is noted to have decreased to 14g/dl, this must be considered significant even though both values are within the normal range for an adult male.

7 7 3.1.1. Definition of Anemia cont’d….. Various diseases and disorders are associated with decreased hemoglobin levels. These include: –Nutritional deficiencies –External or internal blood loss –Increased destruction of RBCs –Ineffective or decreased production of RBCs –Abnormal hemoglobin synthesis –Bone marrow suppression by toxins, chemicals, or radiation & replacement by malignant cells –Infection

8 8 3.1.1. Definition of Anemia cont’d….. Functionally anemia is defined as tissue hypoxia (inability of the body to supply tissue with adequate oxygen for proper metabolic function) –There is an abnormal hemoglobin with an increased O 2 affinity resulting in an anemia with normal or raised hemoglobin levels, hematocrit, or RBC count. Generally anemia is not a disease, but rather the expression of an underlying disorder or disease.

9 9 3.1.1. Definition of Anemia cont’d….. Anemia may develop: –When RBC loss or destruction exceeds the maximal capacity of bone marrow RBC production or –When bone marrow production is impaired

10 10 3.1.2.Hematologic Response to Anemia Tissue hypoxia causes increased renal release of erythropoietin (EPO) to accelerate bone marrow erythropoiesis The normal bone marrow can increase its activity 7-8 times normal –Marrow becomes hypercellular

11 11 Signs of Accelerated Bone Marrow Erythropoiesis The marrow becomes hypercellular due to a marked increase in RBC precursors (called erythroid hyperplasia) and the M:E ratio falls. Nucleated RBCs may be released into the blood circulation along with the outpouring of reticulocytes –NRBC number tends to correlate with the severity of anemia –Increased polychromasia on the Wright's- stained blood smear is seen due to increased number of circulating Retics.

12 12 If demand exceeds maximal bone marrow activity, RBC production may occur in extramedullary sites, liver, spleen (hepatosplenomegaly).

13 13 3.1.3. Physiologic Response to Anemia Ability to adapt to anemia depends on: –Age and underlying disease –Cardio/pulmonary function –Rate at which anemia develops (BM can compensate easier if the onset of anemia is slow), –Underlying disease

14 14 3.1.4. Clinical features: Symptoms of hypoxia: decreased oxygen delivery to the tissues/organs causes: –fatigue, faintness, weakness, dizziness, headaches, dyspnea, poor exercise tolerance, leg cramps.

15 15 Signs of anemia general signs include pallor of mucous membrane, which occur if the Hgb concentration is less than 9g/dl, specific signs are associated with particular types of anemia, for example, jaundice in hemolytic anemia, leg ulcer in sickle cell anemia 3.1.4.Clinical features cont’d….:

16 16 3.1.5. Diagnosis of anemia Before making a diagnosis of anemia, one must consider: –Age –Sex –Geographic location –Presence or absence of lung disease

17 17 3.1.5. Diagnosis of anemia cont’d…… How does one make a clinical diagnosis of anemia? A. Patient history –Dietary habits –Medication –Possible exposure to chemicals and/or toxins –Description and duration of symptoms

18 18 3.1.5. Diagnosis of anemia cont’d…… –Tiredness –Muscle fatigue and weakness –Headache and vertigo (dizziness) –Dyspnia (difficult or labored breathing) from exertion –G I problems –Overt signs of blood loss such as hematuria (blood in urine) or black stools Patient history cont………..

19 19 3.1.5. Diagnosis of anemia cont’d…. B. Physical exam –General findings Hepato or splenomegaly Heart abnormalities Skin pallor –Specific findings In vitamin B 12 deficiency there may be signs of malnutrition and neurological changes In iron deficiency there may be severe pallor, a smooth tongue, and esophageal webs In hemolytic anemias there may be jaundice due to the increased levels of bilirubin from increased RBC destruction

20 20 3.1.5. Diagnosis of anemia cont’d…… C. Lab investigations 1.A complete blood count, CBC –RBC count –Hematocrit (Hct) or packed cell volume –Hemoglobin determination –RBC indices calculation –Reticulocyte count 2.Blood smear examination to evaluate: –Poikilocytosis –Leukocytes or Platelets abnormalities

21 21 3.1.5. Diagnosis of anemia cont’d….. Lab investigation cont’d…… 3.A bone marrow smear and biopsy to observe: –Maturation of RBC and WBC series –Ratio of myeloid to erythroid series –Abundance of iron stores (ringed sideroblasts) –Presence or absence of granulomas or tumor cells –Red to yellow ratio –Presence of megakaryocytes

22 22 4. Hemoglobin electrophoresis 3.1.5. Diagnosis of anemia cont’d …..

