The following are partial results from the patient’s complete blood count (CBC):

WBC (109/L)Hb (g/dL)MCV (fL)PLT (109/L)

What’s what: WBC, white blood cell count; Hb, hemoglobin; MCV, mean cell volume; MCHC, mean cellular hemoglobin concentration; RDW-SD, red cell distribution width-standard deviation; platelets, PLT; Normal values: WBC 5-10 x 109/L, RBC 4-6 x 1012/L, Hb 12-16 g/dL, Hct 35-47%, MCV 80-100 fL, MCHC 32-36 g/dL, RDW-SD < 45 fL, platelets (PLT) 150-450 x 109/L

True or false: this patient likely has iron deficiency anemia.

For reasons that will be discussed on the next slide, a diagnosis of iron deficiency is unlikely.

The Mentzer index is one of many formulas that have been proposed for differentiating iron deficiency anemia from thalassemia minor/trait. What is the Mentzer index?

MCV/RBC count

Mentzer index ratio:

  • > 13 suggests iron deficiency
  • < 13 suggests thalassemia minor/trait

When you see the patient, the red cell count is 6.06 x 1012/L.

MCH, mean corpuscular hemoglobin

MCHC, mean corpuscular hemoglobin concentration

Mean corpuscular hemoglobin (MCH) in thalassemia minor/trait:

The mean corpuscular hemoglobin (MCH) correlates with the mean cell volume (MCV). Since the MCV is low in this patient, the MCH will also be low.

Mean corpuscular hemoglobin concentration (MCHC) in thalassemia minor/trait:

One of the distinguishing features between iron deficiency and thalassemia is that the red cells in iron deficiency tend to have low mean corpuscular hemoglobin concentration (MCHC), which corresponds to increased central pallor, while those in thalassemia minor tend to have normal MCHC and normal central pallor.

Here are more data from the complete blood count (CBC):

Hb (g/dL)MCV (fL)MCH (pg)MCHC (g/dL)RDW-CVRDW-SD

What’s what: WBC, white blood cell count; Hb, hemoglobin; MCV, mean cell volume; MCHC, mean cellular hemoglobin concentration; RDW-SD, red cell distribution width-standard deviation; platelets, PLT; Normal values: WBC 5-10 x 109/L, RBC 4-6 x 1012/L, Hb 12-16 g/dL, Hct 35-47%, MCV 80-100 fL, MCHC 32-36 g/dL, RDW-CV <16%; RDW-SD < 45 fL, platelets (PLT) 150-450 x 109/L


  • Mean corpuscular hemoglobin (MCH) is low.
  • Mean corpuscular hemoglobin concentration (MCHC) is normal.

RDW-CV, red cell distribution width coefficient of variation

RDW-SD, red cell distribution width standard deviation

Which schematic is most likely to represent the patient’s peripheral smear?

Correct! Small red cells (low mean cell volume [MCV]) that are well hemoglobinized (normal mean corpuscular hemoglobin concentration [MCHC]) and show little variation in cell size.
Hypochromic, microcytic red blood cells – iron deficiency
Hypochromic, normocytic red blood cells – does not really correlate with any condition.
Normochromic red blood cells with anisocytosis
Hypochromic red blood cells with anisocytosis – iron deficiency anemia

If you were able to access the patient’s complete blood counts in the past, what would they likely show?

Normal red cell indices
Low mean cell volume (MCV)
Low mean corpuscular hemoglobin concentration (MCHC)
Severe anemia

In fact, complete blood counts dating back 9 years show a mean cell volume of 60-64 fL without anemia.

What do you expect his serum ferritin to be?

However, as we will discuss, even thalassemia minor may be associated with increased iron absorption and elevated ferritin.
This might apply (see answer to B).

Here are the results of the patient’s iron indices:

Are these results consistent with iron overload?

Yes, because the ferritin is so high
No, because the total iron binding capacity (TIBC) is normal
Yes, because the ferritin and transferrin saturation (TSAT) are both high
TSAT = 142/293, about 50%.
No because the ferritin is not high enough

The results of the hemoglobin (Hb) electrophoresis (Sebia Hydrase alkaline method) were the following:

How would you interpret these results?

The patient has no hemoglobin
There is no normal HbA
The patient does not have HbCC disease
There must be another species of Hb

In fact, the diagnosis was HbE disease (HbEE)

Diagnosis was based on Hb electrophoresis using cellulose acetate at alkaline pH, and then agarose gel at acid pH.

Let’s see how Hb electrophoresis can used to discriminate between different hemoglobinopathies. Typically, samples are run on cellulose acetate at alkaline pH, and then on an agarose gel at acid pH. Different types of hemoglobin migrate in distinct patterns on each gel allowing for identification of the relevant hemoglobin:

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Left, cellulose acetate at alkaline pH is the initial procedure. At this pH Hb is negatively charged and moves toward the positively charged anode. Right, next step is the citrate agar or agarose gel at acid pH. Note how the position of HbE changes between the two conditions. A real example of HbEE is shown below. This would have been similar to our patient’s results.
Step 1: Alkaline gel electrophoresis from a patient with homozygous HbE. There is no normal hemoglobin A. There is a variant hemoglobin with the mobility of hemoglobin C (red arrow). The differentials for this variant are hemoglobins E, O-Arab and A2. Source.
Step 2: Acid gel electrophoresis from a patient with homozygous HbE. There is a band with the mobility of hemoglobin A (red arrow). This excludes hemoglobin C (mobility of C) and O-Arab (mobility of hemoglobin S) as potential causes for the variant on the alkaline gel. Source.

