Blood • Blood has red cells(erythrocyctes) 101 • White cells (leukocytes) • Platelets (thrombocytes)

Allan Platt PA-C, MMSc Emory University PA Program Atlanta GA [email protected] www.EmoryPA.org

Blood Components White Blood Cells • Fight infections • Are increased in infections Plasma 54% • Move inside and outside of blood vessels • Are made in the

White cells and platelets 1%

Red Cells 45%

White Blood Cells Platelets WBC - White Blood Cells 4.5 - 11.0 K/uL Low = Leukopenia High = Leukocytosis • Plug holes in the body to stop bleeding WBC Differential • Can help cause blood to clot • - Segs 54 -62% Made in the bone marrow

Neutrophils - Bands 3 -5 % Lymphocytes - Lymphs 25 - 33% Monocytes - Monos 3 - 7% Eosinophils - Eos 1 - 3% Fibrin Basophils - Basos 0 - 0.75% Atypical Lymphs 0

1 Red Blood Cells Red Blood Cells • Carry oxygen from the lungs • Carry carbon dioxide back to the lungs • Also carry CO and NO • Normally live 120 days • Contains the protein • Made from iron, folic acid, vitamin B12 • Made in the bone marrow

Microscope View Red Blood Cells - Shape

Red cells travel through very narrow blood vessels

Incidence of and World wide http://www.who.int/genomics/public/geneticdiseases/en/index2.html#SCA Red Blood Cells

• Red Cell Flow

Blood Vessel

2 Red Blood Cells - Hemoglobin Globin Synthesis in utero

Oxygen Normal Hemoglobin A has 2 alpha and 2 beta globin chains with 4 iron binding sites

Hemoglobin FA – Biochemistry – Normal Newborn Hemoglobin A – Biochemistry – Normal Adult

Chromosome 16 Chromosome 11 Chromosome 16 Chromosome 11 alpha alpha gamma gamma delta beta Mom alpha alpha gamma gamma delta beta Mom

alpha alpha gamma gamma delta beta Dad alpha alpha gamma gamma delta beta Dad

alpha alpha alpha alpha beta beta 50 – 20 % Hemoglobin A beta beta 95 - 97% Hemoglobin A

Hb F (newborn): 50% to 80% alpha alpha alpha alpha 1 – 2 % Hemoglobin F or Fetal gamma gamma (6 months): 8% gamma gamma (over 6 months): 1% to 2%

alpha alpha 2% Hemoglobin A2 alpha alpha 2 - 3% Hemoglobin A2 delta delta delta delta

Red Blood Cells - Marrow Red Blood Cells - Retics • , or Retics are young red cells just released from the bone marrow. The Retic count is the best Red cells, white indicator about how the marrow factory is doing. cells and platelets are made in the bone marrow

3 Food with iron Red Blood Cells Red Blood Cells - Recycled Red cells are Red cells are recycled in the and vitamins spleen and liver. The iron made in the is digested and protein are stored and bone marrow bilirubin is released.

Liver

Red cells live 120 days in the circulation

Spleen

Hemoglobin Recycled Red Blood Cells - The Kidney Bilirubin must be Directed through the Liver to be Erythropoietin is made by Conjugated the kidney as a signal to Hemoglobin LDH - Lactate the bone marrow to make Indirect Dehydrogenase more red cells Bilirubin Iron – Fe bound to Transferrin Kidney - Liver Hemoglobinuria

Direct Bilirubin

Increased levels blocks absorption of Hepcidin The History Iron and cell release - inflammation IL6 • Weakness Hormone made in the • Tiredness - Fatigue liver • Dyspnea • Dizzy – non vertigo Decreased levels • Palpitations increase iron • New angina absorption and release from cells – Erythropoetin, low iron

4 The History -2 The History - 3

• - History of melena, abdominal pain, Aspirin or non-steroidal anti- • - History of abuse - consider deficiency or liver disease. If inflammatory agents (NSAIDs) use, past peptic ulcer disease , then moonshine use or lead paint/pipe exposure, consider lead toxicity. consider GI bleeding, platelet dysfunction. • - Family history of blood cell or bleeding disorder: consider Sickle • - In females the menstrual history quantifying the amount of Cell disease, G6PD,Thalassemia, Hemophilia, von Willebrand bloodloss ,or possible pregnancy should be obtained. • - History of jaundice, transfusion, new medication, infection - • - History of pica or abnormal craving for ice, clay, starch...; dysphagia consider hemolytic process then consider iron deficiency. • - History of weight loss, Cancer, HIV, rheumatoid arthritis, thyroid • - Poor diet, then consider iron or folate deficiency, and general disease, renal disease -then consider secondary cause • malnutrition • - History of fever and chills, cough, dyspnea, then consider • - History of gastric surgery, distal paresthesias, gait problems - Infection. consider B12 deficiency •