23 23 3.1.5. Diagnosis of anemia cont’d….. Lab investigation cont’d…… 5. Antiglobulin testing 6. Osmotic fragility test

24 24 3.2. Methods of Anemia Classification Several schemes of classifying anemias exist 1.Morphologic –Based on RBC morphology –Anemia is divided into three groups mainly on the basis of the MCV (RBC indices) 2.Pathophysiologic –Anemia is divided using three main causes/mechanisms I.Impaired erythrocyte formation (Aplastic anemia, IDA, sideroblastic anemia, anemia of chronic diseases, megaloblastic anemia) –Retic count is low –The bone marrow fails to respond appropriately due to disease or lack of essential supplies

25 25 Methods of Classification cont’d III.Increased blood loss (Acute, Chronic) –Retic count is typically high –Anemia results when red cell loss exceeds the bone marrow’s capacity to increase its activity IV.Increased destruction of RBCs (hemolytic anemias) –Retic count is typically high –Anemia results when red cell destruction exceeds the bone marrow’s capacity to increase its activity

26 26 Methods of Classification cont’d Morphologic Categories of Anemia 1.Normocytic Normochromic anemia (normal red cell indices) Blood loss anemia Hemolytic anemia Aplastic anemia Chronic diseaes Renal insufficciency

27 27 Morphologic Categories of Anemia Microcytic hypochromic ( low red cell indices) Iron deficiency anemia Sideroblastic anemia Lead poisoning Thallassemia Chronic diseases Methods of Classification cont’d

28 28 Morphologic Categories of Anemia Macrocytic Normochromic ( high MCV and MCH, normal MCHC) Megaloblastic anemia Liver disease Post splenectomy Hypothyroidism Stress erythropoiesis Methods of Classification cont’d

29 29 1 Microcytic/hypochromic 3 1 2 2 Macrocytic/Normochromic 3 Normocytic/Normochromic Morphologic Categories of Anemia N.B. The nucleus of a small lymphocyte (shown by the arrow) is used as a reference to a normal red cell size

30 30 3.2.2. Pathophysiological classification 1. B. 2. A.

31 31 1. Microcytic- Hypochromic Anemia

32 32 Microcytic- Hypochromic Anemia Many RBCs smaller than nucleus of normal lymphocytes Increased central pallor. Includes –Iron deficiency anemia –Thalassemia –Anemia of chronic disease –Sideroblastic anemia –Lead poisoning

33 33 IronProtoporphyrin Heme Globin + Hemoglobin Iron deficiency Chronic inflammation or malignant (ACD) Thalassemia (  or  ) Sideroblastic anemia The Cause of Microcytic-Hypochromic Anemia

34 34 RBC maturation in microcytic anemias Normoblastic RBC maturation  normocytic red cells Abbott Manual Microcytic/Hypochromic Anemias Normal RBC maturation is shown for comparison

35 35 A. Iron Deficiency Anemia (IDA) Is a condition in which the total body iron content is decreased below a normal level This results in a reduced red blood cell and hemoglobin production More than half of all anemias are due to iron deficiency.

36 36 Iron Deficiency Anemia (IDA) Causes: –Nutritional deficiency –Malabsorption (insufficient or defective absorption) –Inefficient transport, storage or utilization of iron –Increased need –Chronic blood loss (GI bleeding, ulcer, heavy menstruation, etc)

37 37 DIETARY SOURCES OF IRON Inorganic Iron eg lentils Organic iron eg beef DAILY IRON REQUIREMENT 10-15mg/day (5-10% absorbed)

38 38 Daily Iron cycle (Fig)

39 39 Adult men0.5-1 Post menopausal female 0.5-1 Menstruating female 1-2 Pregnant female1.5-3 Children1.1 Female (age 12-15) 1.6-2.6 Estimated daily iron requirements Units are mg/day

40 40 Iron absorption, Transport and storage Iron absorbed from duodenum and jejunum in the GIT Moves via circulation to the bone marrow Incorporated with protoporpyirin in mitochondria of the erythroid precursor to make Heme

41 41 There are three proteins important for transporting and storage of iron: –Transferrin, –Transferrin receptors and –Ferritin Transport: Transferrin: transports iron from the plasma to the erythroblasts in the marrow for erythropoiesis The transferrin will bind to transferrin receptor on the erythrocyte membrane

42 42 Storage Hgb contains about two third of the body iron At the end of their life, RBCs are broken down in the macrophage of reticuloendothelial system and then iron is released from Hgb enters plasma and provided to transferrin. Some stored in reticuloendothelial cell as ferritin soluble protein – iron complex) and hemosiderin (37%) (degraded form of ferritin insoluble) iron is also found in muscles as myoglobin and in other cells as iron containing enzymes

43 43 Amount of iron Male Female % in average adult (g)(g) of total Hb2.41.765 ferritin & hemosiderin 1.00.330 Myoglobin0.150.123.5 Heme enzyme0.020.150.5 Transferrin-bound0.0040.0030.1 iron The distribution of body iron

44 44 Iron Deficiency Anemia (IDA) Sequence of iron depletion When iron loss or use exceeds absorption, there is a sequence of iron depletion in the body: 1.Storage iron decreases/ low serum ferritin; serum iron & TIBC are normal, no anemia, normal red cells. 2.Serum iron decreases/TIBC increases (increased transferrin); no anemia, normal red cells. 3.Anemia with microcytic/hypochromic red cells = IDA.

