The Normal Range of Osmotic Fragility of Red Blood Cells

With the technique of Parpart et al (1947), the normal range for osmotic fragility of red blood cells has been estimated to include 5%–45% haemolysis at a salt concentration corresponding to 4.5 g NaCl/1 (Dacie 1954). This estimate may be questioned, however. Thus, nearly 20% of the data obtained from 50 presumably healthy subjects fell outside these limits. Furthermore, the distribution pattern was very asymmetric with erroneously small standard deviation. On the other hand, if the technical conditions (salt concentration, buffer) were properly adjusted, nearly all the observations were located within the straight part of the s-shaped haemolysis curve and scattered symmetrically around the mean. Under these conditions, the normal range (mean ± 2 SD) included 22%–88% haemolysis. Moreover, this wider range included about 95% of the observations, even adapted to the original experimental situation. The present estimates should therefore replace earlier statements in the literature.     

The Osmotic Fragility of Red Blood Cells: A Re-Evaluation of Technical Conditions

The osmotic fragility of red blood cells is influenced by even modest environmental changes. Consequently, the technical procedure must be strictly standardized. This implies that temperature equilibrium of the buffered salt solutions should be reached prior to the addition of blood. Furthermore, since erroneous statements concerning the composition of phosphate buffers regularly used to secure correct pH of the salt solutions have repeatedly appeared in the literature, pH control of such solutions prior to use becomes essential.     

Osmotic Erythrocyte Fragility and ABO Blood Groups

In a study of 1008 blood donors a reduced frequency of blood group A and an increased frequency of blood group O were observed in those with increased osmotic fragility of their red cells.     

Does Preincubation of the Red Blood Cells Contribute to the Capability of the Osmotic Fragility Test to Detect very Mild Forms of Hereditary Spherocytosis?

During incubation for 24 h at 37°C, erythrocytes from patients with hereditary spherocytosis (HS) undergo a greater increase in osmotic fragility than do normal cells, and this procedure has been recommended for differentiating more clearly between patients with very mild HS and normal subjects. The greater effect of preincubation on erythrocytes from patients with HS was confirmed, but, except in cases demonstrating a markedly increased osmotic fragility before incubation, this effect was outweighed by a simultaneous loss of test precision. It therefore seems that preincubation does not significantly contribute to the capability of the osmotic fragility test to detect very mild forms of HS.     

Lineation of the Osmotic Fragility Curve of Erythrocytes

Lineation of the osmotic fragility curve by the method of Detraglia et al (1974) may be performed in most samples from a hospital population. Using the lineation procedure, the osmotic fragility may be tested by only 2 solutions of known osmolarity without any great loss of precision or accuracy. The osmotic fragility curve may be described by 2 values: C₅₀ = the concentration at which 50% of the erythrocytes are haemolyzed and C₅₀ - C₈₀ = the decrease in concentration raising the fraction of haemolysis from 0.50 to 0.80.     

Failure of Ouabain to Influence the Osmotic Fragility in Hereditary Spherocytosis

No effect of 3 μmol ouabain on the osmotic fragility of red blood cells in subjects suspected of being carriers of hereditary spherocytosis, as well as in patients with overt disease could be demonstrated. These results are in disagreement with a recent report. Some possible explanations for these discrepant results are discussed. It is concluded that ouabain probably adds little to the diagnostic capability of the osmotic fragility test.     

Increased Osmotic Fragility of Erythrocytes in Essential Hypertension

The correlation between hypertension and the osmotic fragility of erythrocytes was examined. High osmotic fragility of erythrocytes was observed in patients with essential hypertension and normotensive subjects with family history of hypertension, compared with normotensive controls without family history of hypertension. In patients with secondary hypertension, the osmotic fragility of erythrocytes was not significantly different from that of normotensive controls without family history of hypertension. The membrane fragility had no correlation with the level of blood pressure or dietary salt intake. Thus, the osmotic fragility of erythrocytes might reflect functional or structural abnormalities of cell membranes, and could be one of the genetic markers of the hypertensive predisposition.     

