1. Approach to Lows of the CBC Lauren Gerard, MD, FRCP Hematologist Royal Victoria Hospital gerardl@rvh.on.ca Huronia Nurse Practitioner Conference September 26, 2014

2. Objectives  Review the “CBC” and blood films  To develop a differential diagnosis of anemia  To appreciate blood work requiring immediate attention  Red flags….when to refer!

3. Parts of the CBC  RBC = # of RBCs per liter  MCV = mean cell volume Normal for adult 80 – 100 Normal in pediatrics slightly lower  MCH = avg. amount of Hb in an individual cell  Decreased in hypochromic anemias  Total mass of Hb / # RBCs in volume of blood  MCHC = avg. concentration of Hb in a cell  Increased in HS and Hb SS  RDW = reflection of anisocytosis Ddx: IDA, reticulocytosis, blood transfusion etc Normal = uniform population of cells  HCT = measures the volume of red blood cells compared to the total blood volume (red blood cells and plasma)

4. Parts of the CBC: Hemoglobin  Automated cell counter from a tube of well-mixed EDTA-anticoagulated blood filled to a predetermined level.  RBCs are lysed, all forms of hemoglobins are converted to the colored protein cyanomethemoglobin and measured by a colorimeter.  False readings: inadequate sample (due to insufficient volume or inadequate anticoagulation)  Hemoglobin is quantified based on its absorption characteristics. False elevations: Hyperlipidemias Hyperbilirubinemia high white blood cell count high serum protein can

5. Indications for Peripheral Blood Film  Anemia NYD, Jaundice NYD  Thrombocytopenia or neutropenia  Features of MPD of LPD  DIC (n.b. acutely fragments may be absent)  Suspicion of parasitic disease

6. General Approach to the Anemia  Acute vs. Chronic Look at previous CBCs  Anemia alone or multiple cell lines affected Multiple cell lines affected is more concerning for primary bone marrow disorder  Detailed PMH/Social/family history Anemia can result from a number of other systemic causes

7. Approach to Anemia  Start with the MCV!  Microcytic< 80 fL  Normocytic80-100 fL  Macrocytic> 100 fL  What determines size of red cell? Hemoglobin is negative regulator of cell division Less hemoglobin = extra division = smaller cells

8. Anemia MCV Low (Microcytic ) High (Macrocytic) Measure B12, (folate)Ferritin Low (<20) Fe Def Establish Cause Normal ACD or Hb-opathy Low Normal Replace Establish Cause Consider Bone Marrow Biopsy Reticulocyte Count LowHigh ACD Renal Disease BM Failure Hemolyisis Or Blood Loss Normal

9. Anemia MCV Low (Microcytic ) High (Macrocytic) Measure B12, (folate)Ferritin Low (<20) Fe Def Establish Cause Normal ACD or Hb-opathy Low Normal Replace Establish Cause Consider Bone Marrow Biopsy Reticulocyte Count LowHigh ACD Renal Disease BM Failure Hemolyisis Or Blood Loss Normal

10. Microcytic Anemias

11. Case # 1  31 female with Hemoglobin 88g/L  Symptoms: Fatigue, otherwise well  PMH: IBS G2P2  Meds: None  Family Hx: None  Exam: Normal

12. CBC (incl platelet Count) Hemoglobin 88130 - 180 g/LL WBC Count 104.0 - 11.0 x10E9/L Platelet Count 488150 - 400 x10E9/LH Neutrophils 7.02.0 - 7.5 x10E9/L Lymphocytes 3.01.0 - 4.0 x10E9/L Monocytes 1.20 - 1.0 x10E9/LH Eosinophils 0.00 - 0.7 x10E9/L Basophils 0.00 - 0.3 x10E9/L Hematocrit.2940.390 - 0.540 L/LL RBC Count 4.714.00 - 6.00 x10E12/L MCV 62.478 - 96 fLCRITICAL RDW 18.810.0 - 14.5 %H MCH 18.728.0 - 32.0 pgCRITICAL MCHC 299310 - 360 g/LL MPV 9.55.0 - 15.0 fL