45 45 CLINICAL FEATURES IRON DEFICIENCY Symptoms eg. fatigue, dizziness, headache Signs eg. pallor, Tongue atrophy/ glossitis - raw and sore, angular cheilosis (Stomatitis) Koilonychia Glossitis Angular Cheilosis or Stomatitis

46 46 Clinical signs and symptoms –Spoon ‑ shaped nails (koilonychia), brittle nails and hair.

47 47 Lab Investigation of IDA Iron tests ►Used to differentiate microcytic hypochromic anemia's or detect iron overload (hemochromatosis) –Iron circulates bound to the transport protein transferrin Transferrin is normally ~33% saturated with iron Iron tests include serum iron, Total Iron Binding Capacity (TIBC), serum ferritin

48 48 Lab Investigation cont’d Serum iron level – measures the amount of iron bound to transferrin –Does not include the free form of iron Total Iron Binding Capacity (TIBC) –Is an indirect measure of the amount of transferrin protein in the serum –Inversely proportional to the serum iron level If serum iron is decreased, total iron binding capacity of transferrin increased (transferrin has more empty space to carry iron)

49 49 Lab Investigation cont’d Serum ferritin – indirectly reflects storage iron in tissues –found in trace amount in plasma –It is in equilibrium with the body stores –Variation in the quantity of iron in the storing compartment is reflected by plasma ferritin concentration e.g. Plasma ferritin is decreases in IDA Plasma ferritin increases in ACD Limitation: During infection or inflammation Serum Ferritin increases like other acute phase proteins, and then it is not an accurate indicator in such situations.

50 50 Bone marrow iron (Tissue iron) Tissue biopsy of bone marrow Prussian blue stain Type of iron is hemosiderin

51 51 ABSENT IRON STORES IN BONE MARROW IN IRON DEFICIENCY Iron deficiencyNormal control

52 52 Iron Deficiency Anemia Lab findings –Low RBC, Hgb, Hct –Low MCV, MCH, MCHC –Normal WBC and PLT Blood smear

53 53 Iron Deficiency Anemia RBC morphology –Hypochromia –Microcytosis –Anisocytosis –Poikilocytosis Pencil cells (cigar cells) Target cells –no RBC inclusions Iron parameters –Low serum iron, –High TIBC, –Low serum ferritin Blood smear

54 54 Wright’s stained blood smear Ovalocytes - Pencil forms No RBC inclusions Iron Deficiency

55 55 IDA cont’d Treatment –Identify the underlying cause –Oral iron is given; see increased Retic count post-therapy. –May see dimorphism following treatment a dual red cell population with older microcytic red cells along with the newly produced normocytic red cells.

56 56 B. Sideroblastic Anemia (SA) This group of anemias are characterized by defective protoporphyrin synthesis (blocks) resulting in iron loading and a hypochromic anemia due to deficient hemoglobin synthesis. Block(s) in protoporphyrin synthesis leads to iron overload and microcytic/hypochromic anemia

57 57 Sideroblastic Anemia (SA)

58 58 Terms: Siderocytes are mature RBCs in the blood containing iron granules called Pappenheimer bodies....abnormal. Sideroblasts are immature nucleated RBCs in the bone marrow containing small amounts of iron in the cytoplasm....normal.

59 59 SA Sideroblastic anemia is characterized by the –Accumulation of iron in the mitochondria of immature nucleated RBCs in the bone marrow; –Iron forms a ring around the nucleus  these are called ringed sideroblasts....abnormal. The iron accumulation in the mitochondria is the result of blocks in the protoporphyrin pathway.

60 60 SA cont’d Lab findings: Microcytic/hypochromic red cells, low MCV and MCHC; variable anemia, low retic. RBC inclusions: Basophilic stippling and Pappenheimer bodies (siderocytes). (May see target cells). High serum iron and high serum ferritin (stores); low TIBC and high % saturation. *Decreased transferrin synthesis occurs in iron overload states. Bone marrow: ringed syderoblasts (Hall mark of Sideroblastic Anemia)

61 61 RBC with iron Wright’s stain NRBC with iron Prussian blue stain NRBC with ring of iron Prussian blue stain Pappenheimer bodies Blood Bone marrow Sideroblast Ringed Sideroblast Sideroblastic Anemia (SA)

62 62 Pappenheimer bodies Wright’s stain Blood Basophilic stippling/stippled RBCs Blood Pappenheimer bodies Prussian blue iron stain Blood Sideroblastic Anemia (SA)

63 63 100x Ringed Sideroblasts Prussian blue iron stain Bone marrow 10x Increased stainable iron Prussian blue iron stain Bone marrow Sideroblastic Anemia (SA) Bone marrow findings (if done): 1.*Ringed sideroblasts demonstrated with Prussian blue stain. 2.Increased stainable iron in macrophages.

