Environmental Stability of Hemoglobin Samples for HPLC Analysis on the Bio-Rad D10 Platform.

ABSTRACT

High Performance Liquid Chromatography (HPLC) is an important method in analysis of normal and abnormal hemoglobins. We carried out a laboratory based study to assess the stability of hemoglobins in two important environmental conditions, room temperature (18-250C) and cold storage conditions (2-8 degrees). The study showed that samples for analysis on the Bio-Rad
D10 are stable at room temperature for up to 3 days and up to 4 days when stored at 2-8C.

INTRODUCTION

Cation-exchange high-performance liquid chromatography (HPLC) has become a preferred method to identify and quantify abnormal hemoglobin variants. In HPLC, hemoglobins are adsorbed to a negatively charged resin column (stationary phase) and then eluted from the column by a positively charged solution that is added in increasing concentration and competes for binding to the negatively charged resin (Gupta et al. 2009; Higgins* 2000). Hemoglobins are thus differentially eluted at a rate related to their affinity for the column and detected by a photometer. The resulting elution patterns are graphically represented
in defined windows based on their relative time to appearance in the eluate (retention time). The size of the hemoglobin fractions can be quantified by computing the area under the curve (AUC) for each peak (Wild & Bain 2004; Basset et al. 1982). D-10 Hemoglobin Testing System, here in referred as the D10 (Bio-Rad Laboratories, Inc, Clinical Diagnostics Group, Hercules, California, 94547 USA) provides comprehensive, fully automated hemoglobin testing in a compact footprint. The equipment from Bio-Rad combines both diabetes and β-thalassemia testing on a single platform that offers simple, efficient operation for clinics, physician offices or clinical laboratories (Bio-Rad; n.d.).

BACKGROUND

The Aga Khan Hospital Kisumu Laboratory is an ISO15189 accredited medical laboratory in Kisumu City in Kisumu County (AKHK). The laboratory serves the residents of western Kenya region and fully supports hemoglobin electrophoresis testing for its affiliated 13 small medical centres distributed throughout the Western and Rift Valley regions in Kenya. Each day samples for hemoglobin analysis are transported to the main laboratory in Kisumu for analysis. This is a laboratory based study that aimed at assessing the stability of stability of hemoglobin subtypes in clinical samples for analysed on the Bio-Rad HPLC D10 Platform
when samples are stored either at room or cold conditions. The study was carried at Aga Khan Hospital Main Laboratory in Kisumu, Kenya from June 24th to July 2nd 2019.

METHODOLOGY

Ten freshly collected samples were collected from health blood volunteers (healthy staff working at the Main Laboratory). The samples were split into 2 batches that were then stored differentially, one batch at room temperature and another batch at fridge maintained at temperature of 18-250C and 2-8 o C respectively. Each of these samples were aliquoted into 10 smaller portions for ease of analysis. Each batch of samples from the room temperature and fridge storage were analysed at 12 pm each day following manufacturer’s instructions. The analysis was carried out for a period 6 days (days 1, 2, 3, 4, 5 and 7) and for a period of 5 days (days 1, 2, 3, 4 and 5 for samples stored at room temperature). The instrument quality controls and calibrations were followed according to manufacturer’s guidelines. The results were captured on excel sheet and analyzed using graphical methods for stability and trends.

RESULTS

When stored at room temperature, the P3 window on the chromatogram showed an increase starting on Day 3 suggesting increased degradation of hemoglobins. When samples are stored at 2-80C the increase in P3 window started on Day 4 of storage. Glycated hemoglobin A1C remained fairly stable during the period of storage, both at room and fridge temperatures (Day 7 for 2-8 degrees and Day 5 at room temperature). Hemoglobin A (HbA) values showed a bigger decrease of about 8.5% on Day 5 when stored at room temperature compared to a 3.2% decrease when stored at fridge temperature. Hemoglobin A2 values remained fairly stable both at room fridge and room temperatures during the period of study.

CONCLUSION

Hemoglobin samples are stable for analysis for a period of 3 days when stored at room conditions (18-250C) and for 4 days when stored under fridge conditions. The manufacturer’s claim of stability up to 4 days was confirmed during this study (Source: Bio-Rad D10 Instrument Manual). After 3 days of storage at room conditions and 4 days at fridge storage, the hemoglobin degradation rapidly increased with decrease in Hemoglobin A values and increased in P3 window values. The study also noted that glycated hemoglobin (A1C) and hemoglobin A2 was remained fairly stable when stayed at both room temperature for up to 5 days and up to 7 days at fridge temperature.