Physical Exam Sclera

Spoon Nails – Fe Def. Glossitis and Chelosis – Fe and B12

5 Physical Exam LAB- INITIAL SCREENING TESTS • GENERAL INSPECTION- clubbing in TB or lung cancer • Skin- Hypothyroid, SLE, Bruises, lesions, petechiae or purpura. • Weight - Loss in Cancer, HIV, Chronic disease, gain in hypothyroid

• VITAL SIGNS- Pulse: Tachycardia from increased cardiac output • Urinalysis- Hematuria/proteinuria in renal disease • Respirations: Tachypnea from decreased oxygen transport hemoglobinuria in . • BP: Orthostatic if volume depleted • CBC, red cell morphology and white blood cell differential, • Temp: Fever in infections and drug or transfusion reactions, count • HEENT- Eye: Jaundice if hemolysis, pallor in palpebral conjunctiva • Chemistry profile (LDH, Bilirubin- Direct and Indirect, BUN, • Mouth: Glossitis and angular stomatitis in iron or B12 deficiency Creatinine, GPT), • NECK- Thyroid enlargement or nodules, lymph nodes • Hemoglobin Electrophoresis if hereditary is • HEART- Increased output/murmur- consider high output failure suspected • LUNG- consider infection, lesion • ABDOMINAL- Liver/spleen size, masses, tenderness, surgical scars • IF BLEEDING - Platelet Count, PT, aPTT, PFA • RECTAL- Stool guaiac, prostate exam in men • PELVIC/BREAST- Uterine abnormality, Pap smear, Breast nodule • LYMPHNODES- consider lymphoma,leukemia,infection,connective tissue Disease • NEUROLOGIC- Decreased vibratory and position sense in B12 deficiency

CBC values by age CBC- Red Cell Measures PARAMETER NORMAL ADULT COMMENTS HB - Hemoglobin Male= 15.5 +/- 2 mg/dl Low = Anemia Female = 13.5 +/- 2 High = HCT - Hematocrit Male= 46.0 +/- 6% " Female= 41.0 +/- 6% " RBC - Red Blood Male = 4.3 - 5.9 Million/uL High in Thalassemia Cell Count Female = 4.0 - 5.2 "

Red Cell Indices MCH, MCHC Red Cell Indices MCV - RDW MCH - Mean Corpuscular 27 -32 pg Low = Hypochromic MCV - 80 - 94 fl Hemoglobin High = Hyperchromic Low = Microcytosis High = MCHC - Mean Corpuscular 30 - 36 gm/dl Low = R/O Fe def. RDW - Red Cell Distribution Width 11.5 - 14.5 Variation in RBC size Hemoglobin Concentration High = Spherocytosis

6 RBC Morphology Red Cell Morphology SIGNIFICANCE Platelets Burr Cells Uremia, Low K, artifact, Ca stomach, PUD Spur Cell Post-splenectomy, Alcoholic liver disease Stomatocyte Hereditary, Alcoholic liver disease, • Platelet Count 150 - 400 K cell/uL Spherocyte Hereditary, Immune , Low = Thrombocytopenia water dilution, post-transfusion Shistocyte - helmet TTP, DIC, vasculitis, glomerulonephritis, High = Thrombocytosis heart valve, burns Eliptocyte - Ovalocyte Hereditary, Thalassemia, Fe Def., Myelophthistic, megaloblastic Sickle Cells Sickle cell disease Target Cells , Microcytes Thalassemia, Iron Def., Lead Toxic, Macrocytes B12 of Folate Def. Parasites Malaria, Babesiosis, Bartonellosis

Retics or Reticulocyte count Correcting the Retic • absolute reticulocyte count (measured) • reticulocyte (%) = absolute number of reticulocytes ÷ number • Retic - Reticulocyte Count 0.5 -1.5 % of RBC × 100 Low in anemia = low marrow output • reticulocyte index = % reticulocytes × actual hematocrit ÷ High = RBC loss normal hematocrit • corrected reticulocyte index (corrects for appropriate bone marrow release of reticulocytes) = reticulocyte index ÷ maturation factor • maturation factor = 3.25 – (actual hematocrit ÷ 20) – if hematocrit 45, maturation factor = 1 – if hematocrit 35, maturation factor = 1.5 – if hematocrit 25, maturation factor = 2 – if hematocrit 15, maturation factor = 2.5

