1. Clinical laboratory diagnostics of anemias

2. Actuality of theme: Anemia is very often met in 20% women of the developed countries and in 50% women of the non- developed countries; Anemia is very often met in 20% women of the developed countries and in 50% women of the non- developed countries; More than 50% patients with chronic diseases and tumors; More than 50% patients with chronic diseases and tumors; Considerably worsens quality of life and capacity. Considerably worsens quality of life and capacity.

3. Anemia - is the decreasing of hemoglobin and red blood cells level in the unit of blood volume Reduction in one or more of the major red blood cell (RBC) measurements: Hemoglobin concentration Hematocrit RBC count From data of WHO: a hematocrit less than 40 in men and 37 in women, or hemoglobin less than 130 g/l in men and less than 120 g/l in women. Anemia - is the decreasing of hemoglobin and red blood cells level in the unit of blood volume Reduction in one or more of the major red blood cell (RBC) measurements: Hemoglobin concentration Hematocrit RBC count From data of WHO: a hematocrit less than 40 in men and 37 in women, or hemoglobin less than 130 g/l in men and less than 120 g/l in women.

4. Anemia Decrease in the number of circulating red Decrease in the number of circulating red blood cells Most common hematologic disorder by far Most common hematologic disorder by far Almost always a secondary disorder Almost always a secondary disorder As such, critical for internist to know how As such, critical for internist to know how to evaluate/determine cause

5. Anemia Symptoms Because a low red blood cell count decreases oxygen delivery to every tissue in the body, anemia may cause a variety of signs and symptoms. It can also make almost any other underlying medical condition worse. If anemia is mild, it may not cause any symptoms. If anemia is slowly ongoing (chronic), the body may adapt and compensate for the change; in this case there may not be any symptoms until the anemia becomes more severe. Because a low red blood cell count decreases oxygen delivery to every tissue in the body, anemia may cause a variety of signs and symptoms. It can also make almost any other underlying medical condition worse. If anemia is mild, it may not cause any symptoms. If anemia is slowly ongoing (chronic), the body may adapt and compensate for the change; in this case there may not be any symptoms until the anemia becomes more severe.

6. Signs of anemia : Signs of anemia : Black and tarry stools (sticky and foul smelling)Black and tarry stools (sticky and foul smelling) Maroon, or visibly bloody stoolsMaroon, or visibly bloody stools Rapid heart rateRapid heart rate Rapid breathingRapid breathing Pale or cold skinPale or cold skin Yellow skin called jaundiceYellow skin called jaundice Low blood pressureLow blood pressure Heart murmurHeart murmur Enlargement of the spleenEnlargement of the spleen Signs and symptoms

7. Symptoms of anemia may include the following: FatigueFatigue Chest painChest pain Abdominal painAbdominal pain Weight lossWeight loss WeaknessWeakness Dizziness and passing out, especially upon standingDizziness and passing out, especially upon standing Signs and symptoms

8. Anemia Symptoms  Fatigue  decreased energy  weakness  lightheadedness  palpitations (feeling of the heart racing or beating irregularly) palpitations  looking pale

9. Symptoms of severe anemia may include:  chest pain, angina, or heart attack chest painanginaheart attack chest painanginaheart attack  dizziness dizziness  fainting or passing out fainting  rapid heart rate

10. Symptoms of severe anemia may include: Change in stool color, including black and tarry stools (sticky and foul smelling), maroon-colored, or visibly bloody stools if the anemia is due to blood loss through the gastrointestinal tract. Change in stool color, including black and tarry stools (sticky and foul smelling), maroon-colored, or visibly bloody stools if the anemia is due to blood loss through the gastrointestinal tract. Change in stool color Change in stool color rapid heart rate rapid heart rate low blood pressure low blood pressure low blood pressure low blood pressure rapid breathing rapid breathing pale or cold skin pale or cold skin yellow skin called jaundice if anemia is due to red blood cell breakdown yellow skin called jaundice if anemia is due to red blood cell breakdown heart murmur heart murmur enlargement of the spleen with certain causes of anemia enlargement of the spleen with certain causes of anemia

11. Depending on the level of hemoglobin in the blood anemia is divided on: - mild degree (Hb 110-90 g/l), - moderate degree (Hb 89-70 g/l), - severe degree (Hb less than 69 g/l). Depending on the size of RBC and their saturation by hemoglobin (from data of colour index - CI) anemia is divided on: - Normocytic anemia (can be normochromic anemia: colour of RBC is normal and CI is 0,86-1,1); - Microcytic anemia (can be hypo- or normochromic): microcytosis, anizopoykilocytosis, hypochromia, CI < 0,7; - Macrocytic anemia: macrocytosis, megalocytosis, CI > 1,1.

