Aging and red blood cell membrane: a study of centenarians.

Successful aging, characterized by little or no loss in physiological functions, should be the usual aging process in centenarians. It is known that well-preserved physiological functions depend on the proper functioning of cell systems. In this article we focus on cell membrane integrity and study the red blood cell membrane to evaluate the effect of physiological aging in centenarians. Fifteen healthy, self-sufficient centenarians, mean age 103 years, were examined by assessing hemocytometric values and some relevant characteristics of the erythrocyte membrane, i.e., the cholesterol/phospholipid molar ratio, the distribution of phospholipid classes and their fatty acid composition, the integral and skeletal protein profiles. The centenarians showed a significant decrease in the red blood cell count (p < 0.0002), hemoglobin (p < 0.0002), and hematocrit (p < 0.0005). The red blood cell membrane showed a significantly increased cholesterol/phospholipid molar ratio (p < 0.01), with a concomitant increase in polyunsaturated fatty acids in phosphatidylcholine (p < 0.001) and, to a lesser extent, in phosphatidylethanolamine. The electrophoretic pattern of membrane proteins was qualitatively normal compared to controls but the densitometric analysis showed a significant increase in the integral protein band 4.2 (p < 0.05) and in the skeletal protein actin (p < 0.001). Extreme longevity seems to be associated with a substantial integrity of the erythrocyte membrane. Moreover, the evident increase in polyunsaturated fatty acids and in actin are likely to improve the membrane fluidity and to strengthen the membrane structure.

Evaluation of the blue formazan spot test for screening glucose 6 phosphate dehydrogenase deficiency.

Several screening tests for glucose 6 phosphate dehydrogenase (G6PD) deficiency have been reported thus far, and a standardized method of testing was proposed by the International Council for Standardization in Hematology (ICSH). The screening test used in any particular laboratory depends upon a number of factors such as cost, time required, temperature, humidity, and availability of reagents. In this study, a direct comparison between three different G6PD screening methods has been undertaken. In 71 cases (50 hematologically normal volunteers, 9 hemizygous G6PD-deficient males, and 12 heterozygous deficient females), the blue formazan spot test (BFST) was compared with the conventional methemoglobin reduction test (HiRT) and the ICSH-recommended fluorescent spot test (FST-ICSH). In all cases, the results obtained with the three screening tests were correlated with the enzyme activity assayed spectrophotometrically. In hemizygous G6PD-deficient males, all cases were equally detected with the three methods: BFST (4.7-6.64, controls: 11.1-13.4), BMRT (score +3 in all 9 cases), and FST (no fluorescence in 9 cases). In heterozygous G6PD-deficient females, two methods detected 7 out of 12 cases (BFST: 8.71-11.75, controls: 11.1-13.4; and BMRT: score +3 in 7 cases), whereas the FST-ICSH missed all 12 cases that presented a variable degree of fluorescence. Although the sensitivity for G6PD-deficient carrier detection is the same for the BMRT and the BFST, the latter has the advantage of being semiquantitative and not merely qualitative. Unfortunately, none of the three screening tests compared here allowed the detection of the 100% heterozygote carrier state of G6PD deficiency.

Hemoglobinometry: A comparison between the hemiglobincyanide method and the Coulter S counter.

The relative accuracy and precision of the Coulter S Counter have been evaluated in comparison with manual hemiglobincyanide determination according to the recommendations of the International Committee for Standardization in Hematology. Some improvements in the manual procedure, such as centrifugation of hemiglobincyanide solutions and the use of the detergent Triton X-100, were also tested. The Coulter S Counter generally gives higher precision in comparison with the manual method. Nevertheles, Coulter S determinations are systematically lower due to both constant and proportional errors. The available data ranged between hemoglobin values of 11.5 and 18.5%, giving differences of 0-8% between hemoglobin values determined by the Coulter S Counter and the hemiglobincyanide method.

Junctional sites of erythrocyte skeletal proteins are specific targets of tert-butylhydroperoxide oxidative damage.

