モノクローナル抗体を用いたＭａｔｕｈａｓｉ‐Ｏｇａｔａ ｐｈｅｎｏｍｅｎｏｎの解析

Gaining an accurate type for patients’ red blood cells that have a positive direct antiglobulin test (DAT) is problematic using serological methods. Attempting to type red cells heavily coated with immunoglobulin, complement or both by an indirect antiglobulin technique will be falsely positive because of the in vivo coating. Currently, only limited direct saline agglutinating reagents (often IgM monoclonals) are available. Alternatively, chemical treatment can be applied to remove IgG antibodies (allo-antibodies or auto-antibodies) from the DAT-positive red blood cells. Chloroquine diphosphate1 is a chemical used in blood transfusion laboratories to dissociate IgG from red blood cells without compromising the integrity of the red cell membrane antigens by neutralisation of the charged groups on amino acids that govern the tertiary structure of antibody molecules (i.e. R groups that are involved with intermolecular bonds). However, chloroquine diphosphate may not totally remove the coating of antibody from red cells and it is not able to remove complement component 3 (C3) coating. ZZAP2 is a combination of an enzyme/reducing agent composed of dithiothreitol and papain; it removes antibody coating by disruption of membrane-bound IgG through the combined action of a protease and a thiol reagent. EDTA/citric acid3 removes antibodies from red blood cells by lowering the pH of antigen and antibody proteins such that they both become protonated. The antigens and antibodies lose their ability to attract one another through electrostatic bonding and may be forced apart by repulsion of like charges. The tertiary structure of proteins may be affected, hydrogen (H+) ions attracted to hydroxyl (OH−) groups on aspartic acid and glutamic acid resulting in molecular unfolding with consequential loss of structural complementarity between antibody and antibody. Several other methods are also available for removing antibody to allow serological testing; however, the red blood cells are not suitable for typing after these treatments because there is damage to the cells as a result of loss or denaturing of some red blood cell antigens. This leads to invalid typing results with regards to some MNS, Duffy, Kell and Yt system antigens2–9. Murine monoclonal antibodies are known not to cross species specificity. These antibodies have been successfully used to type DAT-positive patients’ red blood cells9,10. A number of murine antibodies from the New York Blood Center (New York, USA) were used in those studies, including MIMA-8 (anti-Jsb), MIMA-19 (anti-Fya), MIMA-22 and −23 (anti-K), MIMA-21 and −27 (anti-Kpa), and MIMA-174 and −180 (anti-s) (unpublished data). The antigen blocking phenomenon is not uncommon in examples of human anti-D and -K11,12. MIMA-19 (anti-Fya), a very specific antibody which can be used in the testing of human DAT-positive red cells, was shown by Lee et al. to be blocked by a high level of anti-Fya. Failure to recognise this false negative result obtained using the MIMA-19 would have led to a false negative type being reported, even though the murine monoclonal antibodies were used as described by the Authors previously. We report a case of a false negative Fya type when using a murine monoclonal anti-Fya (MIMA-19) due to blocking by a maternal anti-Fya.

Innate immune lectins shape anti-blood group antibody development and transfusion outcomes

Specific and Cross-reacting Antibodies in ABO Heterospecific Twin Pregnancy

Density increment and decreased survival of rat red blood cells induced by cadmium