Monocyte-Induced Increase in Osmotic Fragility of Human Red Cells Sensitized with Anti-D Alloantibodies

The mechanism by which human monocytes increase the osmotic fragility of red cells sensitized with Rhesus alloantibodies anti-D was studied in vitro. Both the increase in osmotic fragility and the lysis of red cells by monocytes were enhanced by cytochalasin B and were inhibited by hydrocortisone. These effects were similar to the effects of these agents on lysosomal enzyme release by monocytes. However, hydrocortisone was completely ineffective when added 1 h after mixing monocytes and sensitized red cells. This indicates that the damage responsible for the fragility increase and lysis is completed within 1 h and suggests that it is due to lysosomal enzymes released by the monocytes. Since for the full expression of the osmotic fragility increase and lysis an incubation time much longer than 1 h is required, it appears that the latter phenomena are the non-specific sequelae of damage inflicted upon the red cell by released lysosomal enzymes.     

Effect of Ouabain on Osmotic Resistance and Monovalent Cation Transport of Red Cells in Hereditary Spherocytosis

The effect of ouabain on the osmotic resistance of red cells from 17 splenectomized patients with congenital or hereditary spherocytosis (HS), from 5 of their relatives suspected of having a subclinical form of the disease and from unsplenectomized and splenectomized normal controls was studied. In red cells from the HS patients and the splenectomized controls a small ouabain-induced decrease in osmotic resistance was seen, whereas in the unsplenectomized controls no such change was observed. The osmotic resistance of red cells from the 5 relatives, on the other hand, was significantly decreased by ouabain. The usefulness of ouabain in unmasking HS in its subclinical form was further tested by studying monovalent cation influx into red cells. The active influx of ⁸⁶Rb+ into red cells from HS sufferers was significantly increased. A similar degree of ouabain-induced inhibition of ⁸⁶Rb+ transport was seen in all groups studied. The passive influx of ²²Na+, which was also increased in HS red cells, was not affected by ouabain in any of the four groups.     

High Prevalence of e Antigen among Healthy Blood Donors Carrying Hepatitis B Surface Antigen in Japan

Abstract. Hepatitis B surface antigen (HB_sAg) was detected by an immune adherence haemagglutination method in the serum samples of 292 voluntary, apparently healthy blood donors at four regional blood centres in Japan. Their serum samples were concentrated 3-fold and tested for e antigen (e Ag) and antibody to e (anti-e) by immunodiffusion. The e Ag was found in 41 samples (14.0%) and anti-e in 57 (18.6%). When 100 randomly selected serum samples containing HB_sAg were tested as they were (unconcentrated), and at 3- and 5-fold concentrations, e Ag was detected in 3,16 and 27, respectively, and anti-e in 10,21 and 26. Subtypes of HB_sAg were similar in carriers with e Ag and with anti-e. There is a high prevalence of e Ag in healthy individuals in Japan. There are also high rates of vertical transmission of hepatitis B virus from mothers to children, as well as a high incidence in the past of post-transfusion hepatitis. This is a further evidence that e antigen is a marker for the infectivity of hepatitis B virus in carriers.     

