Partially oxygenated sickled cells: sickle-shaped red cells found in circulating blood of patients with sickle cell disease.

Abstract

A previously uncharacterized type of sickled cell was found in venous blood ofpatients with sickle cell disease when blood was collected without exposure to air and fixed immediately with 1% glutaraldehyde solution equilibrated with 5% oxygen. These cells were either elongated, resembling irreversibly sickled cells (ISCs), or nonelongated, with a raisinlike shape. Both types assumed a normal discoidal shape upon full oxygenation. Since these cells exist only under partially oxygenated conditions, they are described as partially oxygenated sickled cells (POSCs). POSCs are morphologically distinct from partially deoxygenated sickled cells formed during deoxygenation by having rounded edges, while the latter have sharp edges. Transmission electron microscopy of POSCs revealed various amounts of misaligned Hb S polymers. Investigations in vitro demonstrated the formation of POSC-like cells by partial oxygenation of deoxygenated cells. Since POSCs contain intracellular fibers and sickle readily upon deoxygenation, they may have clinical and pathological significance.Sickle cell disease was so named because of the abnormal morphology of red blood cells (RBCs) observed by microscopic examination offixed and stained blood smears obtained from affected patients (1). Subsequently, relationships were found between oxygen and RBC sickling (2) and between intracellular polymerization of abnormal hemoglobin and cell deformation (3-5). Demonstration of sickling of RBCs from patients with sickle cell disease (SS cells) in vitro is highly dependent upon the method of deoxygenation. For example, classic sickle-or crescent-shaped cells are formed after slow deoxygenation (6-9), whereas SS cells with a granular or mosaic appearance result from rapid deoxygenation with nitrogen (6) or sodium dithionite (7). These differences may be explained by the number and size of domains of polymerized Hb S formed in the cells (10). During morphologic studies of SS cells, we noticed that the number and shape of sickled cells in venous blood were altered by the oxygenation of blood during and after blood collection. To avoid postcollection artifacts, we developed a method to collect blood under venous oxygen pressure without exposure to air. This enabled us to discover two additional types of reversibly sickled cells, elongated sickled cells with a shape like irreversibly sickled cells (ISCs) and shrunken cells with a raisin-like appearance. Since these cells exist only under partially oxygenated conditions, they are described as partially oxygenated sickled cells (POSCs) (11). We report our investigation of the morphologic properties and analysis by electron microscopy ofintracellular polymers of POSCs found in venous blood of patients with sickle cell disease. MATERIALS AND METHODSPreparation of Blood Samples. To minimize sickling during blood drawing (12), blood was drawn from the antecubital vein after release of the tourniquet. A conventional 5-ml plastic disposable syringe was used by the technique similar to that used for collectio...

Exposure of blood from patients with sickle cell disease to air changes the morphological, oxygen‐binding, and sickling properties of sickled erythrocytes

“…First, since there was no technology to collect blood samples under physiological oxygen pressures for further studies, most RSCs were fixed in fixing solution at the time of blood collection. Second, when blood is collected by the ordinary method (Exp-blood), cells are exposed to oxygen in the needle, syringe, and Vacutainer, and most RSCs are oxygenated, causing them to assume a normal discoidal shape [1,2]. As we will describe in this paper, partial deoxygenation of once-oxygenated blood to venous oxygen pressure does not recover in vivo properties of SS cells.…”

“…Previously, we found that venous blood samples collected from steady-state patients with sickle cell disease (SCD) under venous oxygen pressure without exposure to air (UnExp-blood) contained many reversibly sickled cells (RSCs) with blunt edges [1,2]. Although the percentage varied widely among patients (4-78%), it remained within a narrow range in individual patients during the steady state [3].…”

Blood samples collected under venous oxygen pressure from patients with sickle cell disease contain a significant number of a new type of reversibly sickled cells: Constancy of the percentage of sickled cells in individual patients during steady state

“…UnExp-blood was collected from the antecubital vein for routine blood tests [9,10]. To prevent in vivo cell sickling caused by stagnation of blood [11], the tourniquet was released after the needle was inserted into the vein and before the blood was drawn.…”

“…Although the sickled cells found in UnExp-blood had a shape similar to ISCs, most of the sickled cells were determined to be POSCs because they converted to discocytes upon exposure to air or carbon monoxide [10,19]. Blood samples collected by the standard method do not contain a large number of POSCs because they are oxygenated to discocytes by the oxygen in the volume of air in the needle, syringe, and Vacutainer [9,10]. As shown herein, although the percentage of POSCs varied widely among patients, the percentage and morphological characteristics of POSCs in individual patients were maintained in a narrow range during steady state.…”

“…The constancy of the percentage of sickled cells in individual steady-state patients suggests one or both of the following two possibilities: (1) that most of the modulating factors, including capillary size (diameter and length), lung capacity, blood pH, Hb F level, and 2,3-DPG level [3,9], are relatively constant in individual patients during steady state; (2) that the morphology of reversibly sickled cells does not necessarily change in response to small changes in the microenvironment that occur in vivo, due to semi-irreversible stretching of the sickled cell membrane. We assume that both of these factors are involved in maintaining the percentage and morphology of sickled cells within a narrow range in steady-state patients.…”