Cation-exchange high-performance liquid chromatography for variant hemoglobins and HbF/A2: What must hematopathologists know about methodology?

Abstract

Cation-exchange high-performance liquid chromatography (CE-HPLC) is a widely used laboratory test to detect variant hemoglobins as well as quantify hemoglobins F and A2 for the diagnosis of thalassemia syndromes. It's versatility, speed, reproducibility and convenience have made CE-HPLC the method of choice to initially screen for hemoglobin disorders. Despite its popularity, several methodological aspects of the technology remain obscure to pathologists and this may have consequences in specific situations. This paper discusses the basic principles of the technique, the initial quality control steps and the interpretation of various controls and variables that are available on the instrument output. Subsequent sections are devoted to methodological considerations that arise during reporting of cases. For instance, common problems of misidentified peaks, totals crossing 100%, causes of total area being above or below acceptable limits and the importance of pre-integration region peaks are dealt with. Ultimately, CE-HPLC remains an investigation, the reporting of which combines in-depth knowledge of the biological basics with more than a working knowledge of the technological aspects of the technique.

Paper-based microchip electrophoresis for point-of-care hemoglobin testing

“…This sharing of detection window or peak overlapping is also a challenge for the reference standard method, HPLC as well as its alternatives. 93,95,96 For example, in HPLC, hemoglobin S elutes in the S window with 28 other hemoglobin variants, including 10 other β-chain variants. 97 A similar issue occurs in the A 2 window for HPLC, where hemoglobin E and 18 other hemoglobin variants elude in the same window, and 13 of these 18 variants are β-chain variants.…”

Multispectral Imaging for MicroChip Electrophoresis Enables Point-of-Care Newborn Hemoglobin Variant Screening

“…For example, it is challenging to discern Hb C and Hb A 2 , even using a laboratory platform of capillary zone electrophoresis because these hemoglobin variants demonstrate partially overlapped peak within the same detection window [35, 36]. This peak overlapping is also a challenge for the reference standard HPLC as well as its alternatives [35, 37, 38]. Notably, Hb C and Hb E are known to co-migrate in paper-based hemoglobin electrophoresis.…”

HbH disease due to compound heterozygosity for hemoglobins Zürich‐Albisrieden and Sallanches

“…He had normal range HbF, mildly reduced HbA2 (1.8%), and two small preintegration peaks (retention time < 1 min, Figure 1A, blue arrow). Differential diagnoses of such peaks include bilirubin (typically a single, sharp, early peak), acetylated HbF (unlikely in this case as HbF was 0.6%), Hb Barts (unlikely in an adult), and HbH . Supravital staining revealed erythrocytes with golf‐ball‐like HbH inclusions as well as several Heinz bodies (Figure 1B).…”

“…Our patient, in addition to expanding the list of genotypes resulting in HbH disease and being the first reported instance of this rare combination worldwide, also highlights other diagnostically relevant points. His small preintegration HbH peaks, missed by the earlier laboratory, remind hematopathologists that correlation of HPLCs with the clinical background, erythrocyte indices, and low‐cost investigations such as HbH preparation and heat/isopropanolol instability tests may be of high yield in such cases . The inappropriate immunosuppressive therapy due to the single 2+ DCT in a recently transfused patient who may have developed clinically insignificant alloantibodies also highlights the importance of evaluating weak or low‐grade positive DCT results from the perspective of the complete clinical scenario .…”

“…, Cation-exchange high-performance liquid chromatography (CE-HPLC) shows two small preintegration peaks (blue arrow) and a mildly reduced HbA2. B, New methylene blue stained supravital preparation shows red cells with Heinz bodies (yellow arrows) and rare red cells with golf-ball-like inclusions (red arrow) along with reticulocytosis remind hematopathologists that correlation of HPLCs with the clinical background, erythrocyte indices, and low-cost investigations such as HbH preparation and heat/isopropanolol instability tests may be of high yield in such cases 2. The inappropriate immunosuppressive therapy due to the single 2+ DCT in a recently transfused patient who may have developed clinically insignificant alloantibodies also highlights the importance of evaluating weak or low-grade positive DCT results from the perspective of the complete clinical scenario 10.…”