Increased Monoester Lipase Activity of Red Blood Cells in Alcoholism

Monoester lipase activity was assayed by a radiochemical assay in the red blood cells (RBC) from 50 chronic alcoholic patients within 48 hr after discontinuation of chronic alcohol intake and from 40 nonalcoholic control subjects. The mean value of lipase activity was increased to 1213 +/- 229 milliunits/10(12) RBC in the alcoholics as compared with a value of 997 +/- 120 milliunits/10(12) RBC in the controls (p less than 0.001). The lipase increase was associated with increased values of the mean cellular volume of RBC. A subgroup (64%) of 32 alcoholic subjects with macrocytosis (mean cellular volume greater than 96 femtoliters) showed the highest mean lipase activity (1276 +/- 224 milliunits/10(12) RBC) as compared with a value of 1101 +/- 196 milliunits/10(12) RBC in the normocytic alcoholic subgroup (p less than 0.05). This latter subgroup had a mean value higher (p less than 0.05) than that in the control group. The relationship between values of mean cellular volume and lipase activity was not of predictive value in individual cases. The enzymatic increase was not related to a direct effect of alcohol on the intact RBC. It is postulated that this alteration might result from changes in the chemical and/or physical state of the plasma membrane induced by ethanol during RBC formation. In any event, the increased lipolytic activity of RBC represents a new biological characteristic of alcoholic subjects. Its determination might represent a noninvasive way of evaluating the influence of alcohol on a tissue parameter.     

Comparison of Solid-Phase Antibody Screening Tests with Pooled Red Cells in Blood Donors

Solid-phase systems are very sensitive for detection of erythrocyte alloantibodies in serum and suitable for large scale donor screening. Serum samples of 10,008 blood donors were screened by three solid-phase tests (Capture R Ready Screen, Solidscreen II, and Solidscreen II-Donor) with pooled red cells derived from two donors. The prevalence of antierythrocyte IgG and IgM antibodies in donor sera was 0.56% (IgG antibodies: 0.42%). The test efficiency for IgG antibodies was 1.40 with Capture R Ready Screen (test sensitivity 97.6%, test specificity 99.39%), 1.78 with Solidscreen II (95.1, 99.88%), and 1.95 with Solidscreen II-Donor (92.7, 99.98%). The IgG antibody titers differed significantly between all tests including a gel matrix test: Capture R > ID-Gel > Solidscreen II > Solidscreen II-Donor. Previously characterized antibodies that were not detected after long-term follow-up by any of the three solid-phase tests had a prevalence of 0.10%. All three solid-phase tests detected the alloantibodies, which were of higher titers and considered clinically relevant in blood components. The significant difference in antibody titers between the tests was not matched by a similar variance in the detection of donors with antibodies. Even with sensitive solid-phase tests, many antibodies may not be detected after long-term follow-up.     

Increased Potassium Permeability by Calcium in Hypochromic Red Blood Cells

The red blood cell (RBC) content of Na⁺ and K⁺ were measured both on fresh cells from normal, heterozygous β-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37° C, either in presence or in absence of Calcium (Ca²⁺). Ca²⁺ did not increase membrane permeability to Na⁺, but increased the K⁺ loss, both from normal cells and to a greater degree much more from hypochromic cells. Glucose largely prevented the K⁺ loss from hypochromic cells incubated either in absence or in presence of Ca²⁺, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K⁺ in hypochromic cells incubated for 24 h at 37° C, possibly removing Ca²⁺ bound to the cell membrane. The results suggest that Ca²⁺ does not represent the primary cause of K⁺ leak in hypochromic cells, but it is able to enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down.     

Production of Spherocytosis and Increased Osmotic Fragility in Normal Erythrocytes by Sodium Stress

Red cells from normal human subjects were exposed to hypertonic sodium chloride solutions for varying periods of time. The cell changes commonly observed in hereditary spherocytosis were induced, including microspherocyte formation with corresponding increase in osmotic fragility. Solutions of magnesium sulphate of equal osmolarity did not produce these changes. These observations lend support to the theory that excessive sodium permeability is concerned in the pathogenesis of hereditary spherocytosis. Increased influx of sodium and water into erythrocytes leads to spherocytosis, but only increased efflux of sodium leads to irreversible microspherocytosis.     