13. What is your Differential of Microcytic Anemia?  Thalassemia  Anemic of Chronic Disease (ACD)  Hepcidin prevents eggress of iron from enterocytes  Reduced RBC lifespan  Inflammatory cytokines interfere w/ Epo prodn  Iron Deficiency  Chronic blood loss (GI, hemolysis, menstruation)  Nutritional deficiency  Malabsorption (Celiac, H. Pylori, gastric resection, achlorydia)  Pregnancy, lactation  Hookworm infestation  Lead  Sideroblastic Anemia

14. Case #1  Ferritin 10  Iron deficiency anemia (ferritin < 30) Ferritin <18 LR 41 Ferritin 18-45LR 3.12 Ferritin >100LR 0.13  But in the setting of inflammation In inflammation, ferritin may increase 3-fold Iron studies (TSAT) < 20% may be helpful Guyatt et al. Am J Medicine 1990;88:205-209

15. Serum Iron  TIBC = %Transferrin Saturation (TIBC = total iron binding capacity) Normal (20-40%) Iron def:  serum Fe  TIBC  %saturation ACD:  serum Fe N or  TIBC  %saturation Serum Iron Cannot R/O Fe def TIBC (Transferrin)

16. The Blood Film http://meds.queensu.ca/medicine/deptmed/hemonc/macro/slide14.html Red Blood Cell Platelet White Blood Cell

17. Hypochromic (1/3), microcytic Low Hb Low MCV High RDW Pencil cell Case #1

18.  Once you have diagnosed iron deficiency, what is next most important issue to answer? A. Does the patient have malabsorption of iron or a poor diet? B. Does the patient have a source of blood loss? C. Does the patient have chronic vs acute iron deficiency? D. Is there concomitant thalassemia?

19. Fe deficiency - Causes  Too much OUT Increased blood loss Increased iron requirements: use of ESAs, pregnancy, post-bleeding recovery  Too little IN Inadequate absorption:  Loss of enterocytes: resection, celiac disease, IBD  Antacids, H.pylori, excess dietary bran, tannins, phytates, starch, competition with other metals rare mutations: DMT1, transferrin, TMPRSS6

20. Causes of Iron Deficiency Always think about, look for, and treat the underlying cause!

21. Case #1 – Iron Deficiency Anemia  History: No GI causes of blood loss Normal diet (not vegetarian) 2 pregnancies last 4 years Heavy periods since menarche  Changes pad/tampon q1-2 hours x 6 days  Family History: Mother & aunt– heavy periods, easing bruising Brother – excessive bleeding with dental extraction

22. Treatment of Iron Deficiency  Treat underlying cause if possible  Iron replacement BID to TID dosing Ferrous gluconate30mg/300mg Ferrous sulfate60mg/300mg Ferrous fumarate100mg/300mg Proferrin11mg po BID Feramax150mg po OD  Take on empty stomach with vitamin C  At least 3 months…

23. Poor Response to oral Iron  Lack of response within 4 to 6 weeks of therapy with a minimum of 100 mg po od elemental dose Expect reticulocytosis in 7 days Increase in Hgb by 10g/L in 14 - 28 days SO LONG AS NO BLEEDING: normalize Hgb in 6 to 8 weeks  Review: Compliance/Side effects (especially constipation) Food inhibitors of iron absorption:  Oats, bran, rye  Tea  Calcium  Rule Out: H.Pylori (19% of cases) Celiac disease – even in absence of symptoms (5%) Atrophic gastritis (26%) Achloryhydric gastric atrophy – acid is required to solubilize and reduce dietary iron On going blood loss

24. IV Iron  If oral iron is not tolerated, is not effective or severe anemia Intravenous Iron  Iron Sucrose (Venofer)  Ferumoxytol (Feraheme)  Iron Dextran (Infufer, Dexiron)

25. Case #1 Summary  Iron deficiency anemia  resolved completely with oral iron supplementation  Management of heavy menses: Mirena IUD placement  VWD Investigations VWF antigen – 20%, Activity- 20%, Factor VIII 50% Consistent with mild VWD type 1