Although the vertebrate erythrocyte is a very specialized type of cell it has provided material for a whole variety of general studies which are important not only for an understanding of red cell function, but also of the nature, structure and properties of cell membranes. More recently, suspensions of these cells have been used to investigate ionic, and other, mechanisms which are important in the respiratory function of vertebrate blood (Nikinmaa, 1982; Cossins & Richardson, 1985). The nucleated nature of non-mammalian red cells endows them with longer life and metabolic properties more directly related to other cells (Schindler & de Vries, 1986). Regulation of the volume of these cells, including the role of catecholamines, has consequences in relation to the concentration of modifying chemicals within the cell which have important influences on the affinity, and other aspects, of haemoglobin gas transport properties (Borgese et al. 1987; Nikinmaa & Huestis, 1984; Weber, 1982).Because of the complex nature of blood plasma, especially in relation to the varying concentrations of circulating catecholamines and other chemicals, many investigators have tended to replace the plasma with a suitable suspending medium. Assessment of the suitability of such media has utilized a variety of criteria including haematocrit and mean cell volume (MCV). Although these features are important, they have not included tests for the maintenance of normal mechanical properties of the red cells, except for the absence of haemolysis. As the remarkable deformability properties of vertebrate red cells are one of their most important adaptations to functioning at the microcirculatory level, it would seem appropriate that this property should be tested to establish the extent to which any red cell suspension may claim to be fully representative of whole blood and, hence, whether any studies of special properties of the red cells using such suspensions truly reflect their condition in the normal circulation. It was on this basis that a recent study of trout red blood cell suspensions was made (Hughes et al. 1986), confirming the suitability of Cortland saline (Wolf, 1963) as a suitable medium for this fish.The aim of the present experiments was to investigate the effects of equilibration with different gas mixtures on the deformability of carp (Cyprinus carpió) erythrocytes. Although the results indicated the presence of such effects, the most significant finding was that in all cases blood equilibrated in tonometers differed from blood which had not been tonometered. Preliminary attempts to produce red cell suspensions with the same filtration properties as whole blood have not been successful.The present study is mainly based upon experiments carried out at 20 °C using blood samples taken from chronically cannulated carp using methods described by Hughes et al. (1983). The blood was taken into heparinized syringes and used in several types of experiments. These included studies of whole blood, comparing samples before and after tonometry (intermittent rotating IL type 237) with different gas mixtures which reproduced normoxic ( 55 mmHg, 3·5 mmHg; 1 mmHg = 133·3Pa) and asphyxie ( 7mmHg, 14mmHg) conditions in carp dorsal aorta blood. Other samples were used in parallel studies with suspension of the red cells made in the following medium (mmol 1−1): KC1, 4·0; NaCl, 124·0; sodium L-lactate, 0·6; CaCl2, 3·6; NaHCO3, 12·6; KH2PO4, 1·0; MgSO4.7H2O, 1·0; D-glucose.H2O, 3·5: pH7·85; 20°C: osmolarity 268 mosmol 1−1. These suspensions were also used for pilot studies on the oxygen consumption of carp red cells.Haematocrit (microcentrifuge) and red cell count (Coulter counter) were routinely determined in duplicate, and were adjusted to a standard haematocrit value of 20%. After modifying the composition of the suspending medium to that given above, and always making it up freshly, suspensions were obtained in which haemolysis was absent.To test the filtrability of whole blood and red cell suspensions the apparatus developed by Kikuchi et al. (1983), as modified by Hughes et al. (1987), was used to determine the timecourse of blood flow through Nuclepore filters containing pores of 8 or 5 μm. From the recordings a slope value was used to calculate the filtration time of the 0·3-ml sample. All samples used in the filtration tests were heparinized to the same extent (500i.u. ml−1 blood or suspending medium). The filtration time of the suspending medium alone was determined before each measurement and a new filter used each time. Filtration times for carp plasma were also determined, as was the content of total protein using a refractometer. The latter measurements were confirmed by direct chemical analysis (Lowry et al. 1951).Cannulation time was very brief with rapid recovery of all specimens when returned to the aquaria. Nevertheless, clear changes in haematocrit and MCV were observed. All experiments showed a significant decrease (P<0·05) in both haematocrit and MCV during postoperative recovery. Some of this occurred within 90 min but it was most marked after 24 h. Pore passage time for single red blood cells (PPT) sometimes decreased during this period, as was to be expected from the corresponding reductions in haematocrit and MCV. In this respect results with carp blood were less consistent than in comparable studies using rainbow trout (Hughes et al. 1986) where PPT increased in spite of decreasing MCV and haematocrit. However, most of the carp showed some increase in PPT during the 24-h recovery period.Comparisons