Anemia Diagnosis Diagnostic Pathway

• Loosing red cells (high retic count) Reticulocyte Production Index – Bleeding <2 Decreased Production >2 Increased Loss – Hemolysis (High indirect Bili and LDH) Red Cell Indicies MCV Hemolysis Bleeding • Not making enough – (low retic count) >94 80-94 <80 – Low materials – Fe, B12, Folate Macro Normo Micro Extrinsic Intrinsic

– Low epo (Kidney disease) Coombs CoombsMembrane Hb Enzyme Positive Negative – Marrow problem (replaced, toxin….) Drug Warm Cold Antibody Antibody

7 Anemia – low Hb/Hct Lab work-up Low Retic count anemias BPH = Bleeding/Production/Hemolysis

Anemic- Lab – Next look at the MCV on the CBC CBC, Retic, RBC morphology, Metabolic Profile, • UA MCV < 80 Microcytic

Retic Production Retic Production • MCV 80 – 94 Normocytic index < 2 – Marrow Index >2 RBC Loss Production Problem Bleeding or Hemolysis Check MCV • MCV > 94 Macrocytic

MCV <80 – Increased Indirect MCV 80 – 100 MCV > 100 Microcytic Bilirubin and LDH = Normocytic Macrocytic Order Iron Order West Sed Hemolysis Order B12, RBC Bleeding studies, HbELP, Rate, TSH, Renal and serum Folate Order Coombs, Hinze Lead Level Hepatic, Preg Test Body stain, HbELP

Microcytic Tests Microcytic • MICROCYTIC = "TICS" TESTS TO ORDER: • Serum Iron

• T-Thalassemias • TIBC = Transferrin binding sites • I-Iron Deficiency • C-Chronic Inflammation • % Saturation = Transferrin saturation with Iron • S-Sideroblastic - lead, drug, or hereditary • Ferritin = Storage Iron

• HBELP = Hemoglobin Electrophoresis

• Lead level if exposed

Microcytic workup Ferritin by age TICS – Thalassemia, Iron Deficiency, Chronic inflammation, Sideroblastic (Lead)

Iron studies (Ferritin) Low

No, West Sed rate No, Lead Level Yes = Iron No, Abnormal elevated- elevated – Deficiency HbELP? • Too Low – Iron deficiency Inflammatory Block Chelation therapy • Too high >2,000 Iron overload (20 – 25 Yes = Thalassemia – Work up for Refer to Refer to Chronic Blood loss Diet Hematologist for hematologist if transfused units) Need to chelate iron – GI, Menses Bone Marrow Bx severe

8 Thalassemia Syndromes. Iron deficiency • Hereditary – Alpha or Beta chain • Low Serum iron, Low • Decrease Hemoglobin A Ferritin, High TIBC • Hemoglobin ELP • Find out why –GI bleed, abnormal and normal Ferritin are diagnostic menses, diet • Hemolysis (increased • Treat FeSO4 300mg tid indirect Bili and LDH) • Add vitamin C to • Target Cells increase absorbtion • Supportive therapy or BMT • Follow up Retic increase 1 week, Ferritin 1 month

Chronic Inflammation Sideroblastic • Block of normal iron stores transport to bone • Ring sideroblasts in bone marrow factory marrow • Normal Ferritin, serum iron and TIBC are low • Serum iron is increased with a low saturation and TIBC normal resulting in a high saturation. • 30% Microcytic, 70% Normocytic Serum ferritin is • High Sed rate or c-reactive protein increased • Basophillic stippling • Treat inflammation – RA, SLE, HIV…. • Lead toxicity is suspect

Normocytic Anemia Normocytic Tests

• NORMOCYTIC = "NORMAL SIZE" • Blood Urea Nitrogen (BUN), Creatinine, • N-Normal Pregnancy SGOT, Alkaline Phosphatase, Bilirubin, • O-Over hydration, Drowning Erythrocyte Sedimentation Rate (ESR), • R-Renal Disease • M-Myelophthistic – Marrow replaced Urinalysis, and Thyroid profile • A-Acute Blood Loss • L-Liver Disease • Renal Function tests • Pregnancy Test • SI-Systemic Infection/Inflammation • Z-Zero Production- • Bone Marrow Biopsy • E-Endocrine: Hypothyroid, hypoadrenal, decreased androgen

9 Normocytic workup Normocytic - Renal Failure “NORMAL SIZE” • Anemia caused by decrease erythropoetin production causing decreased bone marrow Check BUN/Creat/ Liver, UA, West Sed Rate, Preg Test production • Can monitor erythropoetin levels West Sed rate BUN/Creat elevated TSH elevated = AST/ALT/AlkP – Pregnancy test + elevated- Pancytopenia or abnormal UA Hypothyroid Liver disease Inflammatory Block Prenatal care • Treat with epoetin alfa injections weekly or

