Hereditary Pyropoikilocytosis Workup

The diagnosis of HPP relies on identifying abnormal red blood cell (RBC) morphology on peripheral blood smear and identifying characteristic membrane biomechanical properties using osmotic gradient ektacytometry. ^{[5, 6]} The blood smear is characterized by elliptocytes, as seen in hereditary elliptocytosis, together with the following:

• Microcytic red cells
• Polychromasia
• Bizarrely shaped cells with fragmentation, poikilocytes, pyknocytes, and microspherocytes

The increasingly recognized genetic heterogeneity of RBC membrane disorders underlines the problem of a very complex differential diagnosis. Expanding from limited studies of candidate genes to wider panels of genes has become possible with targeted next-generation sequencing (t-NGS). Recent studies have established t-NGS as a comprehensive and invaluable diagnostic tool that can provide a correct diagnosis, allowing clinicians to proceed with careful management of these patients. ^[7]

One of the most important aspects of the use of t-NGS gene panels in clinical practice is their ability to be easily upgradable in view of novel discoveries. Despite their wide use in clinical practice, the major drawback of current NGS applications is the difficulty in determining the pathogenicity of the numerous identified variants. One of the ways to overcome this limitation is the simultaneous evaluation of all family members, which helps establish the inheritance pattern of the identified variants and thus to delineate their pathogenetic role, although functional characterizations are often necessary. ^[1]

The International Council for Standardization in Haematology (ICSH) has established guidelines for the diagnosis of nonimmune hereditary red cell membrane disorders, including hereditary pyropoikilocytosis (HPP). The guidelines note that diagnosis can often be made by reviewing the patient's clinical/family history, blood count results, reticulocyte count, red cell morphology, and chemistry results, together with tests available through hematology laboratories. ^[5]

A complete blood count and a peripheral blood smear are the most important studies to obtain when testing for HPP.  The peripheral blood smear of a patient with HPP demonstrates bizarre forms, anisocytosis, fragments, micropoikilocytosis, microspherocytosis, and budding red blood cells. ^[6] See the image below.

A reticulocyte count and, possibly, other markers of hemolysis may be of use in these cases. Reticulocytosis should occur, depending on how well the patient's bone marrow is able to respond. The mean corpuscular volume (MCV), reported in femtoliters, may be as low as 25-55 fL.

Further testing through a hematology laboratory can provide further confirmation of the diagnosis. Results in hereditary pyropoikilocytosis include the following ^[5] :

• Increased osmotic fragility
• Decreased acid glycerol lysis time
• Marked decrease in the maximum value of the deformability index (DI _max), with a distorted trapezoidal profile, on osmotic gradient ektacytometry
• Markedly decreased fluorescence on the eosin-5′-maleimide (EMA) binding test

In addition, thermal sensitivity testing shows fragmentation of the red blood cells at temperatures as low as 45°C.

Molecular analysis of membrane protein genes can be performed, but generally does not add extra information in cases where the patient's family is already known to have the red cell disorder. However, DNA sequencing may be warranted in some cases (eg, cases of suspected de novo mutation). ^[5]

Ektacytometry

Osmotic gradient ektacytometry has been the reference technique for diagnosis of RBC membrane disorders, but its limited availability has severely restricted its use. ^[8] An ektacytometer is a laser-diffraction viscometer in which deformability is measured as a continuous function of the osmolality of the suspending medium. 

On ektacytomettry, the Omin point is the osmolality at which the minimum deformability index is reached and is related to the surface area–to-volume ratio of the cell. The Hyper point is the osmolality at which the minimum deformability index reaches half of its maximum value. The Hyper point is related to the internal viscosity of the cell and to its mechanical properties. ^[9]

In hereditary elliptocytosis, ektacytometry shows decreased maximum deformability characterized by a trapezoidal curve with normal Omin and Hyper points. In hereditary pyropoikilocytosis, the maximum deformability is decreased and the Omin and Hyper points are shifted towards the left. ^[1]

Evaluate the ratio of spectrin to band 3 to determine the spectrin level so that this entity can be differentiated from homozygous hemolytic elliptocytosis.

Ultrasound of the biliary tract may show gallstones, especially in patients with symptomatic cholelithiasis. If ultrasonography is nondiagnostic, a nuclear scan can be done to work up gallbladder disease.