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

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

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

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

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.)

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

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

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

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

Common Causes of Microcytic Anemia Iron deficiency Anemia of chronic disease Thalassemia minor

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

Ddx of Microcytic/hypochromic anemia

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

b-thalassemia major

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

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

IRON STUDIES IN HYPOCHROMIC ANEMIAS

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

Case 1

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

Case 1 Iron studies Diagnosis: Iron deficiency anemia 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

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%

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?

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

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

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

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

Case 3 20 month old male with severe anemia.  

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%

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)

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

Case 3 Isoelectric focusing

Isoelectric focusing gel electrophoresis

Case 3 Diagnosis Alpha-thalassemia

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

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

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

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

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%

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

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

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

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?

IPF and platelet transfusion assessment

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

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

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

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 2.6-20.4%

Immature Reticulcyte Fraction

www.beckmancoulter.com/literature/ClinDiag/recticliterature.pdf

Ret-He or CHr Measure of hemoglobin in the reticulocyte Nl 29.8-40.4 pg Measure of adequacy of iron availability Monitor early response to iron therapy Early diagnosis of iron deficiency in pediatric population