12. Approaches to Anemia Kinetic approach Kinetic approach  Decreased RBC production  Increased RBC destruction  Blood loss Morphologic approach Morphologic approach  Macrocytic  Normocytic  Microcytic

13. Decreased RBC Production Nutrient deficiency Nutrient deficiency Dietary, malabsorption Bone marrow disorders/suppression Bone marrow disorders/suppression Anemia of chronic diseases Low levels of trophic hormones Low levels of trophic hormones Epo, thyroid hormone, androgens Epo, thyroid hormone, androgens

14. Increased RBC Destruction

15. Blood Loss Most common cause of anemia Most common cause of anemia Fe deficiency almost always due to blood loss Fe deficiency almost always due to blood loss Obvious bleeding Obvious bleeding Occult bleeding Occult bleeding Induced bleeding Induced bleeding Operative blood loss Operative blood loss

16. Clinical classification of anemia is based on morphological prinsiple - determination of mean corpuscular volume (MCV) of red blood cells MСV- is determined by automatic laboratory analyzers or by such formula: MСV = (hematocrit x10) / amount of RBC (in mln/l).

17. Definition of MEAN CORPUSCULAR VOLUME : the volume of the average red blood cell in a given blood sample that is found by multiplying the hematocrit by 10 and dividing by the estimated number of red blood cells—abbreviation MCV

18. First use size (MCV) to sort the Differential Dx MCV MicroNormoMacro

20. Normocytic anemia (MCV - 80-100) A loss or destruction of RBC is increased -Acute bleeding -Early iron deficiency - Hemolytic anemia - Hypersplenism Decreasing of RBC synthesis -Anemia of chronic diseases (most commonly) -Endocrine dysfunctions -Renal insufficiency Pathology of bone marrow (for example, action of medications, infection, aplastic anemia, myelodysplastic syndrome, multiple myeloma and other infiltrative diseases).

21. Anemia of Chronic Disease Common Common Develops over 1 to 2 months Develops over 1 to 2 months Non-progressive Non-progressive Usually mild to moderate Usually mild to moderate – but hematocrit < 0.20 occasionally – but hematocrit < 0.20 occasionally 30% mildly microcytic 30% mildly microcytic WBC, platelets normal or increased WBC, platelets normal or increased

22. ANEMIA OF CHRONIC DISEASE Thyroid disease Thyroid disease Collagen Vascular Disease Collagen Vascular Disease – Rheumatoid Arthritis – Rheumatoid Arthritis – Systemic Lupus Erythematosus – Systemic Lupus Erythematosus – Polymyositis – Polymyositis – Polyarteritis Nodosa – Polyarteritis Nodosa Inflammatory Bowel Disease Inflammatory Bowel Disease – Ulcerative Colitis – Ulcerative Colitis – Crohn’s Disease – Crohn’s Disease Malignancy Malignancy Chronic Infectious Diseases Chronic Infectious Diseases – Osteomyelitis – Osteomyelitis – Tuberculosis – Tuberculosis Familial Mediterranean Fever Familial Mediterranean Fever Renal Failure Renal Failure

23. Normocytic Anemia (MCV 80-100 fl) Type of anemia Blood filmFerritinFeTIBCMarrow Fe stores Chronic disease* Normochromic, normocytic Nl or ↑↓ ↓Nl or ↑, clumped Early Fe deficien cy Mild anisocytosis hypochromia Nl or ↓↓↑absent *including anemia due to renal disease and AIDS

24. Hemolytic Anemia Anemia of increased destruction Anemia of increased destruction normochromic anemia normochromic anemia – Shortened RBC survival – Shortened RBC survival – Reticulocytosis - Response to increased RBC – Reticulocytosis - Response to increased RBC Destruction Destruction

26. Hemolytic Anemia Coombs’ (DAT) Positive Immune Hemolysis Drug related Hemolysis Transfusion, Infection, Cancer Negative Hemoglobinopathy, G6PD, PK, Spherocytosis, Eliptocytosis, PNH, TTP, DIC

27. Equired hemolytic anemia Reticulocytosis

29. Hereditary disorders include erythrocyte membrane and enzymatic defects and hemoglobin abnormalities. Some hereditary disorders include the following:G6PD deficiency Herediditary spherocytosis Sickle cell anemia Thalassemia

30. Acquired hemolytic conditions can be due to immune disorders, toxic chemicals and drugs, antiviral agents (eg, ribavirin) physical damage, and infections Autoimmune hemolytic anemia (AIHA) may result from warm or cold autoantibody types; rarely, mixed types occur. Most warm autoantibodies are immunoglobulin (Ig) G and can be detected with the direct Coombs test, which is also known as the direct antiglobulin test (DAT)

31. Acquired hemolytic conditions AIHA may occur after allogeneic hematopoietic stem cell transplantation hematopoietic stem cell transplantation Microangiopathic anemia is found in patients with disseminated intravascular coagulation (DIC) or hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. Fragmented erythrocytes (schistocytes) also occur with defective prosthetic cardiac valves.

