RBC disorders

Hemoglobin C

Amino acid substitution in Hb B chains resulting in rod shaped HbC crystals which cause splenic sequestration and hemolytic anemia

Hemoglobin C etiology

Point mutation—lysine substituted from glutamate at 6th position of B chain (+2 charge)

Homozygous Hemoglobin C

2 affected alleles, primarily affects Black people, and Europe/Asians

Homozygous Hemoglobin C etiology

Lysine substituted for glutamate, causing structural change that renders OxyHb unstable, leading to high splenic sequestration, which leads to normo/normo hemolytic anemia

Laboratory diagnosis of Hemoglobin C

Low RBC, Hb, Hct, with normal MCV, MCH, and MCHC. Hb solubility test negative, over 90% HbC/0% HbA/<7% HbF

Microscopic view of hemoglobin C

Citrate agar electrophoresis

Separates C from E and O

Crystal formation in hemoglobin C disease

3% NaCl, crystals in over 90% of cells

Hemoglobin C heterozygous

1 affected allele, primarily affects Black people, Europeans, and Asians

Hemoglobin C heterozygous etiology

Lysine substituted for glutamate, inducing structure change causing oxyHb to become unstable

Laboratory tests for heterozygous hemoglobin C

Normal Hb/40% target cells on smear, 55-65% HbA/2.5% HbA2/35-45% HbC/<1% HbF, HbC crystal formation in hypertonic salt

Hemoglobin E

Point mutation resulting in an amino acid substitution causing a mild anemia

Etiology of Hemoglobin E

Lysine substituted for glutamate at 26th position of B chain

Homozygous hemoglobin E

2 affected alleles, primarily impacts SE Asians and Black people

Etiology of homozygous hemoglobin E

Lysine substituted for glutamate, causing structural change resulting in mild anemia from low RBC survival. Has no polymerization or crystals

Hemoglobin E homozygous tests

Mild anemia, target cells, microcytes. Hb solubility negative, 95% HbE/0% HbA/ 2.5-4% HbA2/<1% HbF

Hemoglobin E heterozygous

1 affected allele, primarily affects SE Asians and Black people

Laboratory tests for heterozygous hemoglobin E

Normal CBC, a few target cells

Hemoglobin O Arab etiology

Lysine substituted for glutamic acid at 121st position of beta chain

Hemoglobin O Arab demographics

Primarily affects SWANA, Mediterranean, and Black African

Hemoglobin SC etiology

HbS and HbC mutate on each beta gene

Hemoglobin SC laboratory testing

Mild to moderate anemia

– Target cells (85%), Sickle cells/HbC crystals or HbSC cells

– HbS = 45%

– HbC = 45%

– HbA = 0%

– HbA2 = 2.5%

– HbF = 1-7%

Hemoglobin D etiology

Point mutation resulting in an amino acid

substitution of a glutamine (+0) for a glutamic acid

(-1) at the 121st position of the beta chain

▪ Total change in charge = +1 (migrates with HbS)

Hemoglobin D affected populations

Indians (from India)

▪ African Americans (<0.2%)

Hemoglobin D homozygous symptoms

Very rare, almost no symptoms

Hemoglobin D homozygous testing

Hb Solubility = Negative

– Some Target Cells

– Hb electrophoresis

▪ Hb D = 95%

▪ HbA = 0%

▪ Hb A2 = 2.5%

▪ HbF = 2.5%

Hemoglobin D heterozygous

No anemia

– Normal blood smear

– Hb electrophoresis

▪ HbA = 50-60%

▪ HbD = 40-50%

▪ HbA2 – 2.5%

▪ HbF = <1%

Hemoglobin M etiology

Amino acid substitution that causes

methemoglobin > irreversible iron oxidized to Fe+3

Hemoglobin M pathogenesis

Cyanosis at birth in alpha HbMs

▪ Cyanosis appears at 4-6 months in beta HbMs

Hemoglobin SD

One mutation codes for HbS and HbD

Alkaline electrophoresis — Hb speeds

HbC – Crawl

– HbS – Slow

– HbF – Fast

– HbA – Accelerate

Molecular approaches for hemoglobinopathies

DNA probing

PCR amplification + sequencing