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. Red Cell Distribution Width (RDW)
XE-Series Principles and Technology
Red Cell Distribution Width (RDW)
Two different mathematical formulas used to describe RBC Anisocytosis (variation in size).
RDW-SD is measured at the 20% relative height of the curve. Statistically this is the point of greatest distribution of any curve.
RDW-CV can be falsely elevated or falsely decrease depending upon the MCV since the MCV is in the denominator.
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 fL
Gauge of microcytic, normocytic or macrocytic anemia
MCH: mean corpuscular hemoglobin
Nl pg
MCHC: mean corpuscular Hgb concentration
Nl 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 %
High in iron deficiency anemia

7. 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 Pts. Hematocrit Value, % Correction Factor 40-45 1.0
35-39
1.5
25-34
2.0
15-24
2.5
<15
3.0

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 Diagnosis: Iron deficiency anemia
Serum iron 6.0 mcg/dL(50-150)
TIBC 483 mcg/dL( )
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 HbE homozygous 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( nl)
TIBC 211 mg/dL ( nl)
% sat 44
Ferritin 402 ng/mL ( 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: %
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

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

51. Immature Reticulocyte Fraction
Reticulocytes divided into 3 fractions based upon the amount of fluorescence detected in the reticulocyte cytoplasm
LFR: Low fluorescence reticulocyte
MFR: Medium fluoresence reticulocyte
HFR: High fluoresence reticulocyte
IRF= MFR + HFR
Nl %

52. Immature Reticulcyte Fraction

54. Ret-He or CHr Measure of hemoglobin in the reticulocyte
Nl pg
Measure of adequacy of iron availability
Monitor early response to iron therapy
Early diagnosis of iron deficiency in pediatric population