26. Case #2:  74 year old man with Hb 92 g/L  PMH: Life long smoker, Type 2 DM, HTN, Dyslipidemia, Coronary artery disease, CABG CHF grade III/IV, Rheumatoid Arthritis, Recurrent UTIs  Medications ASA, Plavix, Metformin, Ramipril, Lasix, Metoprolol

27. CBC (incl platelet Count) Hemoglobin 92130 - 180 g/LL WBC Count 104.0 - 11.0 x10E9/L Platelet Count 250150 - 400 x10E9/L Neutrophils 7.02.0 - 7.5 x10E9/L Lymphocytes 3.01.0 - 4.0 x10E9/L Monocytes 1.20 - 1.0 x10E9/LH Eosinophils 0.00 - 0.7 x10E9/L Basophils 0.00 - 0.3 x10E9/L Hematocrit.2940.390 - 0.540 L/LL RBC Count 4.714.00 - 6.00 x10E12/L MCV 7878 - 96 fLL RDW 1310.0 - 14.5 % MCH 18.728.0 - 32.0 pg MCHC 299310 - 360 g/LL MPV 9.55.0 - 15.0 fL

28. Case #2  Iron Studies Ferritin 120 TIBC – 0.40 Iron Saturation 0.22  Blood Film Microcytic hypochromic

30. Clues to Microcytic Anemia MCV < 80 fl Serum Iron TIBC / Serum Iron BM Perls stain Iron Def. Anemia ↓↓ ↑↑ / LOW0 Chronic Infection ↓↓↓↓/ Low or Normal + + Thalassemia↑↑N + + + + HemoglobinopathyNN + + Lead poisoning NN + + Sideroblastic↑↑N + + + +

31. Anemia of Chronic Disease 1) Iron restricted erythropoiesis 2) Blunted erythropoietin response Andrews. Blood 2008;112:219 IL-6 Lipopolysaccharides Inflammation ↓Oral absorption Iron trapping within RES

32. Anemia of Chronic Disease  Presents as normocytic or microcytic anemia  Usually does not cause “severe” anemia  Can be challenging to differentiate from iron deficiency  Treatment Treat underlying cause (if possible) Treatment of Anemia (if symptomatic)  Consider IV iron + Erythropoeitin

33. Macrocytic Anemias

34. Case #3  45F with Hb 108 MCV 112  PMH Hyperthyroism - treated with radioactive iodine Ulcerative colitis (mild)  Meds: Synthoid  Social Hx: Non-smoker, minimal ETOH

35. What is the DDx of Macrocytic anemia?  Round Liver disease Hypothyroid Alcohol Reticulocytosis Drugs  Oval Myelodysplastic syndrome  Megaloblastic B12 Deficiency Folate Deficiency Drugs  Folate: Methotrexate  DNA: Hydroxyurea, azathioprine, AZT

36. Investigations of Macrocytosis  Liver disease  Hypothyroid  Alcohol  Drugs  Reticulocytosis  Myelodysplastic syndrome  Megaloblastic (B12, folate, other drugs) Blood film Liver enzymes, INR, albumin TSH Alcohol and Drug history Retic count CBC  look at other cell lines Serum B12 (RBC folate)

37. Case #3  CBC Only anemia with macrocytis  Liver enzymes - normal  Unconjugated bilirubin 41  LDH: 450 (elevated)  TSH 3 (normal)  Vitamin B12 < 40 pmol/L  RBC folate – not performed

38. Low Hb MCV 120 Hypersegmented neutrophil (>5 lobes) Seen with Megaloblastic Anemias Case #3

39. B12 Deficiency  Needed for DNA and RNA production in nucleus  Deficiency results in asynchronous maturation of cytoplasm with nuclear arrest in G2 phase – thus megaloblasts form  Diagnosis Serum Cobalamin level < 150 pmol/L on 2 occasions Or Serum cobalamin level 0.4 umol/l and homocysteine > 13 umol/L  Etiology Pernicious Anemia - Immune destruction of parietal cells that make IF  Anti-parietal Abs  Anti-IF Abs  (Schilling test)  Dietary deficiency  Achlorydia  Exocrine failure  Ileal resection  Crohn’sdisease

