A Guide to Red Blood Cell Indices

1. A Guide to Red Blood Cell Indices Mary Ann Thompson, M.D., Ph.D. May 8, 2013

2. Complete blood count (CBC) • WBC # • RBC # • Hemoglobin • Hematocrit • RBC indices: MCV, MCH, MCHC, RDW • Platelets

3. RBC indices • MCV: mean corpuscular volume • Hct (%) x 10/ RBC ct (x106/ml) • MCH: mean corpuscular hemoglobin • Hgb (g/dL) x 10/ RBC ct (x106/ml) • MCHC: mean corpuscular Hgb conc. • Hgb (g/dL) x 100/ Hct (%) • RDW: rbc distribution width

4. XE-Series Principles and Technology Red Cell Distribution Width (RDW) RDW-SD is measured at the 20% relative height of the curve RDW-CV can be falsely elevated or decreased depending on the MCV since MCV is in the denominator

5. RBC indices MCV: mean corpuscular volume Nl 81-98 fL Gauge of microcytic, normocytic or macrocytic anemia MCH: mean corpuscular hemoglobin Nl 27-32 pg MCHC: mean corpuscular Hgb concentration Nl 31-35 g/dL High in spherocytosis High if Hgb spuriously high due to interference (turbidity, TPN, etc.)

6. RBC indices RDW: rbc distribution width RDW-SD nl 37.4-52.4 fL RDW-CD nl 11.1-14.3% High in iron deficiency anemia

7. Corrected Reticulocyte Count • Corrected Reticulocyte Count = Percent Observed Reticulocytes x Patient’s Hematocrit) / 45 • Reticulocyte Production Index= • [(Percent Reticulocyte x Hematocrit Value)/45] x [1/Correction Factor]

8. Correction Factor

9. Causes of Microcytic/hypochromic Anemia • Deficient Hemoglobin Synthesis • Iron • Protoporphyrin • Globin

10. Common Causes of Microcytic Anemia • Iron deficiency • Anemia of chronic disease • Thalassemia minor

11. Causes of Microcytic Anemia • Disorders of iron metabolism • Iron deficiency • Anemia of chronic disease • Disorders of heme synthesis: Sideroblastic anemia • Hereditary • Acquired idiopathic (myelodysplasia) • Acquired toxic (lead, drugs, alcohol) • Disorders of globin synthesis: Thalassemias

12. Ddx of Microcytic/hypochromic anemia

14. Ddx of Thalassemia minor vs. Iron deficiency anemia • In thalassemia, MCV and MCHC < than those in same degree of iron deficiency anemia • Microcytosis with RBC > 6 x 106/ul suggests thalassemia • MCV/RBC <13 in Thalassemia minor, >13 in iron deficiency anemia • Basophilic stippling and target cells common in thalassemia • RDW nl in thalassemia minor, high in iron deficiency anemia

15. b-thalassemia major

16. Detection of b Thalassemia trait • Rathod, D. et al Usefulness of cell counter-based parameters and formulas in detection of b-thalassemia trait in areas of high prevalence AJCP 128: 585, 2007 • Sensitivity and specificity of parameters and formulas for detecting b-thal and b-thal + IDA • Winner: Shine and Lal Index: MCVxMCVxMCH/100 • For b-thal sensitivity 99.01%, specificity 93.3% with cut-off of <1,530, for b-thal + IDA sensitivity 94.8%, specificity 70% • Runners-up • MCV • MCH • Srivastava index: MCH/RBC • Mentzler index: MCV/RBC

17. Ddx of Iron Deficiency Anemia vs. Anemia of Chronic Disease (ACD) • Serum iron and % saturation low in both • TIBC hi in iron deficiency, low in ACD • Serum Ferritin low in iron deficiency, nl or high in ACD • Marrow stores absent in iron deficiency, high in ACD

18. IRON STUDIES IN HYPOCHROMIC ANEMIAS

19. Case 1 • 18 month old male admitted for FUO • CBC: • WBC 22.9K/ml • PCV 20% • Hgb 5.3 g/dL • Platelets 700 K/ml • Differential: 40% neutrophils, 47% lymphocytes, 7% monocytes

20. Case 1

21. Case 1 • RBC indices: • MCV 52.1 fL • MCH 24.2 pg • MCHC 27.3 g/dL • RDW 21.7% • RBC 3.76 mil/ml • 2% reticulocytes

22. Case 1 • Iron studies • Serum iron 6.0 mcg/dL(50-150) • TIBC 483 mcg/dL(250-450) • Ferritin 6.1 ng/mL(10-300) • Diagnosis: Iron deficiency anemia

23. Case 2: Lab values • WBC = 9.1 K/ml • Hb = 9.6 g/dL • PCV = 30% • Plts = 481 K/ml • RBC = 6.68 mil/ml • MCV = 45 • MCH = 14.4 • RDW = 27.3 • Reticulocyte = 1.0%

25. Case 2 • 1 year ago RBC indices were normal • Thalassemia vs. Iron deficiency ? • Iron deficiency • History • High RDW • Increased plts • MCV/RBC = 8.87—thalassemia?

