Labs

The following is the complete blood count (CBC) the day you see the patient:

WBC (109/L)Hb (g/dL)MCV (fL)MCHC (g/dL)RDW-SD (fL)PLT (109/L)
9.48.98630.164.3240

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

The diagnostic algorithm for anemia begins with a mean cell volume and reticulocyte count:

The following was the patient’s reticulocyte count:

The absolute reticulocyte count, which is what we want to focus on is:

0.45 x 1012/L = 450 x 109/L

Therefore, the patient has a hyperproliferative normocytic, normochromic anemia.

The patient was told she has thalassemia trait. However, her mean cell volume (MCV) is 86 fL. Does the normal MCV rule out thalassemia? If not, why?

a
Yes
b
No
She has many reticulocytes, which are larger than mature red cells. The presence of these larger reticulocytes might “cancel out” the population of small thalassemic red cells, leading to a normal mean cell volume (MCV). This would be consistent with her elevated red cell distribution width (RDW).

We will take a look at the peripheral smear shortly. For now, consider the following questions:

If the patient has hemolytic anemia, what might you expect to find?

a
Low serum haptoglobin
b
High serum LDH
c
High serum direct bilirubin

It is the indirect bilirubin that increases in hemolysis.

d
High serum AST
e
High serum creatiinine

The following are the results of the hemolysis screen:

Hemolytic anemia may be classified in several ways. One classification is intravascular vs. extravascular. The isolated elevation in indirect bilirubin without any increase in AST and LDH is most consistent with:

a
Intravascular hemolysis
b
Extravascular hemolysis
AST and LDH are released by red cells when they are lysed in the circulation (intravascular hemolysis). Bilirubin is formed from breakdown of hemoglobin when red cells are engulfed by macrophages (extravascular hemolysis).

Now we can conclude that the patient has a hemolytic anemia, most likely congenital given her age and long history of anemia. What are possible causes of congenital hemolytic anemia?

a
Paroxysmal nocturnal hemoglobinuria
This is an acquired condition.
b
Sickle cell disease
c
Hereditary spherocytosis
d
Autoimmune hemolytic anemia
This is an acquired condition.
e
Pyruvate kinase deficiency

The following table includes the more common causes of congenital hemolytic anemia. Fill in the peripheral smear findings (in addition to polychromatophilia from reticulocytosis) and relevant diagnostic tests.

ConditionPeripheral smearAdditional tests
Sickle cell disease
Thalassemia minor

Hereditary spherocytosis
Hereditary elliptocytosis
G6PD deficiency
Pyruvate kinase deficiency

The following table includes the more common causes of congenital hemolytic anemia. Fill in the peripheral smear findings (in addition to polychromatophilia from reticulocytosis) and relevant diagnostic tests.

ConditionPeripheral smearAdditional tests
Sickle cell diseaseSickle cells, nucleated red cells, Howell-Jolly bodiesHemoglobin electrophoresis
Thalassemia minorMicrocytic red cellsHemoglobin electrophoresis, DNA sequencing
Hereditary spherocytosisSpherocytesEMA binding, osmotic fragility
Hereditary elliptocytosisElliptocytesOsmotic gradient ektacytometry 
G6PD deficiencyPitted red cellsEnzyme activity
Pyruvate kinase deficiencyAcanthocyte-like cellsEnzyme activity, DNA sequencing

The following is similar to the peripheral smear from the patient:

Spherocytes – hereditary spherocytosis
Note the presence of numerous spherocytes. Spherocytes are formed when there is loss of part of the red blood cell membrane. This may occur in the setting of immune-mediated hemolysis or congenital red cell membrane defects such as hereditary spherocytosis. Spherocytes are smaller than normal red blood cells and lack central pallor. They are less deformable and less able to navigate through small vessels, leading to increased destruction in the spleen. Source.

What is the next test you would like to perform?

a
Osmotic fragility test
b
Pyruvate kinase activity
c
G6PD activity
d
Hemoglobin electrophoresis

Osmotic fragility test

Specimens for red cell osmotic fragility tests are anticoagulated with EDTA. Osmotic lysis is performed using sodium chloride (NaCl) solution, 0.50 g/dL. An incubated fragility test is performed following 24-hour incubation at 37 degrees C at the following NaCl concentrations: 0.60, 0.65, and 0.75 g/dL. Results are reported and interpreted.

Spherocytes are osmotically fragile cells that rupture more easily in a hypotonic solution than do normal RBCs. Because they have a low surface area: volume ratio, they lyse at a higher osmolarity than do normal red cells. Cells that have a larger surface area: volume ratio, such as target cells or hypochromic cells are more resistant to lysing. After incubation, an increase in hemolysis is seen in spherocytes. Hereditary spherocytosis typically has greater number of spherocytes than other causes of spherocytosis. Therefore, the degree of lysis is usually more pronounced, but this is not always the case. Some rare disorders can also cause marked fragility and hereditary spherocytosis cases can display moderate fragility.

Source.

These are the results of the patient’s osmotic fragility study:

How would you interpret these data?

a
Normal
b
Increased osmotic fragility
c
Decreased osmotic fragility

The following is the interpretation of this patient’s osmotic fragility test:

JaypeeDigital | eBook Reader
Example of an osmotic curve in a normal individual and in a patient with hereditary spherocytosis. Note that for any given osmolality, there is more hemolysis in the HS sample. Source.

Collectively, the data support a diagnosis of hereditary spherocytosis.

What do you expect the mean cell hemoglobin concentration to be in patients with hereditary spherocytosis?

a
Decreased
b
Normal
c
Increased
See next slide.

The increased mean cell hemoglobin concentration is evident from the loss of central pallor in spherocytic cells. Consider the following schematic of red cells. Which of the following questions is/are correct?

a
A has increased mean cell hemoglobin
b
A has decreased mean cell hemoglobin
c
A has increased mean cell hemoglobin concentration
d
C has increased mean cell hemoglobin concentration
e
C has increased mean cell hemoglobin

A recent study examining red cell indices in patients with various hemoglobinopathies found that hereditary spherocytosis is indeed associated with an elevated mean corpuscular hemoglobin concentration (MCHC). It is interesting to note that the mean cell volume (MCV) is not decreased in patients with hereditary spherocytosis. One might expect that red cells that lose surface membrane would become smaller, and indeed they appear smaller on peripheral smears. It is possible that the normal MCV reflects a population of smaller than normal spherocytes and a population of larger reticulocytes.

Here again is the patient’s complete blood count.

WBC (109/L)Hb (g/dL)MCV (fL)MCHC (g/dL)RDW-SD (fL)PLT (109/L)
9.48.98630.164.3240

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

Note that the mean corpuscular hemoglobin concentration (MCHC) is actually on the low side. As we saw from the last slide, this is not typical for hereditary spherocytosis.

As we will see in About the Condition, hereditary spherocytosis can be classified according to its clinical severity:

% CasesHemoglobin (g/dL)Reticulocytes (%)Bilirubin (mg/dL)
HS trait N/ANormalNormalNormal
Mild HS20-30%11-153-61-2
Moderate HS 60%-75%8-12> 6> 2
Severe HS5%6-8> 10> 3

Let’s look at this patient’s parameters again:

WBC (109/L)Hb (g/dL)MCV (fL)MCHC (g/dL)RDW-SD (fL)PLT (109/L)
9.48.98630.164.3740

How would you classify the patient?

a
HS trait
b
Mild HS
c
Moderate-severe HS
She has some elements of severe (the reticulocyte count and bilirubin) and a Hb in the moderate range.