were made between results obtained using blood samples immediately following their removal from the fish (= whole blood) and samples from the same blood after it had been kept in tonometers for varying periods. No changes were observed if more than 1 ml was placed in the tonometer. However, tonometry of a smaller volume for longer than 20 min was associated with an increase in PPT and also significant reductions (10%) in total plasma proteins (Table 1). This reduction in plasma protein was observed even after 10 min of tonometry but did not seem to occur when more than 1 ml of blood was placed in the tonometer. Further experiments involving tonometry under asphyxie conditions, either with or without the addition of adrenaline (ICT6 molI−1), gave comparable results, i.e. reduction in plasma proteins and increase in PPT with longer tonometry. An indication was also obtained (paired t-test: 0·10> P>0·05) that adrenaline reduces the pore passage time when compared with blood tonometered under asphyxie conditions alone (Table 1). No significant effect of adrenaline was found when the blood was tonometered with air.Although fresh suspensions had been prepared and gave repeatable results when used in oxygen consumption measurements, it was found that these suspensions filtered very badly in spite of many attempts to modify the suspension procedure in ways comparable to those used for rainbow trout (Hughes et al. 1986). These included adequate heparinization and repeated passage through a syringe needle (18 gauge) to reduce red cell aggregates. Pore passage times of all suspensions were very long so that even 8 μm filters became blocked quite quickly. Estimates of the filtration rate could only be obtained at the very beginning of the recordings.The results obtained here confirm other research showing that the filtration technique can give useful information regarding the condition of both whole blood and red cell suspensions. The intention at the beginning of the study was to investigate filtration properties of carp red cell suspensions equilibrated with different gas mixtures and compare them with whole blood. However, at an early stage it became apparent that the filtration times of the suspensions used were so long that such a study was not possible. Furthermore, parallel experiments using whole blood revealed properties of great interest and are reported here.Changes in deformability following anaesthesia have been observed in rainbow trout where PPT decreased in spite of red cell swelling (Hughes et al. 1986). In carp this effect was observed in some specimens, but in others PPT increased with the increase in MCV. This difference may reflect variability in hypoxic levels during surgery and also that carp are not affected as much as trout when subjected to the same degree of hypoxia. A very striking effect, however, was the decrease in filtration time of blood tonometered with adrenaline and under asphyxie conditions relative to blood with asphyxia alone. Furthermore, such an effect was not observed with normoxic blood. These effects are related to comparable studies (Fuchs & Albers, 1988) showing an effect of adrenaline on MCV which occurred under conditions of hypercapnic hypoxia but not normoxia.In all cases tonometered blood showed an increase in pore passage time relative to blood which had not been tonometered and its filtration time determined immediately after sampling from the fish. It is this effect which is mainly responsible for the significant difference which analysis of variance showed between columns in Table 1. Tonometered blood also showed a significant reduction in total protein compared with identical samples which had not been tonometered.It is concluded: (i) that red cells in blood tonometered under the particular conditions of these experiments show different mechanical properties to those of whole blood; (ii) that adrenaline decreases the filtration rate of whole blood but only when equilibrated with low-oxygen and high-CO2 mixtures; and (iii) that red cell suspensions which otherwise appeared to maintain respiratory properties similar to those of whole blood evidently have changed mechanical properties as they do not pass through filters containing pores of 8 μm whereas whole blood samples can do so with little difficulty.Prolonged tonometry is commonly used in some methods for determining the oxygen dissociation characteristics of whole blood and/or acid-base properties, and such determinations might easily be influenced by modifications to the condition of the red blood cells. As yet no studies have been made comparing these characteristics following tonometry under different conditions. It has been pointed out (Albers, 1972), that the volume ratio of gas to blood must be adequate and depends on the type of tonometer. It should be emphasized that no comparisons have been made between the relative merits of the many types of tonometer used in different laboratories.The use of red blood cell suspensions is often encouraged to reduce some of the variability conferred by blood plasma. As a result of this study it seems possible that adverse changes in the condition of the red cells may occur which must be weighed against these advantages. It is suggested that routine monitoring of filtration characteristics might be incorporated in future studies using red cell suspensions to provide an index of the mechanical condition of the material. More extended studies on suspensions of carp red cells are planned to assess the factors which tend to reduce their filtrability.