64 64 Types of Sideroblastic anemia cont’d Primary ‑ cause unknown (can't identify blocks) and are not reversible....called Idiopathic or primary Sideroblastic anemia. 1.Elderly, responds to no treatment. Requires transfusion support if severe anemia. 2.Characterized by a dimorphic red cell population - micro/hypo red cells with 3.normocytic and/or macrocytic red cells....MCV is variable and RDW is high. 4.Primary type of sideroblastic anemia is one of myelodysplastic syndromes called Refractory Anemia with Ringed Sideroblasts; may terminate in leukemia

65 65 Wright’s stained blood smear Stippled RBCs – Lead poisoning Secondary Types of SA Alcohol inhibits vitamin B6/pyridoxine Anti-tuberculosis drugs inhibit vitamin B6 Lead causes multiple blocks –Inhaled or ingested –Abnormal lead level –Neurologic problems –Lead line (gums) –Chelation therapy (EDTA).

66 66

67 67 C. Anemia of chronic disease Anemia of chronic disease (ACD) – inability to use iron and decreased response to EPO –Very common anemia, #2 Associated with systemic disease, including chronic inflammatory conditions: –Rheumatoid arthritis –Chronic renal disease –Thyroid disease –Malignancies –Tuberculosis –Chronic fungal infections etc

68 68 ACD pathogenesis Lactoferrin is an iron biding protein in the granules of neutrophils Its avidity for iron is grater than transferrin During infection or Inflammation, neutrophil- lactoferrin released into plasma and Scavenges available iron Bind to macrophage and liver cells (because they have receptor for lactoferrin Cytokines: Produced by macrophages during inflammation and contribute to ACD by inhibiting erythropoiesis

69 69 Lab Diagnosis Blood findings –Early stage: normocytic normochromic –Late stage: hypochromic microcytic, –Low serum iron, low TIBC, normal or high serum ferritin Leukocytosis Abundant storage of iron in macrophage (Prusian blue)

70 70 Target cells/Codocytes Beta Alpha D. Thalassemias Inherited decrease in alpha or beta globin chain synthesis needed for Hgb A; quantitative defect –All have microcytic/hypochromic RBCs and target cells Genetic mutations classified by: –↓ beta chains = beta thalassemia…Greek/Italian –↓ alpha chains = alpha thalassemia…Asian

71 71 Haemoglobin Molecule Hgb A = 2α & 2β Hgb A 2 = 2α & 2δ Hgb F = 2α & 2γ Consists of 4 globin chains + 4 heme groups Normally, each individual inherits 2α, 1β, 1γ, and 1δ gene from each parent.....so 4α, 2β, 2γ, and 2δ genes are inherited. 97% 2% 1%

72 72 Thalassemia Impaired alpha or beta globin synthesis results in an unbalanced number of chains produced that leads to: –RBC destruction in beta Thalassemia major –Production of compensatory Hgb types in beta thals –Formation of unstable or non-functional Hgb types in alpha thals

73 73 Thalassemia Severity ranges from lethal, to severe transfusion-dependency, to no clinical abnormalities; severity depends on the number and type of abnormal globin genes inherited. 1.Major  severe anemia; no α (or β) chains are produced, so cannot make normal hemoglobin (s). 2.Minor/trait  mild anemia; slight decrease in normal hemoglobin types made.

74 74 Heinz bodies Excess alpha chains Supravital stain Beta Thal Major (Homozygous) Both beta genes abnormal –Marked decrease/absence of beta chains leads to alpha chain excess…no Hgb A is produced –Rigid RBCs with Heinz bodies destroyed in bone marrow and blood (ineffective erythropoiesis)

75 75 Stippled NRBC NRBC Target cell Wright’s stained blood smear HJB Beta Thal Major (Homozygous) Clinical findings Lab findings –Severe anemia, target cells, nucleated red cells –RBC inclusions –No hemoglobin A; compensatory Hgb F

76 76 Pap bodies NRBC Transfused RBC Target cell Hypercellular Bone Marrow (10x) Blood smear Howell-Jolly body Target cells Blood smear Transfused RBC Beta Thal Major (Homozygous) Treatment –Transfusion –Splenectomy –Iron chelation

77 77 Wright’s stained blood smear Stippled RBC Target cell Beta Thal Minor (Heterozygous) One abnormal beta gene –Slight decreased rate of beta chain production –Blood picture can look similar to iron deficiency Lab findings –Mild anemia, target cells, no nRBCs, stippled RBCs –No Heinz bodies –Normal iron tests –Compensates with Hgb A2 Ovalocytes

78 78 Alpha Thal Major/Homozygous Deletion of all 4 alpha genes results in complete absence of alpha chain production –No normal hemoglobin types made Known as Barts Hydrops Fetalis –Die of hypoxia….Bart’s Hgb

79 79 Target cells Wright’s stain blood smear Heinz bodies Excess beta chains Supravital stain Alpha Thal Intermedia = Hgb H Disease Three alpha genes deleted –Moderate decrease in alpha chains leads to beta chain excess…unstable Hgb H –Moderate anemia