The oxidative denaturation of the erythrocyte membrane, which is considered a major cause of the haemolytic process, was evaluated upon 'in vitro' oxidative stress with tertbutylhydroperoxide. Biochemical and ultrastructural analyses were performed to point out the effect of this substance on the skeletal network, which is mainly responsible for red cell shape and viability. Moreover, cell morphology was observed by scanning electron microscopy and membrane rigidity assessed by EPR measurements. The most relevant features of the membrane denaturation were, (i) lipid peroxidation, as assessed by malonidialdehyde production, (ii) spectrin and ankyrin degradation with simultaneous globin binding to the membrane, as evidenced by electrophoretic pattern of red cell ghosts. These phenomena were related to the drug concentration in the incubation medium, and accompanied by depletion of intracellular reduced glutathione. The denaturation of protein components hindered the release of spectrin in a hypotonic extraction medium and could be only partially reversed by dithiothreitol. The extensive membrane protein and lipid degradation, at high drug concentration, was coherent with a marked increase of membrane order (membrane 'rigidity'). No clustering of intramembrane proteins was shown by the transmission electron microscopy images. At the same time scanning electron microscopy demonstrated shrinking and disco-stomatocytic deformation of erythrocytes. Ultrastructural analysis of the membrane skeleton by fluorescence-labelling of spectrin and actin, allowed to point out that exposure to t-BHP caused the marginalization of spectrin and the rearrangement of actin molecules with formation of micro aggregates, so that a detachment of actin from the spectrin network was suggested. In addition to the generalized damage of red cell membrane, tertbutylhydroperoxide was found to induce a specific alteration of the skeletal network at the horizontal junction sites involving spectrin, actin, and protein 4.1 and thus to modify the cytoskeletal assembly. This effect on the membrane skeletal components was consistent with the hypothesis that oxidative stress plays a key role in the haemolytic process.

A new monoclonal antibody to an age sensitive band 3 transmembrane segment.

The appearance of band 3 structural modifications related to aging could be evidenced by means of monoclonal antibodies against senescence antigen. Hence in the attempt to provide an immunological marker of erythrocyte aging, we raised a monoclonal antibody against native band 3 (B6 MoAb), which seems to detect differences in the band 3 molecule from erythrocytes of different ages separated by density gradient. Densitometric evaluation of immunoblotting patterns indicates that the in vivo aging is associated with band 3 monomer degradation. The Percoll separated fractions show a significant increase of those proteolytic fragments that bind the B6 antibody. Finally, protease digestions of unsealed membrane ghosts have been performed to test the binding site of the B6 antibody on the band 3 molecule. The data show that the B6 antibody binds a 19 KDa chymotryptic-tryptic fragment which corresponds to a segment of the looped membrane domain whose steric structure appears to be sensitive to age.

A pilot programme of external quality assessment for general haematology in Italy.

In the years 1984-1989 the Istituto Superiore di Sanità organized an EQAS for haematology (SVEQE) in Italy. A series of trials for haemocytometry, abnormal haemoglobins, HbA2, HbF, red cell G6PD and peripheral blood films, were carried out with the participation of 126 hospital laboratories, in different regions. SVEQE was an educative programme, aiming at promotion of quality assurance (QA) in laboratory haematology. At the same time an attempt was made to survey the analytical methods and instruments and to estimate the "state of the art" by the dispersion of all results. Participant laboratories were not scored for their performances. The operative protocol was harmonized to the guidelines established by WHO and ICSH; the trial specimens were prepared from normal or pathologic blood samples provided by blood banks or hospital departments. The trials for haemocytometry demonstrated a wide use of completely automated analyzers and in a steady state of performance during about five years. CVs, mainly for WBV and PLT, were somewhat higher than in other countries, where national QA systems have been established for a long time. Such discrepancies were not surprising in a pilot programme and were likely to be caused by inadequate internal quality control. The exercises for abnormal haemoglobins, HbA2, HbF and G6PD pointed out the need of using standardized methods according to the recommendations of ICSH. A large number of participating laboratories took part in the trial for blood cell morphology, being convinced of the educative function of this exercise; it is important to continue with systematic surveys, even including rare haematological disorders amongst the selected cases.

[Glucose-6-phosphate dehydrogenase deficiency and hereditary hemolytic anemia]. / Deficit di glucosio-6-fosfato deidrogenasi ed anemia emolitica ereditaria.

G6PD deficiency is the most common enzymopathy in the world. The highest frequency values are found in tropical Africa, in the Middle East, in some areas of the Mediterranean, in tropical and sub-tropical Asia and in Oceania. This genetic defect shows sex linked inheritance and a marked heterogeneity. At least 400 abnormal variants with different biochemical characteristics and about 100 diverse mutations have been identified. In most cases the phenotypic expression is a marked decrease in erythrocyte G6PD activity. The most common clinical consequences are neonatal jaundice and sporadic haemolytic crises caused by a number of drugs, by infections or by ingestion of fava beans. A few cases of chronic non-spherocytic haemolytic anaemia associated with rare molecular variants have been reported. Early diagnosis, education and epidemiologic surveillance have been proved to be cornerstones in the prevention of the haemolytic disease. Therefore they should be taken into account in the national health programmes, especially in the countries with high prevalence rates.