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Red blood cell size is important for adherence of blood platelets to artery subendothelium

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; Plasma transfusion is one of the basic treatments in patients with major blood loss. The anti-A and anti-B antibodies contained in the plasma demand ABO blood group compatibility. This is limiting the use of plasma in emergency situations and can cause a shortage in the supply of plasma of certain blood groups. We developed a method for anti-A and anti-B depletion by adsorbing plasma isoagglutinins using red blood cells. &lt;b&gt;&lt;i&gt;Materials and Methods:&lt;/i&gt;&lt;/b&gt; Three units of fresh frozen plasma were thawed after quarantine storage, pooled, and an aliquot of red cell concentrate was added. After 2 h of incubation at room temperature antibody-red-cell complexes were removed by centrifugation, the isoagglutinin-depleted plasma was split into three units and deep frozen. Isoagglutinin titers, free hemoglobin, residual red cells, clotting factor activity, and sterility of plasma units were determined after isoagglutinin depletion and a double freeze-thawing procedure. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; Anti-B titers in group A plasma were reduced from values of 1:64 to 1:1 or lower, anti-A titers in group B plasma decreased from values of 1:128 to at least 1:16. Postprocedure clotting factor activities were preserved with 88.0 ± 7.3% (factor V), 106.9 ± 11.4% (factor VIII), and 84.0 ± 7.5% (factor XI) fulfilling the quality control requirements. No residual red cells were found, but free hemoglobin slightly increased to 53.7 ± 5.2 μmol/L. All units were sterile. &lt;b&gt;&lt;i&gt;Discussion:&lt;/i&gt;&lt;/b&gt; We described a method for the production of anti-A- and anti-B-depleted plasma in a closed system that uses standard equipment. The resulting isoagglutinin-depleted plasma may allow for blood group independent plasma transfusion.

Sodium transport in red cells of patients with acute viral hepatitis

The Two Antibody Specificities Within Human Anti-Blood Group B Antibodies

During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood's buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called “sports anemia.” This is not anemia in a clinical sense, because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume (PV). The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g., in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise.

Background and Objectives: Severe hemolytic transfusion reactions due to minor incompatibility are usually associated with the so-called “High titred” antibodies in the plasma of donors especially the O group as it is generally regarded as universal donors. Critical titers of 1:64 or higher was considered high titered antibodies for IgM anti-A and anti-B. Critical titers of 1:256 or higher was considered high titered antibodies for IgG anti-A and anti-B. The objective of this study was to see the prevalence of such high titered antibodies among our donor population and to observe for an association if any with the age, gender, and diet type and probiotic intake of the donors. Methodology: This was a cross-sectional analytical study conducted over 20 months from September 2015 to May 2017. About 560 donors were chosen randomly after screening and counseling and titration of anti-A and anti-B antibodies was done by the double dilution tube agglutination technique. The information on age, gender, diet type, and probiotic intake was gathered from the donor history questionnaire and an association between the factors described and the distribution of critical titers of anti-A and anti-B was elicited. Results: In the present study, the prevalence of high titer anti-A and anti-B IgM antibodies was 36% and 32%, respectively. The prevalence of high titer anti-A and anti-B IgG antibodies was 9.6% and 5.5%, respectively. The median IgM antibody titers reduced with the increasing age of the donors starting 18 years. There was a statistically significant association between a vegetarian diet and high titers. Conclusion: There are a considerable proportion of O group donors with high titers of antibodies and hence a routine pretransfusion screening for such antibodies can prevent the development of hemolytic reactions by segregating units into “high titer” and “low titer” antibodies and issuing only low titer products for out of group transfusions.