80 80 Alpha Thal Minor (Heterozygous) One or two alpha genes deleted (group) –Slight decrease in alpha chain production –Mild or no anemia, few target cells –Essentially normal electrophoresis; many undiagnosed

81 81 Beta Thalassemias

82 82 Alpha Thalassemias

83 83 + HGB Synthesis Defects Differential Diagnosis of Microcytic Anemia

84 84 2. Macrocytic Normocytic Anemias

85 85 Macrocytic Normocytic Anemias Characteristics ?????? Wright’s stained blood smear

86 86 A. MEGALOBLASTIC ANEMIA Vitamin B12 deficiency Folate deficiency Abnormal metabolism of folate and vit B12 B. Non megaloblastic anemia Liver disease Alcoholism Post splenoctomy Neonatal macrocytosis Stress erythropoiesis

87 87 A. Megaloblastic Anemia Macrocytosis due to a deficiency of vitamin B12 or folic acid that causes impaired nuclear maturation –Vitamin B12 & folate are DNA coenzymes necessary for DNA synthesis and normal nuclear maturation –Results in megaloblastic maturation…nucleus lags behind the cytoplasm and leads unbalanced growth called maturation asynchrony Both deficiencies cause enlarged fragile cells –Many cells die in the marrow (ineffective) –Show a similar blood picture and clinical findings Only vitamin B12 deficiency causes neurological symptoms…required for myelin synthesis

88 88 RBC maturation in microcytic anemias…IDA Normoblastic RBC maturation  normocytic red cells Megaloblastic RBC maturation  macrocytic red cells Megaloblastic Anemia

89 89 Lab Findings of Megaloblastic Anemia Mild to severe anemia, –Increased MCV & MCH, normal MCHC –Low RBC, HGB, WBC and PLT counts (fragile cells) due to ineffective hematopoiesis. –Low reticulocyte count – Macrocytic ovalocytes and teardrops; – Marked anisocytosis and poikilocytosis – Schistocytes/microcytes - due to RBC breakage upon leaving the BM – Erythroid hyperplasia - low M:E ratio (1:1) –Iron stores increased.

90 90 Macrocytic Ovalocytes Blood NRBC Blood Howell-Jolly body Teardrop Schistocyte Stippled RBC & Cabot Ring Giant Platelet Pap bodies Hypersegmented Neutrophil >5 lobes

91 91 Vitamin B12 (Cobalamin) Deficiency

92 92 Vitamin B12 deficiency Occur as a result of one of the following conditions 1.Nutritional Coballamin deficiency Strict vegetarianism 2.Abnormal intragasteric events ( i.e. inadequate proteolysis of food Coballamin) atrophic gastritis. 3.Loss or atrophy of gastric mucosa ( deficient IF) total or partial gasterectomy –May develop B12 and iron deficiency with macro and micro red cells…a dual (dimorphic) RBC population pernicious anemia

93 93 Cont….. 4.Abnormal events in the small bowel lumen –Inadequate pancreatic protease –Competing agents like fish tape worms (D.latum) –Disorders of ileac mucosa –Diminished or absent of IF – Coballamin receptor –Drug effects –Metabolic disorders ( coballamin is not used by cells)

94 94 Folate (Folic acid) Deficiency: Deficient intake. Increased needs: pregnancy, infant, rapid cellular proliferation, and cirrhosispregnancy infantcirrhosis Malabsorption (congenital and drug-induced) Inherited DNA Synthesis Disorders: Deficient thiamine and factors (e.g. enzymes) responsible for folate metabolism.DNAthiamine Toxins and Drugs: Two RBC populations Dimorphism Macrocytic RBCs Microcytic RBCs

95 95 1. Pernicious Anemia it is defined as anemia resulting from defective secretion of IF associated with autoimmune attack on the gastric mucosa leading to atrophy of the stomach or Abs that block IF action. Abs block the site of IF where vit B12 binds. The diagnosis is confirmed by low serum B12 level and typically abnormal results of schilling test

96 96 Schilling test Used to diagnose pernicious anemia and determine if IF is available. If absorbed a portion of oral dose of vit B 12 (not used by the body) ---- excreted in urine If not absorbed (malabsorption)…….. Not detected in urine but pass out in feces

97 97 Stage I. To diagnose malabsorption give the patient a radio active cobalt labeled vit B12 orally and Non labeled vit B12 intramuscularly to saturate liver If IF is present in the stomach (no other disease) vit B12 is absorbed and labeled vit B12 detected in urine If absorption is impaired labeled vit B12 not detected in urine instead in stool Diagnose malabsorption if it is not appear in urine by proceeding stage II.