41. B12 Deficiency - Treatment  Identify etiology Refer for OGD – rule out celiac disease  Pernicious Anemia Refer for OGD - Increased risk of gastric cancer (3%)  B12 replacement B12 1000 mcg IM (if severe) Oral supplementation – high dose may be just as effective even if pernicious anemia Watch for rebound hypokalemia Hyperseg PMNs – 14 days Reticulocytosis 3-4 days Normalization of Hgb by 8 weeks  Food: eggs, milk, cheese, milk products, meat, fish, shellfish and poultry

42. Case #4  72 year old woman  PMH Breast cancer  Treated with mastectomy and chemotherapy 8 years ago Awaiting CABG for CAD  Hb 95 MCV 105  Surgeon notes that she was started on iron p.o. for a Hgb of 109 g/ L.

43. CBC (incl platelet Count) Hemoglobin95130 - 180 g/LL WBC Count2.54.0 - 11.0 x10E9/L Platelet Count72150 - 400 x10E9/LL Neutrophils0.82.0 - 7.5 x10E9/LCRITICAL Lymphocytes 1.01.0 - 4.0 x10E9/LL Monocytes0.70 - 1.0 x10E9/L Eosinophils 0.00 - 0.7 x10E9/L Basophils 0.00 - 0.3 x10E9/L Hematocrit.2940.390 - 0.540 L/LL RBC Count 4.714.00 - 6.00 x10E12/L MCV 10578 - 96 fLH RDW 1210.0 - 14.5 % MCH 18.728.0 - 32.0 pgCRITICAL MCHC 299310 - 360 g/LL MPV 9.55.0 - 15.0 fL

44. Case #4 How would you manage this patient? A. Start i.v. iron as she has not had response to oral iron B. Check her RBC folate and vitamin B12 stores; then replace C. Refer to a hematologist for further evaluation D. Liase with anesthesia to ensure usage of cell saver etc.

45. Case #4  Investigations: Liver enzymes – normal INR – 1.0, PTT – 29 B12 – 800, RBC folate – 1000 Reticulocyte Count - 15

46. Low Hb MCV 105 Pelger-Huet anomaly (dumb-bell nucleus) Seen with MDS Case #4

47. Case #4: Summary  Macrocytic anemia with other cytopenias (pancytopenia)  Previous chemotherapy  Low reticulocyte count Suggests primary bone marrow failure  Likely mylodysplastic syndrome (MDS)

48. Normocytic Anemia

49. Reticulocyte Count LowElevated Generalized Disorders Anemia of Chronic Disease Renal dysfunction Early/combined nutritional deficiencies Primary bone marrow disorders Marrow infiltration/infection Aplastic anemia MDS Evidence of Hemolysis? (  LDH,  bilirubin,  haptoglobin) No HemolysisHemolytic Anemia Intrinsic & Extrinsic causes Blood Loss

50. Hemolytic Anemia (  LDH,  bilirubin,  haptoglobin) Intrinsic Causes Memebranopathies Hereditary Spherocytosis Hereditary eliptocytosis Enzymopathies G6PD Deficiency PK Deficiency Hemoglobinopathies Sickle cell Extrinsic Causes Immune Mediated Autoimmune Hemolytic Transfusion reaction Microangiopathic Hemolytic Anemia (MAHA) TTP/HUS DIC Mechanical heart valve

51. Normocytic Anemia Investigations  Reticulocyte count If elevated RBC loss or destruction  Hemolytic Markers LDH, Haptoglobin, Bilirubin  Nutritional Markers Iron indices and B12 (RBC folate)  May have combined deficiency  Direct Antiglobulin Test (DAT) If positive suggests immune hemolysis  Peripheral blood film

52. Low Hb Normal MCV Hemoglobinopathy Sickle Cell

53. G6pd slide Bite Cell G6PD Deficiency

54. Case #5  45 year old woman Hb 105, MCV 92 fl, WBC 4, Plt 300  Otherwise well  Family Hx: Brother had splenectomy as child  Investigations: Reticulocyte count – 300 (elevated) Unconjugated bili – 42, LDH 550, Haptoglobin undetectable Abdo U/S – mild splenomegaly (16 cm) What Additional Investigations Should We Order? Direct Antiglobulin Test (DAT) and Blood Film