Dear Sir, Transfusion therapy is a cornerstone in the treatment of sickle cell anaemia as it improves tissue oxygenation (by increasing the absolute value of haemoglobin and reducing the proportion of haemoglobin S) as well as reducing blood viscosity; chronic transfusion therapy also leads to the suppression of the propensity of endogenous red blood cells to sickle1. The transfusion therapy is based mainly on red blood cell transfusions while experience with the use of erythrocyte exchange (EEX) in this disease is limited2,3. EEX, practised both as acute therapy and as periodic and scheduled treatment, is an “exchange of red blood cells” designed to remove large volumes of sickle cells quickly and replace them with normal red blood cells, without increasing blood viscosity or causing iron overload. The aim of EEX is to reduce and maintain the haemoglobin S <30%, while bringing the values of haemoglobin to approximately 10 g/dL (haematocrit <30%). Acute EEX is considered the treatment of choice in cases in which there is an indication for transfusion therapy but the levels of haemoglobin are ≥10 g/dL, especially in the treatment of stroke3. On the other hand, the purpose of periodic, programmed EEX is to maintain the values of haemoglobin S stably below 30–35%, avoiding iron overload, reducing accesses to hospital and thereby improving the quality of life of patients1. At the Blood Transfusion Service of St. Anna Hospital in Ferrara, we use EEX in the chronic treatment of patients with homozygous sickle cell disease. So far we have followed ten patients (4 males and 6 females), four of whom are children. Most of the patients are African or Albanian. In the period between 2003 to 2013 we performed over 350 EEX procedures, which were carried out in 60–90 minutes with a continuous flow cell separator (Fresenius Kabi AG, Bad Homburg, Germany -COM.TEC®). The red blood cells used for the exchange were leucodepleted, extensively typed (for Rh, Kell, Jk, Fy) and resuspended in SAG-M preservative solution (maximum 5–7 days of storage) with a haematocrit of about 60%. The erythrocyte volume exchanged in each procedure was calculated using a modified method studied at our Transfusion Service to obtain greater removal of haemoglobin S. This method involves three steps carried out with the plasma exchange device of the cell separator. Step 1 - Consisted of connecting, on the cell separator, the reinfusion line of red blood cells with the bag of the waste and removing a volume of patient’s red blood cells calculated to reduce the haematocrit by 10–15%, using saline or human albumin solutions at 4% as replacement fluid. Step 2 - Was isovolaemic exchange of patient’s red blood cells with an equal volume of homologous erythrocytes (20 mL of red blood cells/kg weight). Step 3 - Was to restore the connection of the reinfusion line of red blood cells with the patient and restore the haematocrit to the desired value by by exchanging a volume of the patient’s plasma with the appropriate volume of homologous red blood cells. Compared to standard red cell exchange, the use of this procedure allows a greater percentage reduction of haemoglobin S (60% instead of 50%) using a smaller volume of homologous red blood cells (20 mL red blood cells/kg weight instead of 30 mL/kg weight). Pre-exchange haemoglobin S levels (evaluated after 55–60 days) were always under 50–52%. The procedures were well tolerated and there were no adverse reactions; isovolaemic EEX also safeguarded our patients from the iron overload that can follow transfusion therapy. Only one patient had an episode of erythrocyte alloimmunisation, with anti-M antibody specificity. From a clinical point of view, all the patients treated with this novel EEX are in durable symptomatic remission and none has dangerous complications of their disease. EEX is still a little-known procedure used in the acute or long-term treatment of sickle cell disease2. Our experience leads us to believe, partly in view of the increasing number of citizens of different ethnic groups (including those with high incidences of sickle cell disease) that live in our country3, that EEX should be taken into account for the management of these patients, because of its high tolerability and effective reduction and control of haemoglobin S levels (with excellent control of symptoms and complications) without iron overload. Furthermore, the increased removal of haemoglobin S with the three-step procedure not only saves homologous red blood cells, but could also provide longer periods of therapeutic efficacy thereby reducing accesses to hospital and improving the patients’ quality of life.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Experience in the evaluation of foeto-maternal haemorrhage by flow cytometry.