98 98 Stage II. Differentiate cause of malabsorption Oral dose labeled vit B12 + IF Appear in urine…… pernicious anemia (give IF) If not appear in urine it is other cause of malabsorption

99 99 Performed to determine if the patient suffer from mal absorption of the IF – B12 complex secondary to small intestinal bacterial overgrowth. If this is the condition then tetracycline should normalize vit B12 absorption. Stage III

100 100 Polychromatophilic RBCs Wright’s stain NRBC B. Non-Megaloblastic Anemia Macrocytosis that is NOT due to vitamin B12 or folate deficiency Accelerated erythropoiesis –Regenerating marrow or marked reticulocyte response following recent blood loss

101 101 Stomatocytes, Alcoholic Echinocytes Acanthocytes Target cells Non-Megaloblastic Anemia Liver disease and alcoholism –Complex & multiple problems –Degree of anemia varies, round macrocytes –Target cells/acanthocytes - due to abnormal lipid metabolism. –Echinocytes are also commonly found on the smear in liver disease.

102 102 Blood smear Differential Diagnosis of Macrocytic Anemia Megaloblastic and non-Megaloblastic –Perform B12 and folate levels –Specific morphology

103 103 3. Normocytic Normochromic Anemia

104 104 3. Normocytic Normochromic Anemia It includes Aplastic anemia due to BM failure Blood loss anemia Hemolytic anemia Is a condition in which the size & Hgb content of RBCs is normal but the number of RBCs is decreased.

105 105 A. Aplastic Anemia –Condition of blood pancytopenia caused by bone marrow failure…decreased production of all cell lines and replacement of marrow with fat. –Due to damaged stem cells, damaged bone marrow environment or suppression –No extramedullary hematopoiesis

106 106 Types of aplastic anemia –Primary/idiopathic = 50% –Secondary/acquired….chemicals, drugs, infections, radiation = 50% –Congenital….Fanconi’s Aplasia plus dwarfism, skeletal abnormalities, mental retardation, abnormal skin pigmentation.

107 107 Bone marrow, decreased # precursor cells 10X Normal RBCs No Platelets Blood Lab diagnosis of Aplastic Anemia Normochromic –Normocytic RBC (normal MCV & MCH) Low reticulocyte count & Hgb Pancytopenia No abnormal cells Hypoplasia Bone marrow Normal Serum iron, vitamin B12 and folate levels

108 108 B. Hemolytic anemia Result from an increase in the rate of pre mature red cell destruction. Compensated hemolytic disease Uncompensated hemolytic disease It leads to –Erythropoietic hyperplasia –BM produces red cells 6 to 8X the normal rate –Marked reticulocytosis

109 109 Hemolytic anemia Two main mechanisms for RBC destruction in HA –Intravascular hemolysis: in the circulation –Extravascular hemolysis: in RE system (reticuloendothelial system)

110 110 Extravascular hemolysis Aged RBC 120 day Abnormal RBC During destruction RBC releases Hgb Hgb Exstravascularly removed by Macrophage (RES) in BM, liver and spleen Iron reabsorbed Globin Amino acid Protein synthesis Protoporphyrin Unconjugated bilirubin liver (glucuronic acid) conjugated bilirubin gut reabsorbed & Excreted as urobilin & urobilinogen

111 111 Extravascular hemolysis Lab features –Increased RBC break down Serum bilirubin increase Stool urobilinogen increase Blood urobilinogen increase Urine urobilinogen increase

112 112 Intravascular hemolysis Red cells are destroyed in blood vessels and Hgb is released into the circulation: Free Hgb Saturates plasma haptoglobin Excess free Hgb is filtered by the glomerules (kidney) (if rate of hemolysis saturates renal reabsorption capacity) Free Hgb enters urine Fe is released in bladder tubule Renal tubule loaded with hemosiderin

113 113 Intravascular hemolysis Lab features –Hemoglobinemia and hemoglobinuria –Hemosiderin uria –Reduced/absent serum haptoglobin

114 114 1. Hereditary hemolytic anemia This is a congenital hemolytic anemia. some of which present at birth and other later in life, while still others may remain silent unless a physiological stress is super imposed Result of intrinsic red cell defects –Membrane defect ( Hereditary Shperocytosis, Elliptocytosis and sickle cell anemia) –Metabolic defect : G6PDH and PK defic –Hgb chain defect (hemoglobinopatheis) : sickle cell anemia

115 115 Spherocytes A. Hemolytic Anemias due to Membrane Defects Most common is Hereditary Spherocytosis (HS) –Membrane defect is decreased spectrin and increased permeability of membrane to sodium ions Lab findings –Anemia varies –Few to many spherocytes on smear, high MCHC –Increased OF test

116 116 H Ovalocytosis Normocytic ovalocytes H Ovalocytosis/Elliptocytosis Membrane defect is polarization of cholesterol or hemoglobin at ends and increased sodium permeability Over 25% ovalocytes –Most asymptomatic –Mild anemia in 10- 15%

117 117 H Stomatocytosis Hereditary Stomatocytosis Membrane defect is abnormal permeability to sodium and potassium Caused by edema 20-30% stomatocytes on blood smear –Mild to severe hemolytic anemia

