Types Of Red Blood Cells Research Articles

Factors associated with blood transfusion during intracranial aneurysm surgery

Study objectiveThe purpose of this study was to identify risk factors associated with intraoperative blood transfusions in patients presenting for intracranial aneurysm surgery in the current era of more restrictive transfusion guidelines. DesignRetrospective observational cohort study with stepwise, multivariate binary logistic regression analysis. SettingTertiary care university teaching hospital. PatientsFour hundred seventy-one consecutive patients undergoing intracranial aneurysm surgery at Northwestern Memorial Hospital (Chicago, IL) from 2006 to 2012. InterventionRed blood cell transfusion (retrospective observational). MeasurementsDemographic data, medical comorbidities, hemoglobin levels, Hunt-Hess grades, intracranial aneurysm characteristics, presenting intracranial bleeding states, estimated blood losses, transfused red blood cells, and blood products. Main resultsForty-six patients (9.5%) received intraoperative red blood cell transfusions. Preoperative risk factors associated with transfusions were highly related to aneurysm rupture, including such parameters as older age (P < .001), lower presenting hemoglobin level (P < .001), preoperative rupture (P < .001), and higher Hunt-Hess grade (P < .001). Intraoperative risk factors included larger aneurysm size (>10 mm; P = .03), intraventricular hemorrhage (P < .001), and intracerebral hematoma evacuation (P = .02). Binary logistic regression modeling identified age (P < .001), presenting hemoglobin level (P < .001), larger aneurysm size (>10 mm; P = .003), elevated Hunt-Hess grade (P = .021), and intraoperative rupture (P = .013) as independent predictors of intraoperative red blood cell transfusion. ConclusionThe incidence of intraoperative red blood cell transfusion in intracranial aneurysm surgery in our patient cohort was 9.5%, and the most significant factors associated with transfusion were presenting hemoglobin level less than 11.7 g/dL and age greater than 52 years. It would seem advisable that these patients undergo routine type and cross-matching of red blood cells before intracranial aneurysm surgery.

The experience of extended blood group genotyping by next-generation sequencing (NGS): investigation of patients with sickle-cell disease.

Patients suffering from haemoglobinopathies may be treated by red blood cell (RBC) transfusion on a regular basis and then exposed to multiple antigens with a recurrent, potential risk of alloimmunization routinely prevented by extended RBC antigen cross-matching. While time-consuming and labour-intensive serological analyses are the gold standard for RBC typing, genotyping by current high-throughput molecular tools, including next-generation sequencing (NGS), appears to offer a potent alternative. The potential of extended blood group genotyping (EBGG) by NGS of 17 genes involved in 14 blood group systems was evaluated in a cohort of 48 patients with sickle-cell disease. Sample preparation and sequencing were simplified and automated for future routine implementation. Sequencing data were obtained for all DNA samples with two different sequencing machines. Prediction of phenotypes could be made in 12 blood group systems and partially in two other blood group systems (Rh and MNS). Importantly, predicted phenotypes in the MNS (S/s), Duffy, Kidd and Kell systems matched well with serological data (98·9%), when available. Unreferenced alleles in the ACHE and ART4 genes, respectively, involved in the Yt and Dombrock blood groups, were identified, then contributing to extend the current knowledge of blood group molecular genetics. Overall, we consider that our strategy for NGS-based EBGG, assisted by a simple method for genotyping exons 1 and 2 of the pairs of homologous genes (i.e. RHD/RHCE and GYPA/GYPB), as well as the future support of potent bioinformatics tools, may be implemented for routine diagnosis in specific populations.

Red blood cell components: Meeting the quantitative and qualitative transfusion needs

Red blood cell (RBC) transfusion is a very common therapeutic intervention. However, because of multiple recent studies improving our understanding of appropriate transfusion scenarios, the total number of RBC units transfused per year is actually decreasing in the developed world and there are no longer major shortages of RBC products for general use. Nonetheless, there are an increasing number of "special" uses, which can put strains on the blood supply for particular types of products; these may produce shortages of specific types of RBCs or require collections targeting certain types of donors. This review will focus on several broad topics, including providing some examples of "special" settings that require, or could require, special types of RBC products.