32. Acquired hemolytic conditions Autoimmune hemolytic anemia and hereditary spherocytosis are classified as examples of extravascular hemolysis because the red blood cells are destroyed in the spleen and other reticuloendothelial organs. Intravascular hemolysis occurs in hemolytic anemia due to prosthetic cardiac valves, G6PD deficiency, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, and paroxysmal nocturnal hemoglobinuria (PNH).paroxysmal nocturnal hemoglobinuria (PNH)

33. Peripheral blood smear with sickled cells

34. Spherocytes. One arrow points to a spherocyte; the other, to a normal RBC with a central pallor.

35. Schistocytes (thrombotic thrombocytopenic purpura).

36. Hemolytic anemia due to pyruvate kinase insefficiency. Reticulocytosis

37. Membranopathy Hereditary microspherocytosis - blood

38. Membranopathy Hereditary eliptocytosis – blood

39. - Usually hypochromic as well  Iron-deficiency anemia  Anemia of chronic diseases(rare)  Sideroblastosis  Hereditary anemia (thalassemia)  Lead poisoning  Deficit of copper, poisoning by zinc Microcytic anemia (MCV less than 80)

42. Iron Absorption Mechanism not well understood Mechanism not well understood Only mechanism to regulate stores is Only mechanism to regulate stores is through absorption Occurs in duodenum & upper jejunum Occurs in duodenum & upper jejunum Heme forms better absorbed than free iron Heme forms better absorbed than free iron – Meat forms better absorbed than plant – Meat forms better absorbed than plant

43. Iron Absorption Stomach acid converts ferric (insoluble) to Stomach acid converts ferric (insoluble) to ferrous (absorbable) state Decreased absorption w/ acid blockers Decreased absorption w/ acid blockers Increased w/ citrate & ascorbate Increased w/ citrate & ascorbate(chelators) Increased absorption w/ orange juice Increased absorption w/ orange juice Decreased w/ plant phytates, tannins, soil Decreased w/ plant phytates, tannins, soil clay, & laundry starch – Pica may exacerbate Fe deficiency – Pica may exacerbate Fe deficiency

44. Iron Absorption Increased erythropoesis greatly enhances Increased erythropoesis greatly enhances Fe absorption – Increased erythropoesis (thalassemia, – Increased erythropoesis (thalassemia, sickle cell) may lead to Fe overload independent of transfusions Co-regulated with other metal absorption Co-regulated with other metal absorption – Lead poisoning -> Fe deficiency -> increased – Lead poisoning -> Fe deficiency -> increased lead absorption

45. Iron Deficiency Causes – Blood loss – Blood loss GI tract GI tract Renal (rare) Renal (rare) Pulmonary (rare) Pulmonary (rare) – Insufficient dietary iron – Insufficient dietary iron Decreased absorption Decreased absorption

46. Consequences of Iron Deficiency Hematologic Microcytic, hypochromic anemic Microcytic, hypochromic anemic Low grade hemolysis d/t stiff RBC Low grade hemolysis d/t stiff RBCmembrane Thrombocytosis w/ plts 500-700k Thrombocytosis w/ plts 500-700k – Erythropoietin may cross react w/ plt – Erythropoietin may cross react w/ plt Precursors Precursors

50. Makrocytic anemia (MCV more than 100 fl) - Megaloblastic anemia (vitamin В12 or folic acid deficiency ) - Toxic effect of chemotherapeutic agents (methotrexate) or other medications (zidovudine (AZT), phenytoin) - Pathology of bone marrow - Chronic abuse by alcohol (toxic effect) - Liver disease

51. Macrocytosis (MCV > 100 fl) Common Common – Drugs (cytotoxics, immunosuppressants, AZT, anticonvulsants) – Drugs (cytotoxics, immunosuppressants, AZT, anticonvulsants) – Alcohol – Alcohol – Liver disease – Liver disease – Reticulocytosis – Reticulocytosis –B12/folate deficiency –B12/folate deficiency – Myelodysplastic syndrome – Myelodysplastic syndrome – Marrow infiltration (malignancy, fibrosis) – Marrow infiltration (malignancy, fibrosis) Less common Less common –Aplasia –Aplasia – Cold agglutinins – Cold agglutinins – Hyperglycemia – Hyperglycemia – Hyperleukocytosis – Hyperleukocytosis

