Different Involvement of Band 3 in Red Cell Deformability and Osmotic Fragility—A Comparative GP.Mur Erythrocyte Study

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3–AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3–AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural–functional roles of band 3.

Download Full-text

The effect of the plasticizer di(2-ethylhexyl)phthalate on red cell deformability

Blood ◽

10.1182/blood.v70.1.319.bloodjournal701319 ◽

1987 ◽

Vol 70 (1) ◽

pp. 319-323

Author(s):

RS Labow ◽

RT Card ◽

G Rock

Keyword(s):

Storage Period ◽

Osmotic Fragility ◽

Osmotic Gradient ◽

Red Cell ◽

Cell Deformability ◽

Red Cell Deformability ◽

Plastic Bag ◽

Elongation Index ◽

Plastic Containers ◽

Ethylhexyl Phthalate

Red cell concentrates (RCC) are stored for 35 to 42 days in plastic containers manufactured with the liquid plasticizer di(2- ethylhexyl)phthalate (DEHP). DEHP leaches from the polyvinylchloride (PVC) plastic bag, then binds to and stabilizes the RC membrane. This study was undertaken to determine the deformability of the RC membrane using an osmotic gradient ektacytometer and to relate these measurements to the concentration of DEHP in the stored RCC. Pooled RCC was aliquoted into PL146 (PVC), PL732 (polyolefin), and PL732 (with added DEHP) bags with samples removed weekly for analysis of osmotic fragility, deformability, and DEHP concentration. The adenosine triphosphate (ATP) content was also measured. The increase in osmotic fragility during storage was greater when RCC was stored without DEHP. In addition, there was a decrease in the maximum elongation index (El max) when there was decreased DEHP in the storage bag. The osmolarity (Omax) at which El max occurred, as well as the Omin, the osmolarity at which minimum elongation (El min) occurred was higher in the PL732 container than in the PL146 or in the PL732 to which DEHP had been added. These changes could be reversed by addition of DEHP at the beginning of the storage period, showing a direct correlation between DEHP concentration during storage and RC membrane flexibility. By a better understanding of the mechanism of DEHP protection, it might be possible to substitute a less toxic stabilizing compound.

Download Full-text

Red Cell Band 3 Content Evaluation By Eosin Maleimide (EMA) Fluorescence: Beyond Diagnosis of Dominant Hereditary Spherocytosis (HS)

Blood ◽

10.1182/blood.v126.23.3343.3343 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3343-3343

Author(s):

Ahmar Urooj Zaidi ◽

Miguel Herrera-Martinez ◽

Gerard W. Goyette ◽

Steven Buck ◽

Manisha Gadgeel ◽

...

Keyword(s):

Conflicts Of Interest ◽

Band 3 ◽

Glycolytic Enzyme ◽

Small Cells ◽

Deficiency Anemia ◽

Osmotic Gradient ◽

Red Cell ◽

Scatter Plot ◽

Cell Deformability ◽

Red Cell Deformability

Abstract Estimation of band 3 content by EMA fluorescence intensity is rapidly replacing conventional osmotic fragility test as a tool for laboratory confirmation of dominant HS, especially in UK, Europe and certain other countries outside of US (e.g India, Argentina). Its use in US still anecdotal. We have systematically evaluated the utility of EMA test as a screening tool for anemias of various causes and compared the results with those obtained with osmotic gradient ektacytometry-osmoscans, a standard tool in our research laboratory for the last 30 years. Our experience with osmoscans are previously published (Johnson et al, Ravindranath et al) and show distinctive patterns in HS, hereditary elliptocytosis (HE), Southeast Asian Ovalocytosis (SAO), hereditary pyropoikilocytosis (HPP), red cell enzymopathies and hemoglobinopathies. Here we compare the results obtained with osmoscans and EMA testing. An important difference with the two methodologies is that osmoscans measure red cell deformability of all the red cells in the sample across an osmotic gradient from 100-500 mOsmoles (mOsm) while the EMA test reflects the band 3 content of individual red cell. Thus the EMA test is band 3 content as well as cell size dependent. The Omin of osmoscan (lowest osmotic strength at which the cells are still intact and deformable) and the MCF (mean channel of fluorescence) correlate well in dominant HS (usually from mutations in ankyrin, band 3, beta spectrin [SPTB]) in which band 3 is decreased. However, not all samples exhibiting low MCF are due to HS. In fact we observed the lowest MCF values in 3 MCF cases of HPP (MCF 224.4, range 199.7 to 250.8); 34 % reduction of MCF in two Filipino individuals with SAO (319.1, 359.4) and MCF of 390 +/-76 in 19 dominant HS cases versus control values of 514 +/-15, and variably low values in iron deficiency anemia, thalassemia traits, Hb C and cases of autoimmune hemolytic anemias (AIHA). Diagnosis of neonatal HS and distinction form spherocytosis associated with ABO incompatibility remains a challenge. HE cases show distinctive bimodal pattern. A previously unsuspected signature of glycolytic enzyme defects has emerged on scatter plots and histograms with a distinctive tail of small cells - presumably the ATP depleted dense spiculated cells in 3 cases of pyruvate kinase (PK) deficiency and a new case of phosphoglycerte kinase (PGK1) deficiency. In two cases with HS associated with SPTA1 mutations, the MCF was minimally decreased. Representative patterns are shown below. Conclusions Thus while all dominant HS cases have low MCF on EMA scan, low MCF per se is not diagnostic of HS. The EMA scan results should be interpreted with caution and both the histograms and dot plots should be analyzed in the context of the clinical picture and morphology; >30% reduction in MCF should raise suspicion of SAO or HPP. Figure 1. Osmoscan (upper panel) and EMA histogram (lower panel) for HS, HPP, SAO and HE. PKD scatter plot (upper panel) and EMA histogram (lower panel) Figure 1. Osmoscan (upper panel) and EMA histogram (lower panel) for HS, HPP, SAO and HE. PKD scatter plot (upper panel) and EMA histogram (lower panel) Disclosures No relevant conflicts of interest to declare.