118 118 H Acanthocytosis = Abetalipoproteinemia H Acanthocytosis Defect is increased membrane cholesterol due to abnormal plasma lipids Numerous acanthocytes on smear –Mild anemia –Also known as abetalipoproteinemia

119 119 Hypotonic Osmotic Fragility Test (OF) Most commonly used to diagnose Hereditary Spherocytosis –Red cells are placed in hypotonic solutions

120 120 Osmotic Fragility Test (OF) Decreased Surface: Volume Ratio “Easy to Lyse” Increased Surface: Volume Ratio “Hard to Lyse”

121 121 B. Defect red cell metabolism (Enzyme defect) G6PD deficiency –G6PD is the only source of NADPH in red cell –NADPH is reduced for the production of reduced Glutathione. –Hgb and RBC membrane are usually protected from oxidant stress by reduced glutathione (GSH) –In G6PD deficiency NADPH and GSH synthesis is impaired, rendering the red cells vulnerable to oxidant stress. –Most individuals with G6PD are asymptomatic except during oxidant stress resulting from drugs or other causes.

122 122 B. Hemolytic Anemias due to Enzyme Defects Inherited enzyme deficiencies that lead to premature RBC death

123 123 PK Deficiency Echinocytes Hemolytic Anemias due to Enzyme Defects PK deficiency –↓ATP impairs cation pump –Severe hemolytic anemia –Echinocytes G-6-PD Deficiency –Unable to protect Hgb due to decreased NADPH –No clinical problems unless exposed to oxidants –Exposure to oxidants induce Heinz body formation and RBC destruction Normal RBCs if no exposure to oxidant G-6-PD Deficiency

124 124 G-6-PD deficiency after exposure to oxidant Heinz bodies - denatured Hgb Supravital stain G-6-PD deficiency Hemolytic episode Damaged RBCs Wright’s stain G-6-PD Deficiency Blood findings after oxidant exposure: –Mod to severe anemia –Schistocytes, spherocytes due to pitting out of Heinz bodies by spleen Enzyme assay

125 125 Target cells/Codocytes C. Normocytic anemias due to hemoglobinopathies Inherited hemoglobin defect with production of structurally abnormal globin chains; –All have target cells Beta chain amino acid substitution = variant Hgb –Hgb S = valine substituted for glutamic acid @ 6th of ß –Hgb C = lysine substituted for glutamic acid @ 6th of ß

126 126 HGB S Disease (Hgb SS) Sickle cell Target cell Hemoglobin S Disorders –Two sickle cell genes inherited (both beta chains are abnormal) –Symptomatic after 6 months of age Lab findings –Severe anemia –Targets, sickle cells –NRBCs, inclusions –No Hgb A, >80% Hgb S, ↑ F A. Hemoglobin S disease/Sickle cell anemia/Hgb SS

127 127 Target cells only NO Sickle cells HGB S Trait (Hgb SA) Hemoglobin S Disorders –One sickle cell gene inherited Lab findings –Asymptomatic, targets only –No anemia or sickle cells –~60% Hgb A, ~40% Hgb S Potential problems if hypoxic B. Hemoglobin S trait/Sickle cell trait/Hgb SA

128 128 C crystals HGB C Disease (Hgb CC) Target cell Hemoglobin C Disorders Lab findings –Mild anemia –Many target cells –Intracellular C crystals –No Hgb A, >90% Hgb C –Decreased OF A. Hemoglobin C disease/Hgb CC Two C genes inherited (both β chains are abnormal) C crystals polymerize differently and look like blocky Hgb packed rods in the red cells....intracellular.

129 129 HGB C Trait (Hgb CA) Target cells only NO C crystals Hemoglobin C Disorders B. Hemoglobin C trait/Hgb CA –One C gene inherited Lab findings –Asymptomatic, no anemia –Targets, no C crystals –~60% Hgb A, ~40% Hgb C –Normal Hgb A2 and F

130 130 SC Crystals Target cells HGB SC Disease (Hgb S & Hgb C) Hemoglobin SC Disease Lab findings –Intermediate in severity between Hgb SS & SA –Several target cells –Many SC crystals –No Hgb A, ~50% Hgb S, ~50% Hgb C, ↑ F Hemoglobin SC disease/Hgb SC One sickle gene and one C gene inherited Double heterozygote ‑ inherit sickle gene (S) from one parent and C gene from other parent; Both β chains are abnormal

131 131 2. Acquired hemolytic anemia A variety of acquired conditions result in shortened survival of previously normal red cells. These include immune mediated destruction, red cell fragmentation disorders, acquired membrane defects, spleen effects Result of extrinsic causes –Immune HA; warm HIHA, cold AIHA –Drug associated –Infection associated

132 132 Spherocytes & polychromasia Blood Warm Autoimmune HA (WAIHA) Altered immune response causes production of an IgG warm autoantibody against ‘self’ RBC antigens –Antibody/complement attaches to RBC antigen…partially phagocytosed (loss of membrane)  spherocytes Cause: Primary (idiopathic) or secondary to disease