55. Spherocytes – round RBC lacking central pallor Spherocytes DDx: Immune mediated Hereditary spherocytosis Case #5

56. Case #6  57 year old woman, previously well  Brought to ED feeling “generally unwell”, headache and husband noted mild confusion  Physical exam – normal  Investigations: Hb 98 g/L, MCV 94fL, WBC – normal, Plts 34 x 10 9/L CT head - normal

57. Case #6  What is your next step in this patients management? A) Discharge patient and reassure that likely related to a viral illness B) Discharge to be seen in hematology clinic within 2 – 4 weeks C) Urgent peripheral blood film D) Start on B12 supplement

58. Case #6  What would you like to know on history?  What investigations would you order?  Investigations: Reticulocyte count 408 LDH 568 Haptoglobin < 0.06 Indirect bilirubin 55 Direct Antibody test: negative PT 11.0 sec, PTT 31.0 sec Creatinine 120 umol/L Urinalysis: + blood  What is her most likely diagnosis? A. Warm Autoimmune Hemolytic anemia B. Oxidative hemolysis C. Acute blood loss D. Disseminated Intravascular Coagulation E. Thrombotic Thromocytopenia Purpura

59. Fragments Case #6

60. Microangiopathic Hemolytic Anemia  DIC  HUS/ TTP  Gestation-related HELPP AFLP  Malignant HTN  Catastrophic Antiphospholipid Syndrome  Medication induced Endotheliopathies Ticlopidine, Clopidogrel Mitomycin C Calcineurin Inhibitors  Valvulopathies  Kidney rejection  Scleroderma crisis, lupus, APLA crisis  March hemoglobinuria

61. Anemia Algorithm Anemia MCV Microcytic:Normocytic:Macrocytic: Iron Deficiency(next page)Megaloblastic Thalassemia- Folate, B12 Anemia of chronic diseaseDrugs Sideroblastic anemiaMyelodysplasia Alcohol Liver Disease Reticulocytosis Thyroid disease

62. Anemia Algorithm Anemia MCV Microcytic:Normocytic:Macrocytic: Iron Deficiency(next page)Megaloblastic Thalassemia- Folate, B12 Anemia of chronic diseaseDrugs Sideroblastic anemiaMyelodysplasia Alcohol Liver Disease Reticulocytosis Thyroid disease Blood filmBlood film exam Serum ferritin, iron studies(Serum B12) Hgb electrophoresisRBC folate Liver enz, TSH Retic count

63. Normocytic Retic Count Low:High: Generalized Disorders - Anemia of chronic diseaseHemorrhage - Chronic renal failureHemolysis - Nutritional deficiencies(Hematinic) - Toxins, drugs Primary Bone marrow - Hematologic - Infiltration/Infection

64. Normocytic Retic Count Low:High: Generalized Disorders - Anemia of chronic diseaseHemorrhage - Chronic renal failureHemolysis - Nutritional deficiencies(Hematinic) - Toxins, drugs Primary Bone marrow - Hematologic - Infiltration/Infection Blood filmBili, LDH Ferritin, Iron studiesHaptoglobin Creatinine TSH, B12 Exam/Abdom. ultrasound

65. Normocytic Retic Count Low:High: Generalized Disorders - Anemia of chronic diseaseHemorrhage - Chronic renal failureHemolysis - Nutritional deficiencies(Hematinic) - Toxins, drugs Primary Bone marrow - Hematologic - Infiltration/Infection Blood filmBili, LDH Ferritin, Iron studiesHaptoglobin Creatinine TSH, B12 Exam/Abdom. ultrasoundDirect Antiglobulin Test

66. When to refer to Hematology?  Anemia with Thrombocytopenia Leukopenia Macrocytic Anemia Evidence of hemolysis Red cell fragmentation  Anemia with Hb < 100

67. Conclusions  Many types of anemia have a “non- hematologic cause”…look for it and treat it whenever possible  The most important investigation of normocytic anemia is the reticulocyte count.  Peripheral blood film can provide important diagnostic clues.

68. Questions? Email: gerardl@rvh.on.cagerardl@rvh.on.ca