Hereditary haemolytic ovalocytosis with defective erythropoiesis.

Patients belonging to four families with 'atypical elliptocytosis' have been investigated. Clinical, haematological, erythrokinetic and enzymatic characteristics as well as the effect of splenectomy are discussed. These studies appear to define the fundamental features of a particular disorder or a variety of hereditary elliptocytosis; characterized by a genetic autosomal dominant character, moderate degree of RBC eccentricity, erythroid dysplasia with relative marrow failure and incomplete response to splenectomy.

Hematotoxic effects in the rat of a toluene dinitro derivative after short-term exposure.

3,5-Dinitro-4-chloro-alpha,alpha,alpha-trifluorotoluene (DNCTT) is an intermediate in the synthesis of dinitroaniline herbicides and was involved in an episode of ground water pollution in 1977. The compound presents a high environmental persistence, which may have possible implications concerning public health. In one experiment male Sprague-Dawley rats were administered DNCTT for 3 days at a dose level of 150 mg/kg body wt by oral gavage. Groups of rats were sacrificed up to 10 days after the end of the administration, at 2-day intervals. Methemoglobin was increased up to Day 7; white blood cells were also increased both in peripheral blood and in bone marrow smears. Spleen relative weights were observed to increase slightly at Days 7 and 10; microscopic examination revealed marked congestion with an increased density of the spleen's white pulp. In a similar scheduled experiment, but at a dose level of 300 mg/kg body wt, the bone marrow white cell series were not affected initially, but were affected after 3 days at the end of the administration. DNCTT has a definite effect on white cells.

Oxidative erythrocyte membrane damage in hereditary spherocytosis.

The occurrence, in Hereditary Spherocytosis, of an oxidative damage to red blood cell membranes was studied by "in vitro" treatment of the erythrocytes with tert-butylhydroperoxide, methylene blue, or phenylhydrazine. Spherocytes were found to be more sensitive than normal erythrocytes to the action of these drugs. Tert-butylhydroperoxide caused a more intense lipid peroxidation as well as more extensive membrane protein alterations, namely spectrin degradation, formation of high molecular weight aggregates, and globin binding to the membrane. Marked spectrin degradation was also induced by methylene blue and by phenylhydrazine, which differed from each other for their effects on the generation of membrane-bound globin and of intermediate proteolysis products. Spectrin appeared therefore to be, in Hereditary Spherocytosis, a highly sensitive target to oxidative stress, a phenomenon which may, also "in vivo", increase the rate of spectrin loss thus enhancing erythrocyte fragility.

A pilot External Quality Assessment Scheme for haemocytometry in Italy.

BACKGROUND: An Italian EQA scheme for haemocytometry, organized by the Istituto Superiore di Sanità, has been active for about five years (1984-1989). The aims of this programme were to evaluate the state of the art and to introduce in Italy a scheme recommended by ICSH. N. 126 public laboratories from different provinces joined voluntarily the programme and trials for haemoglobinometry (A01, A02), full blood count (B01-B08) and platelet count (D01-D03) were performed. METHODS: Materials for testing consisted of blood lysate, preserved blood preparation containing native red cells and pseudoleukocytes, suspension of fixed platelets. The performances of laboratories was evaluated by consensus values (median, mean and standard deviation) and individual deviation index. RESULTS: The instrument survey demonstrated that fully automated systems had the highest frequency. Non Gaussian distributions of results were often obtained and this was particularly true for WBC, PLT and MCV. The overall variability was lower than 5.5% for Hb, RBC and MCH and lower than 9.3% for other erythrocyte parameters; WBC and PLT counts displayed a higher dispersion (CV* = 9.8% and 25.4%); the spreading of results was strongly reduced in the homogeneous group of Coulter counters. In the course of the programme CV*s didn't show any relevant modification, a steady state performance being apparent. As regards the nature of variability, the random component was prevalent for all parameters, with the exception of MCV. CONCLUSIONS: This pilot programme allowed to demonstrate the practicability of a national EQAS for haemocytometry according to the ICSH guidelines. Materials for testing showed acceptable stability, homogeneity and commutability. As regards analytical equipment as well as analytical variability, hospital centers participating in these EQA trials were comparable with laboratories taking part in similar EQAS of other European countries.