133 133 Ingestion of coated RBC RBC Electron Microscopy Blood Monocyte with ingested RBC RBC Warm Autoimmune HA (WAIHA) Lab findings –Mod to severe anemia, spherocytes, high MCHC –Erythrophagocytosis –Looks similar to H spherocytosis but positive DAT –Increased OF, bilirubin –Erythroid hyperplasia

134 134 50x RBC Agglutination 100x Cold Autoimmune HA (CAIHA) Altered immune response causes production of an IgM cold autoantibody against ‘self’ RBC antigens –Antibody/C3 attaches to RBC antigen  agglutination (lysis by complement or macrophage) Primary (idiopathic) or secondary to disease

135 135 Cold Autoimmune HA (CAIHA) Lab findings –Agglutination of red cells in extremities....ears, toes, nose  tissue damage  gangre –Severity varies with seasons….avoid the cold –IgM antibodies cause RBC agglutination –Reticulocytosis –Positive Direct Antiglobulin Test (detects complement)

136 136 Hemolytic Transfusion Reaction Incompatible blood transfusion –Recipient has antibodies to antigens on the donor red cells received –Donor cells are destroyed ABO worst –Intravascular hemolysis that is complement-induced lysis…immediate –Can be life-threatening

137 137 Hemolytic Disease of the Newborn Caused by maternal IgG antibodies directed against baby RBC antigens –Antibodies cross placenta and destroy fetal red cells HDN due to Rh incompatibility –Rh negative mother forms Rh antibody after exposure –HDN due to Rh Sever anemia Many nucleated red cells HDN due to ABO incompatibility –Mother’s ABO blood type is O; baby is type A or B –HDN due to ABO Mild, no anemia Spherocytosis

138 138 Schistocytes Fibrin Strands RBC RBC fragmentation on fibrin strands Hemolytic Anemias due to Trauma Fragmentation syndromes…most common finding on smear are schistocytes; anemia varies Types of trauma –Mechanical…prosthetic heart valves/cardiac abnormalities –Microangiopathic (MAHA)…small vessels (DIC.........bleeding) –March hemoglobinuria…forceful contact…. Schistocytes

139 139 Normocytic/normochromic Hemolytic Anemias due to Trauma – Fragmentation Syndromes A.When RBCs are exposed to excessive trauma within the cardiovascular system, they may undergo fragmentation and lysis. Schistocytes are the most common finding on the smear; RBC destruction tests are abnormal. Severe trauma causes intravascular hemolysis. B.Three types of trauma: 1. Mechanical trauma A. Prosthetic heart valves or cardiac abnormalities fragment red cells. B. Mod to severe anemia with schistocytes and polychromasia.

140 140 Normocytic/normochromic Hemolytic Anemias due to Trauma – 2. Microangiopathic hemolytic anemia (MAHA) – trauma occurs in small vessels A.Disseminated Intravascular Coagulation (DIC) is a widespread clotting disorder initiated by conditions such as OB obstetrical) complications or sepsis. In DIC, clotting factors and platelets form fibrin  fibrin deposited in the microvessels fragment red cells. See anemia with schistocytes on smear, decreased platelets and depletion coagulation factors  leads to severe bleeding; can be fatal..

141 141 2. Microangiopathic hemolytic anemia (MAHA) cont’d…….. B.Hemolytic uremic syndrome (HUS) - most often occurs in children following GI infection (E. coli); noted for renal failure  fibrin damages kidney; hemolytic anemia with echinocytes and schistocytes, decreased platelets; often requires dialysis; can be fatal

142 142 Normocytic/normochromic Hemolytic Anemias due to Trauma – 3. March Hemoglobinuria A.Transient, occurs after forceful contact of body with hard surfaces....joggers, soldiers after long march, bongo drum players. B.Hemoglobinuria; schistocytes may be present on smear.

143 143 Schistocytes & Spherocytes Hemolytic Anemias due to Infectious Agents, and Thermal Burns Anemia varies, with severe hemolysis –Schistocytes and spherocytes on blood smear –Parasitize RBC, elaborate lytic toxins or cause direct damage to red cell membrane Malaria fever Closteridal infections..release toxins

144 144 Normocytic/normochromic Hemolytic Anemia due to Infectious Agents A. Malarial infection 1. Damage to membrane occurs when parasite is pitted out of red cell by splenic macrophages or the entire RBC is removed; chills and fever as red cells rupture. 2. P. falciparum causes severe hemolysis called Blackwater fever.

145 145 Normocytic/normochromic Hemolytic Anemia due to Infectious Agents B.Clostridial infections - Clostridia elaborate toxins which damage RBC membrane causing severe intravascular hemolysis. Both malaria and clostridial infections are characterized by schistocytes and spherocytes on the blood smear; other infectious organisms can cause hemolysis, e.g. Toxoplasma, Bartonella, Babesia, Ehrlichia.

146 146 END OF ANEMIA