Work up for Renal Refer to No - Repeat CBC, darbepoetin alpha every other week or Disease and Low Hematologist for Retic in 2 week EPO Bone Marrow Bx monthly

Parvo – Fifth’s Disease Macrocytic Anemia • MACROCYTIC = "BIG FAT RED CELLS“ Or my “BF”

• Red cell production shut down in the bone • B-B12 Malabsorbtion • I-Inherited marrow for 5 – 10 days • G-Gastrointestinal disease or surgery • • F-Folic Acid Deficiency • May cause hemolysis • A- • T-Thiamine responsive • May cause severe anemia in patients with • • R-Reticulocytes miscounted as large RBCs chronic hemolysis like Thalassemia and Sickle • E- Endocrine - hypothyroid • D-Dietary cell. Use supportive transfusions. • C-Chemotherapeutic Drugs • E-Erythro Leukemia • L- Liver Disease • L- Lesch-Nyhan Syndrome • S-Splenectomy

Macrocytic Tests Macrocytic Work-up

• The peripheral blood changes include:

• -Anemia with decreased reticulocyte count, -Increased MCV Serum B12 • -Neutropenia with hypersegmented RBC/Serum Folate • Neutrophils • -Thrombocytopenia with large platelets.

B12 nromal/Folate normal - Order B12 low /Folate low = Metylmalonic Acid and B12 Deficiency or both • LABS to order: Homocyteine levels • B12, Serum Folate, RBC Folate • if all normal, consider Metylmalonic Acid and Homocyteine Consider Liver disease, Replace with oral, levels, TSH, and a Bone Marrow Bx. Elevated in early B12 hypothyroid, Drugs, nasal or IM B12 and Deficiency Toxins – Refer for BM Folate Bx

10 B12 Cobalamin Deficiency Folate Deficiency

Physical signs include edema, pallor, jaundice, smooth tongue, • Causes - liver disease, diet vitamin decreased vibratory and position sensation B12 deficiency (needed as co-factor), Hypersegmented polys and drugs such as , Low serum B12 level ethanol, and dilantin. Metformin, Gastric bypass, or PPI as cause? • Lab – low serum and RBC Folate Methylmalonic acid and homocysteine levels • Rx – Folate 1mg po qD Pernicious anemia - anti- intrinsic factor antibodies Schilling's test • Rx - cobalamin 1000 mg I.M., oral, or Nasal Spray

Hemolytic Anemia • HEMOLYTIC = "HEMATOLOGIST" Hemolysis (HIT) • Hereditary (HEM) • H-Hemoglobinopathy: sickle cell disease • - Hemoglobinuria: Paroxysmal Nocturnal Hemoglobinuria – Hemoglobin (sickle cell, thalassemia) • E-Enzyme Deficiency – Enzyme (G6PD deficiency) • M-Medication - drug induced: aldomet, INH • A-Antibodies - Immune attack – Membrane (Spherocytosis, Eliptocytosis) • T-Trauma to the red cells: D.I.C , artificial heart valves • O-Ovalocytosis • Immune attack – Coombs positive (transfusion, • L-Liver disease IgM – cold antibody-infections, IgG warm antibody • O-Osmotic fragility in • and in Hereditary Eliptocytosis – Drug induced, PNH) • G-G6PD Glucose-6-Phosphate Dehydrogenase Deficiency • Trauma– Microangiopathic ( TTP, ITP, HUS, DIC, HIT, • I-Infection: malaria, babesiosis • S-Splenic destruction in hypersplenism HELLP- Eclampsia, Malaria, Splenomegaly) • T-Transfusion • - Thalassemias

Hemolytic Signs Hemolytic Tests • 1. The direct antiglobulin (Coombs') test Direct Coombs • 1. Elevated reticulocyte count, with stable or falling hemoglobin. test looks for antibody on the red cells. The Indirect • Coombs looks for antibody in the serum. • 2. Elevated indirect bilirubin -

• 3. Eevated serum lactate dehydrogenase (LDH)- • 2. Hemoglobin electrophoresis

• 4. Decreased Haptoglobin levels - Haptoglobin binds hemoglobin released in the plasma from red cell breakdown. • 3. Heinz body stain

• 5. Hemoglobinemia and hemoglobinuria • 4. Osmotic fragility • 6. Erythroid hyperplasia in bone marrow in chronic hereditary causes