Characterization of the Increased Binding of Acetaldehyde to Red Blood Cells in Alcoholics

Using equilibrium dialysis, we found that acetaldehyde, at the levels commonly occurring after ethanol ingestion, did not bind detectably to plasma proteins, but there was significant binding to red blood cells, more in alcoholics than in nonalcoholics. The binding to red blood cells was inhibited by pyridoxal phosphate and N-ethylmaleimide, suggesting adduction to amino and thiol groups. Binding kinetics were consistent with at least two sites. The one with the highest affinity for acetaldehyde corresponded to hemoglobin. Its affinity and Bmax were not changed in alcoholics, but these binding sites accounted for only 44% of the sites available in the red blood cells of alcoholics and 80% of those in controls. Moreover, this binding was not inhibited by N-ethylmaleimide. There was no detectable binding to red cell ghosts. Nonprotein binding was then assessed by changes in NADH produced by the addition of protein-free fractions of the cells to an alcohol dehydrogenase system in equilibrium; this revealed a second binder of lower affinity, larger capacity and with sensitivity to both inhibitors. This binding (possibly due to thiazolidine formation with cysteine) was enhanced in alcoholics, whose red blood cell cysteine content was doubled. Levels of red blood cell cysteine and acetaldehyde remained high for 2 weeks after withdrawal. Because of the prolonged persistence after withdrawal, these changes may provide new markers of alcoholism.     

Increased ATP-ase Activity of Lymphocytes after Rosette Formation with Sheep Red Blood Cells

In a number of experiments it was demonstrated that in the E-rosette assay for human T-lymphocytes using sheep red blood cells, the ATP-ase activity of the lymphocytes increased in proportion to the number of rosettes being formed. Also, after increasing the number of rosettes by treatment of the sheep red blood cells (SRBC) with neuraminidase and after diminishing the number of rosettes by treatment of the lymphocytes with antilymphocyte globulin (ALG), change in the ATP-ase activity was proportional to alterations in the number of rosettes being formed. ALG itself stimulated the lymphocyte ATP-ase activity, possibly through activation of the same receptor sites as used by SRBC.     

High Prevalence of HBV Infectivity in Blood Donors Detected by the Dot Blot Hybridisation Assay

Hepatitis B virus (HBV) continues to be a significant cause for post-transfusion hepatitis in India, in spite of the introduction of compulsory hepatitis B surface antigen (HBsAg) screening. To understand the true HBV-infective pool in the blood donor population, HBV DNA was detected by a ³²P-labelled dot blot hybridisation assay in 605 donor units that were negative for HBsAg by a third-generation Elisa. Serum alanine aminotransferase (ALT) was estimated in all these samples and correlated with DNA positivity. The frequency of HBV DNA positivity in HBsAg-negative units was very high (9.91%) and correlated well with the elevation in ALT (p<0.00005). However, the frequency of elevated ALT was high (11.9%), using the locally determined upper limit of normal, and half of the DNA-positive samples had a normal ALT. Thus, ALT is a poor surrogate marker for HBV infectivity and efforts should be made to apply DNA detection systems in blood banks.     

The Prevalence of Celiac Disease in Blood Donors in Iceland

Background Prospective epidemiological studies based on serological methods have shown that celiac disease is more common than previously thought. The aim of this study was to evaluate the prevalence of celiac disease among apparently healthy blood donors in Iceland. Methods Plasma samples were obtained from 813 apparently healthy blood donors at the FSA Hospital Blood Bank in Akureyri, Iceland, between December 2004 and January 2007 and screened for human tissue transglutaminase IgA antibodies. Positive samples were retested and, if the test was again positive, the subject was referred to a gastroenterologist for clinical examination and a duodenoscopy with mucosal biopsies. Results Six subjects tested positive for tissue transglutaminase. The prevalence of biopsy-confirmed celiac disease, according to modified Marsh classification, among apparently healthy blood donors in Iceland was found to be 1:136 (0.74%, 95% confidence interval 1/667–1/75, 0.15–1.33%). Conclusion Prevalence of celiac disease in Iceland is similar to what has been reported in many other countries.