52. Macrocytosis of Alcoholism 25-96% of alcoholics 25-96% of alcoholics MCV elevation usually slight (100-110 fl) MCV elevation usually slight (100-110 fl) Minimal or no anemia Minimal or no anemia Macrocytes round (not oval) Macrocytes round (not oval) Neutrophil hypersegmentation absent Neutrophil hypersegmentation absent Folate stores normal Folate stores normal

53. Megaloblastic Hematopoiesis Marrow failure due to: disrupted DNA synthesis Marrow failure due to: disrupted DNA synthesis & ineffective hematopoiesis Giant precursors and nuclear:cytoplasmic Giant precursors and nuclear:cytoplasmic dyssynchrony in marrow Neutrophil hypersegmentation & macroovalocytes Neutrophil hypersegmentation & macroovalocytes in blood Anemia (and often leukopenia & Anemia (and often leukopenia &thrombocytopenia) Almost always due to Cbl or folate deficiency Almost always due to Cbl or folate deficiency

54. Megaloblastic Anemia Smear Macro-ovalocytic Macro-ovalocytic Polychromasia Polychromasia Hypersegmented neutrophil Hypersegmented neutrophil

55. ANEMIA CAUSES External bleeding: Loss of blood through heavy menstrual bleeding, wounds, as well as stomach ulcers can cause anemia. Iron deficiency: The bone marrow needs iron to make red blood cells. Anemia of chronic disease: Any long-term medical condition can lead to anemia. Kidney disease: The kidneys help the bone marrow to make red blood cells. Pregnancy: Water weight gain during pregnancy dilutes the red blood cells. Poor nutrition: Vitamins and minerals are required to make red blood cells. Alcoholism. Uncommon causes of anemia: bleeding disorders, liver disease, thalassemia, infection, cancer, arthritis, enzyme deficiency, sickle cell disease, hypothyroidism, toxins, or hereditary conditions.

56. The only way to diagnose anemia is with a blood test. Generally, a full blood count is done. Apart from reporting the amount of red blood cells and the hemoglobin level, the automatic counters also measure the size of the red blood cells, which is an important tool in distinguishing between the causes. The only way to diagnose anemia is with a blood test. Generally, a full blood count is done. Apart from reporting the amount of red blood cells and the hemoglobin level, the automatic counters also measure the size of the red blood cells, which is an important tool in distinguishing between the causes.blood testfull blood countred blood cellshemoglobinautomatic countersblood testfull blood countred blood cellshemoglobinautomatic counters Occasionally, other tests are required to further distinguish the cause for anemia. These are discussed with the differential diagnosis. The doctor may also decide to take some other screening blood tests that might identify the cause of fatigue; glucose levels, ESR, ferritin, renal function tests and electrolytes may be part of such a workup. Occasionally, other tests are required to further distinguish the cause for anemia. These are discussed with the differential diagnosis. The doctor may also decide to take some other screening blood tests that might identify the cause of fatigue; glucose levels, ESR, ferritin, renal function tests and electrolytes may be part of such a workup.glucoseESRferritinrenal functionelectrolytesglucoseESRferritinrenal functionelectrolytesDiagnosis

57. Lab tests for anemia may include the following: 1.Complete blood count - Determines the severity of the anemia and is almost always the first test ordered 2.Stool guaiac - Tests for blood in stool 3.Peripheral blood smear - Looks at the red blood cells under a microscope 4.Iron level - Low iron is one of the most common causes of anemia 5.Transferrin level - Looks at a protein that carries iron around the body 6.Ferritin - Looks at the total iron available in the body 7.Folate - A vitamin needed to produce red blood cells, which is low in people with poor eating habits

58. Vitamin B12 - A vitamin needed to produce red blood cells, low in people with poor eating habits Bilirubin - Useful to determine if the red blood cells are being destroyed within the body Lead level - Lead toxicity used to be one of the more common causes of anemia Hemoglobin electrophoresis - Sometimes used when a person has a family history of anemia Reticulocyte count - A measure of new red blood cells produced by the bone marrow Liver function tests - Uncommon tests to determine how the liver is working Bone marrow biopsy - One of the last tests done; looks at production of red blood cellsBone marrow biopsy

59. Thank you for attention!