Download Full-text

The effect of the plasticizer di(2-ethylhexyl)phthalate on red cell deformability

Blood ◽

10.1182/blood.v70.1.319.319 ◽

1987 ◽

Vol 70 (1) ◽

pp. 319-323 ◽

Cited By ~ 24

Author(s):

RS Labow ◽

RT Card ◽

G Rock

Keyword(s):

Storage Period ◽

Osmotic Fragility ◽

Osmotic Gradient ◽

Red Cell ◽

Cell Deformability ◽

Red Cell Deformability ◽

Plastic Bag ◽

Elongation Index ◽

Plastic Containers ◽

Ethylhexyl Phthalate

Abstract Red cell concentrates (RCC) are stored for 35 to 42 days in plastic containers manufactured with the liquid plasticizer di(2- ethylhexyl)phthalate (DEHP). DEHP leaches from the polyvinylchloride (PVC) plastic bag, then binds to and stabilizes the RC membrane. This study was undertaken to determine the deformability of the RC membrane using an osmotic gradient ektacytometer and to relate these measurements to the concentration of DEHP in the stored RCC. Pooled RCC was aliquoted into PL146 (PVC), PL732 (polyolefin), and PL732 (with added DEHP) bags with samples removed weekly for analysis of osmotic fragility, deformability, and DEHP concentration. The adenosine triphosphate (ATP) content was also measured. The increase in osmotic fragility during storage was greater when RCC was stored without DEHP. In addition, there was a decrease in the maximum elongation index (El max) when there was decreased DEHP in the storage bag. The osmolarity (Omax) at which El max occurred, as well as the Omin, the osmolarity at which minimum elongation (El min) occurred was higher in the PL732 container than in the PL146 or in the PL732 to which DEHP had been added. These changes could be reversed by addition of DEHP at the beginning of the storage period, showing a direct correlation between DEHP concentration during storage and RC membrane flexibility. By a better understanding of the mechanism of DEHP protection, it might be possible to substitute a less toxic stabilizing compound.

Download Full-text

The Effects of Acute Smoking on Platelet Behaviour, Fibrinolysis and Haemorheology in Habitual Smokers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660996 ◽

1984 ◽

Vol 51 (01) ◽

pp. 006-008 ◽

Cited By ~ 96

Author(s):

J J F Belch ◽

B M McArdle ◽

P Burns ◽

G D O Lowe ◽

C D Forbes

Keyword(s):

Platelet Aggregation ◽

Factor Viii ◽

Plasminogen Activator ◽

Arterial Thrombosis ◽

Plasma Viscosity ◽

Red Cell ◽

Cell Deformability ◽

Red Cell Deformability ◽

Cigarette Smokers ◽

Smoking Group

SummaryThere is an increased frequency of arterial thrombosis in cigarette smokers. The changes in blood coagulation seen in these subjects have been studied by many workers but results have not always been in agreement. We wished to study the effects of acute .smoking on platelet behaviour, fibrinolysis and haemorheology in ten habitual smokers, and to compare these results with nonsmoking controls. Results show that the smoking group had higher plasma fibrinogen (p <0.04), lower plasminogen (p <0.02) and plasminogen activator (p <0.05), and higher plasma viscosity (p <0.003). The changes seen in cigarette smokers after smoking three cigarettes were an increase in the rate of platelet aggregation to ADP (p <0.02), an increase in α2M, (p <0.02), and factor VIII RAG (p <0.05). Plasma viscosity was decreased (p <0.02) as was red cell deformability (p >0.02).We confirm an increased tendency to hypercoagulability in smokers compared to controls which becomes more pronounced immediately after smoking three cigarettes.

Download Full-text

An Assessment of Red Cell Deformability Using a Simple Filtration Method

10.1055/s-0038-1665788 ◽

1979 ◽

Author(s):

M Drummond ◽

G Lowe ◽

J Belch ◽

C Forbes ◽

J Barbenel

Keyword(s):

Cell Count ◽

Shear Viscosity ◽

White Cell Count ◽

White Cell ◽

Red Cell ◽

Cell Deformability ◽

Red Cell Deformability ◽

Simple Method ◽

Filtration Method ◽

Significant Difference

We investigated the reproducibility and validity of a simple method of measuring red cell deformability (filtration of whole blood through 5 µ sieves) and its relationship to haematocrit, blood viscosity, fibrinogen, white cell count, sex and smoking. The mean coefficient of variation in normals was 3. 7%. Tanned red cells showed marked loss of deformability. Blood filtration rate correlated with haematocrit (r = 0. 99 on dilution of samples, r = 0. 7 in 120 normals and patients). After correction for haematocrit, deformability correlated with high shear viscosity, but not low shear viscosity, fibrinogen or white cell count. In 60 normals there was no significant difference between males and females, or smokers and non-smokers, but in 11 smokers there was an acute fall in deformability after smoking 3 cigarettes (p<0. 05). Reduced deformability was found in acute myocardial infarction (n = 15, p<0. 01) and chronic peripheral arterial disease (n = 15, p<0. 01). The technique is reproducible, detects rigid cells and appears useful in the study of vascular disease.

Download Full-text