Wallach's Interpretation of Diagnostic Tests: Pathways to Arriving at a Clinical Diagnosis (976 page)

   Measurement of HbA
₂
and HbF has great clinical value in the diagnosis as well as in characterization of some Hb structural variants and other hemoglobinopathies.

   Increase in the HbA
₂
level is considered the most characteristic diagnostic feature of β-thalassemia trait and represents an essential test in the screening programs for β-thalassemia prevention.

   Hemoglobin A
₂
prime is a delta chain variant Hb occurring in a small percentage of individuals of African ancestry. On HPLC, Hb A
₂
prime falls in the Hb S window, but its retention time differs from that of Hb S. When quantifying Hb A
₂
for the diagnosis of β-thalassemia heterozygosity, it is essential to add together the A
₂
and A
₂
prime to obtain a “total Hb A
₂
.”

   There are two methods in use to screen for Hb variants:

   HPLC is used as the primary screening tool because it readily quantitates HbA, HbA
₂
, and HbF; in addition, it presumptively identifies three of the most common Hb variants seen in North America: HbS, HbC, and HbD. All other abnormal variants will be flagged and need to be identified by hemoglobin electrophoresis (HE).

   Alkaline and acid HE is used to investigate the whole array of Hb variants. A practical method for HE is cellulose acetate at alkaline pH. Hb molecules in an alkaline solution have a net negative charge and move toward the anode. The method separates HbA, HbA
₂
, HbS, HbF, and HbC. Citrate agar gel electrophoresis at an acid pH separates hemoglobin variants that migrate together on cellulose acetate: HbS from HbD and HbG, HbC from HbE and HbO. Many of the hemoglobin forms that are difficult to differentiate by gel HE can be differentiated by HPLC. For instance, on gel electrophoresis, HbA
₂
is difficult to distinguish from HbC because they migrate together. Testing by HPLC allows the quantification of HbA
₂
in the presence of HbC. The two methods complement each other.

   All Hb variants that are not diagnosed by these two methods require further testing with mass spectroscopy, capillary isoelectric focusing, or sequencing of DNA fragments generated by PCR.

   Interpretation

Increases

   HbA
₂
: megaloblastic anemia, β-thalassemias

   HbF: acquired aplastic anemia, hereditary persistence of fetal Hb, hyperthyroidism, leakage of fetal blood into maternal circulation, leukemia (acute or chronic), myeloproliferative neoplasms, sickle cell disease, thalassemias, β chain substitutions

   HbC (second most common variant in the United States)