RBC electrophoresis with discontinuous power supply – a newly established hemoglobin release test

Abstract

In this paper, we aimed to introduce a newly established red blood cells (RBCs) electrophoresis method hemoglobin release test (HRT) and tried to determine its significance. Human blood samples from beta-thalassemia patients and healthy controls were analyzed with HRT, which was carried out on starch-agarose mixed gel. First, the whole blood samples were electrophoresed for 2 h, then paused for 15 min and ran for 15 min by turns. This "pause-run-pause" experiment was performed for several turns and the total electrophoresis time lasted for about 6 h. The results showed that some other hemoglobin (Hb) components were released from the origin of each sample during the HRT, and the samples from beta-thalassemia patients released more Hb than the healthy controls. This finding demonstrates that Hb may exist differently associated in RBCs, and it may have an important theoretical and clinical significance in Hb and RBC research.

Abnormal increased re-released Hb from RBCs of an intrahepatic bile duct carcinoma patient was detected by electrophoresis release test

“…The increased Hb re-release was firstly observed in ȕ-thalassemia patient [1], and then it was observed in diabetes patients and some general surgical patients [3]. To the contrary, the re-released Hb could also decrease distinctively or disappear from RBCs of hereditary spherocytosis patient.…”

“…Hemolysate was prepared by continuously adding 200 μL saline and 100 μL CCl 4 to the RBCs. After turbulent mixing, the sample was centrifuged at 12 000 rpm for 10 minutes and the upper red hemolysate was pipetted out carefully and stored at 4 for later use [1][2][3]. The 2% starch-agarose mixed gel (4:1) was prepared with TEB buffer (pH 8.6) as described previously [1].…”

“…The electrophoresis was ran at 6 V/cm for 15 minutes, then paused for 15 minutes and ran for another 15 minutes by turns, and the total electrophoresis time was about 2 hours. After electrophoresis, the red bands on the gel were firstly observed with eyes and then sequentially stained with Ponceau Red and Benzidine [1].…”

“…Electrophoresis release test (ERT) has been established by our laboratory [1,2]. During ERT, live red blood cells (RBCs) are added directly onto the starch-agarose mixed gel and the electric current perforates the membrane instantaneously.…”

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test

“…This phenomenon was named the ''HbA 2 phenomenon'' [19][20][21] and an electrophoresis method using live RBCs, the electrophoretic release test (ERT), was developed [21]. Although many people think of this as a simple physical phenomenon, we continue to believe that this is an intrinsic phenomenon that may have some relationship with the interactions between Hbs and other proteins in the RBC.…”