• 7 Abnormal Hemoglobin Electrophoresis • 5. Blood smear

11 Hemolysis Genetic Hemoglobin Issues Retic Production Index > 2, high LDH High indirect Bilirubin • Thalassemia – Normal DNA sequence , Coombs or DAT Reduction in globin production • Alpha - Not enough alpha globin production -

No – HbELP abnormal+ No – RBC Southeast Asian, Indian, southern Chinese, Hemoglobinopathy – Morphology+ No – Heinz body + SS, SC, SD S beta Thal Yes Shistocytes and Low Platelets in DIC, HIT, Middle Eastern and African ancestry Thalassemia TTP, HELLP • Beta – Not enough Beta globin production Greek, Italian, Middle Eastern, Southeast Yes = G6PD Deficiency Warm Antibody Cold Antibody Asian, southern Chinese and African descent

Hemoglobin FA Barts > 4% – Biochemistry Alpha Thal 2 gene Hemoglobin – Biochemistry Major –Cooleys deletion – Alpha Thal Trait Anemia Chromosome 16 Chromosome 11 Chromosome 16 Chromosome 11 alpha alpha gamma gamma delta beta Mom alpha alpha gamma gamma delta beta Mom alpha alpha gamma gamma delta beta Dad alpha alpha gamma gamma delta beta Dad

gamma gamma alpha alpha gamma gamma > 4% Hemoglobin Barts beta beta <84% Hemoglobin A alpha alpha beta beta 10-40% Hemoglobin A alpha alpha >10 - 90% Hemoglobin F gamma gamma alpha alpha 50 - 80% Hemoglobin F or gamma gamma Fetal alpha alpha 2% Hemoglobin A2 alpha alpha >3.5% Hemoglobin A2 delta delta delta delta

Medical Care for Thalassemia Patients Hemoglobinopathy

• If Symptomatic – Get expert care with Sickle Cell Disease – SS, SC, SD, SE, SOarab, S beta Thal Hematologist • Transfusion support and/or BMT Newborn Screening or HbELP • Watch Ferritin/Iron and chelate to prevent Daily Penicillin –birth -6yo overload Hydroxyurea prolongs life, prevents • Give Folic Acid (Folate)1 mg complications • Do not give supplemental iron unless proven Hydration, Oxygen, Temperature, and Folate low and monitor

12 Hemoglobin FSA – Biochemistry – SB+Thal disease Hemoglobin – Biochemistry – Sickle Trait

Chromosome 16 Chromosome 11 Chromosome 16 Chromosome 11 alpha alpha gamma gamma delta Beta -S Mom alpha alpha gamma gamma delta Beta -S Mom

alpha alpha gamma gamma delta Beta Dad alpha alpha gamma gamma delta beta Dad

Normal CBC alpha alpha 65-90% Hemoglobin S alpha alpha Beta - S Beta - S Beta - S Beta - S 47% Hemoglobin S

alpha alpha 5-30% Hemoglobin A alpha alpha Beta Beta beta beta 50% Hemoglobin A 2-10% Hemoglobin F or Fetal alpha alpha alpha alpha 1% Hemoglobin F or Fetal gamma gamma gamma gamma 3.5-6% Hemoglobin A2 alpha alpha alpha alpha 2% Hemoglobin A2 delta delta delta delta

Resource Enzyme - G6PD - Glucose - 6 - Phosphate • World Wide Web Site - The Sickle Cell Information Center – http://www.SCInfo.org Dehydrogenase Deficiency – Information for providers, patients, teachers, employers, administrators X linked genetic – Monthly E-mail Newsletter [email protected] – Listing of Clinics Precipitated by oxidant drugs – Guidelines Heinz body stain show denatured Hb

Avoid medications such as antimalarials, aspirin, sulfa drugs, and avoid eating fava beans.

Membrane problems Spherocytosis Immune Attack and Ovalocytosis • Coombs Test: IgG and Compliment +/- • Transfusion reaction: immediate or delayed • IgM – (IgG Neg Comp +) cold antibody- infections like mycoplasma, EBV, HIV • IgG warm antibody – Drug induced – Antibiotics, Ibuprofen, Autoimmune diseases • PNH Paroxysmal Nocturnal Hemoglobinuria – Red cells attacked by complement Lack of CD55 or CD59 on RBC surface

13 Parasites – Malaria, Babesiosis, Trauma To Red Cells Bartonellosis

• Microangiopathic- Coagulation gone wild, fibrin shredding red cells- TTP, ITP, HUS, DIC, HELLP- Eclampsia • Splenomegaly • Infections within the red cell – Malaria – Mosquito parasite- Tropics – Babesiosis – Tick parasite- New England area – Bartonellosis – Bacteria - Cat Scratch

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