26. Hemoglobin evaluation HbA 75% HbF 2% HbE 23% HbA2 cannot be quantitated since HbE elutes similarly

27. Hemoglobin E • Hb E is b26 (Glu—Lys) • Second most prevalent Hb variant • >80% in Southeast Asians • 19% incidence in refuges from Cambodia and Laos • Mutation creates a cryptic donor splice site that competes with the normal donor splice site

28. Hemoglobin E • Hb AE trait • Clinically silent • Microcytosis, MCV = 65 • Slight erythrocytosis • No significant anemia • 20-35% E due to poor synthesis (due to splice site competition), and mRNA instability • HbE homozygous • Thalassemia-like due to poor synthesis • MCV 55-65

29. Hemoglobin E • Relative proportion of Hb E decreases if concomitant a thalassemia • Poor competition with normal b chains if a chains limiting • Reduced Hb E if iron deficiency

30. Case 3 20 month old male with severe anemia.

32. Case 3 Lab values WBC=21.7K/ul PCV=24% Plts =354K/ul MCV= 49 fL RBC= 4.89 mil/ul MCH= 17 pg, MCHC= 34.6 g/dL RDW= 30.3%, Reticulocyte count= 1.6%

33. Case 3 supplemental data • Iron studies • Iron 93 mg/dL(50-120 nl) • TIBC 211 mg/dL (250-450 nl) • % sat 44 • Ferritin 402 ng/mL (10-300 nl)

34. Case 3 •  HPLC results • Hemoglobin A 90.8% • Hemoglobin A2 1.8% • Hemoglobin F 1.2% • Hemoglobin other 6.2%

35. Case 3 Isoelectric focusing

36. Isoelectric focusing gel electrophoresis

37. Case 3 Diagnosis Alpha-thalassemia

38. Normal Hemoglobins in Adults • HbA: a2b2: ~98% • HbA2: a2d2: 1.5-3.5% • Hb F: a2g2: <1.0%

39. Hereditary Spherocytosis: Labs • Chronic extravascular hemolysis • Increased MCHC (>34 g/dL; due to decreased surface area) • Increased osmotic fragility • Coomb’s test to rule out autoimmune hemolytic anemia as cause of spherocytosis

40. New parameters of the Sysmex XE-5000 • IG: Immature granulocytes (%, #) • Promyelocytes, myelocytes, metamyelocytes • IPF: Immature platelet fraction (%) • IRF: Immature reticulocyte fraction (%) • Ret-He: Hemoglobin content of reticulocytes

41. Immature platelet fraction • IPF measures platelets newly released from bone marrow • Therefore IPF is a measure of the rate of thrombopoiesis www.healthsystem.virginia.edu/internet/hematology

42. Immature platelet fraction • Ratio of immature platelets to total number of platelets • The reticulocyte channel utilizes a fluorescent polymethine dye that recognizes RNA and DNA • Separates reticulocytes from rbcs based on cytoplasmic RNA • Separates immature platelets based on amount of RNA • The highly fluorescent immature platelets are defined as those with the highest 3% intensity of fluorescence • Nl 0.9-7%

43. Reticulocyte channel histograms Forward scatter (cell volume) Fluorescence intensity (RNA/DNA) Mature platelets Immature platelets

44. Immature platelet fraction Briggs, C. et al Br J. Haematol. 126: 93, 2004

45. Uses of the IPF • Evaluation of thrombocytopenic patients • Distinguish between increased platelet destruction and decreased platelet production • Prediction of the recovery phase of thrombocytopenia • Regeneration after chemotherapy • Engraftment after bone marrow transplant

46. Role of the IPF in diagnosis of ITP • Confirm that thrombocytopenia is due to increased destruction rather than ineffective thrombopoiesis • Including IPF in work-up is cost-effective, as it is automatically run in the reticulocyte channel • In atypical cases or cases that are refractory to treatment, will this avoid bone marrow biopsy for the patient?

47. IPF and platelet transfusion assessment

48. Briggs, C. et al., Br J. Haematol. 126: 93, 2004

49. Rise in IPF precedes engraftment Takami, A. et al. Bone Marrow Transplantation 39: 501, 2007

50. Immature Reticulocyte Fraction • “The reticulum” is a mixture of RNA and protein • Fluorescent dye directed against RNA binds to the reticulum. • Reaction uses same reagents as the usual reticulocyte channel