Hematologic data in various hemoglobin E syndromes:

HemoglobinMean cell volumeMCHC
Normal12.5-1687 ± 633 ± 0.9
Hb E trait12.8 ± 1.584 ± 533 ± 1.8
Homozygous HbE 11.4 ± 1.870 ± 433 ± 1.7
Hb E/β0-thalassemia7.8 ± 2.667 ± 628 ± 4.8
MCHC, mean corpuscular hemoglobin concentration. From Hematology Am Soc Hematol Educ Program. 2007;79-83.


The differential diagnosis of microcytosis includes iron deficiency and alpha- or beta-thalassemia. Now we have just learned that HbEE also causes microcytosis. Are there other hemoglobinopathies that we should consider in a patient with microcytosis? Well, let’s step back and consider hemoglobinopathies, a group of recessively inherited genetic conditions affecting the alpha or beta globin chains of hemoglobin. Hemoglobinopathies are the most common autosomal recessive disorder worldwide, with 7% of the global population carrying an abnormal hemoglobin alpha or beta globin chain allele.

Hemoglobinopathies may be split into 2 groups, one leading to quantitative changes in hemoglobin (termed thalassemia), the other to qualitative changes (termed structural variants):

  • Thalassemia syndromes:
    • Quantitative defect caused by decreased expression of one of the two globin chains of the hemoglobin molecule, α (HBA) and β (HBB).
    • Decreased expression can result from:
      • Deletion of the structural gene(s).
      • Mutations that result in decreased RNA synthesis, processing, or stability.
      • Mutations resulting in decreased protein synthesis or stability.
    • The decrease in expression of one of the globin chains results in accumulation of excess polypeptides encoded by the unaffected gene. This chain imbalance causes abnormal RBC maturation, resulting in microcytosis as the characteristic laboratory abnormality.
    • Thalassemia syndromes include:
      • Alpha-thalassemia
      • Beta-thalassemia
      • Hb E
        • As in our patient
        • Hb E actually has a thalassemia phenotype
  • Structural hemoglobin variants:
    • The hemoglobin variants are caused by amino acid substitutions in either globin chain.
    • Clinical disease includes:
      • Thalassemia-like phenotype (HbE)
      • Sickling (HbS)
      • Hemolysis due to unstable hemoglobins
      • Hemoglobins associated with altered oxygen affinity
      • Hemoglobins in which iron cannot be maintained in the ferrous (Fe2+) state
    • The main Hb abnormalities worldwide are:
      • Hb S
      • Hb C
      • Hb E
    • Rarer abnormalities that may have clinical significance include:
      • Hb D
      • Hb OArab
      • Hb Lepore

More than 1,800 hemoglobin variants have been characterized! The good news is that only a small subset are associated with microcytosis. These include:

  • Those with a thalassemia phenotype
    • Beta-thalassemia
    • Alpha-thalassemia
    • HbE
  • HbC (we will discuss the hemoglobinopathy as a cause of microcytosis in another case study.)

All of this is to say, if you working up a patient with microcytosis who you think has thalassemia, you need to consider HbC and HbE in the differential diagnosis, but, thankfully, not any of the other hundreds of hemoglobin variants.

Returning to the patient, recall that his serum ferritin and transferrin saturation are elevated. Would you order genetic tests for hereditary hemochromatosis (HH)?

Probably not since the allele frequency is very low in Southeast Asia and because HH has not been shown to increase ferritin levels in persons with thalassemia minor (see next slide).

A word on beta-thalassemia trait and iron stores

Beta thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload (J Hum Genet (2004) 49:651–655). This may be aggravated with the coinheritance of C282Y or H63D mutation. C282Y is extremely rare in Southeast Asia (our patent was from Cambodia), whereas H63D is more widely distributed.

In a study from Thailand, H63D heterozygotes were found in 5.5% (11/201) of normal subjects and 7.3% (27/370) of thalassemia carriers, leading to mutant allele frequencies of 0.027 and 0.036 in normal subjects and thalassemia carriers, respectively. The proportions of subjects with elevated ferritin were found to be 37.5% (6/16) for those with the H63D mutation and 14.0% (18/129) for those without the mutation in males and 0% (0/11) for for those with the H63D mutation and 3.0% (5/164) for those without the mutation in female subjects, respectively. The H63D heterozygosity has no significant effect on the serum ferritin level and the authors concluded that screening for this HFE mutation in thalassemic patients is not recommended. Similar findings were observed in a subgroup of 14 HbE/-thalassemia patients.

Table 1. Prevalence and 95% confidence interval (CI) of the H63D mutation of the HFE gene and frequencies of the H and D alleles among 370 Thai thalassemia carriers and 201 normal individual
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