Isolation and analysis of mannose/trehalose/maltose specific lectin from jack bean with antibruchid activity

A lectin with insecticidal property against the stored product pest, Callosobruchus maculatus was successfully isolated from the seeds of Canavalia virosa using standard affinity chromatography. The isolated molecule typically behaved like a lectin in its characteristics. It agglutinated indicator red blood cells (RBC) in its native as well as enzyme treated conditions. The enzyme treated RBC types exhibited a very high hemagglutination (HA) titre values and this property of isolated molecule behaved like arcelin, the lectin-like molecules reported from several species of Phaseolus. As a characteristic feature of a lectin, the isolated molecule effectively inhibited the agglutination of indicator RBC types with simple and complex carbohydrates including glycoproteins. This nature of the isolated molecule also relate with characteristic feature of arcelin isoforms in inhibiting HA activity with complex glycoproteins as reported in many studies. Most interestingly, the present study disclosed trehalose as a potent inhibitor of C. virosa lectin. Therefore, feeding insect pests on the lectin like arcelin could serve as antibiosis factor/anti-insect activity. The molecular characteristics of this isolated molecule and its mass studies too revealed its homology with arcelin, arcelin-1, 2 and 6 isoforms of P. vulgaris and lectin from Canavalia cathartica, C. lineata and C. brasiliensis.

Healthy and unhealthy red blood cell detection in human blood smears using neural networks

One of the most common diseases that affect human red blood cells (RBCs) is anaemia. To diagnose anaemia, the following methods are typically employed: an identification process that is based on measuring the level of haemoglobin and the classification of RBCs based on a microscopic examination in blood smears. This paper presents a proposed algorithm for detecting and counting three types of anaemia-infected red blood cells in a microscopic coloured image using circular Hough transform and morphological tools. Anaemia cells include sickle, elliptocytosis, microsite cells and cells with unknown shapes. Additionally, the resulting data from the detection process have been analysed by a prevalent data analysis technique: the neural network. The experimental results for this model have demonstrated high accuracy for analysing healthy/unhealthy cells. This algorithm has achieved a maximum detection of approximately 97.8% of all cells in 21 microscopic images. Effectiveness rates of 100%, 98%, 100%, and 99.3% have been achieved using neural networks for sickle cells, elliptocytosis cells, microsite cells and cells with unknown shapes, respectively.

Comparative evaluation of factors affecting hemagglutinatingactivity of avian influenza (H9) virus

Influenza virus H9 was standardized for hemagglutination assay using different factors such as red blood cell (RBC) types, concentrations, diluent types, and storage times. Avian influenza virus H9 was grown in embryonated chicken eggs and confirmed by spot agglutination. A significant (P 0.05) were found between human blood type O, chicken, and dog RBCs, as well as among rabbit, pigeon, sheep, and parrot. The highest titer (9.00 ± 0.00) with a nonsignificant difference was found using virus stored at 4 °C and -20 °C while 37 °C showed the lowest significant mean hemagglutinin (HA) titer (11.08 ± 188.21). Nonsignificant differences were observed in HA titers against H9 virus stored for 3, 4, 5, and 6 months. Nonsignificant differences were found between the use of normal saline and 0.5% peptone water with the lowest HA titers of 7.83 ± 0.40 and 8.00 ± 0.00, respectively, while the highest HA titer (9.00 ± 0.00) with nonsignificant difference was observed by using HA-HI buffer and phosphate buffer saline as diluents. RBCs with 0.5% and 1% concentrations showed nonsignificant difference in HA titer but significant difference with 0.1% RBCs.

A comprehensive analysis of membrane and morphology of erythrocytes from patients with glucose-6-phosphate dehydrogenase deficiency

Acute hemolytic anemia could be triggered by oxidative stress in the patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the underlying hemolytic mechanism is unknown. To make clear the hemolytic mechanisms, a systematic study on membrane ultrastructure had been undertaken. A comprehensive method was used including atomic force microscopy, scanning electron microscopy, flow cytometer and fluorescence microscopy to analyze the membrane ultrastructure, externalized phosphatidylserine (PS), intracellular Ca(2+) concentration, morphology and the distributions of band 3 protein in G6PD deficient red blood cells (RBCs) after tert-butyl-hydroperoxide (t-BHP) oxidation. The results showed that erythrocyte shrinkage, annexin-V binding to externalized PS on the membrane of early-stage apoptotic cells, the increased membrane roughness and intracellular Ca(2+) concentration, as well as the change of distributions of band 3 protein in RBCs. Compared with the control RBCs, as the concentration of t-BHP up to 0.1mM, the membrane roughness of G6PD deficient RBCs showed significant difference (p<0.05) and as the concentration of t-BHP up to 0.3mM, externalized PS showed significant difference (p<0.05). Furthermore, the population types of RBCs showed dramatic difference between control groups and G6PD deficient groups. Oxidative stress induced more serious erythrocyte apoptosis and resulted in increased roughness of erythrocyte membrane and abnormal distributed band 3 protein in G6PD deficient RBCs. Echinocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with G6PD deficiency, which may shorten the RBCs lifespan. The results in the present study will give an increased understanding for the hemolytic mechanism of G6PD deficiency.

Changing practice: red blood cell typing by molecular methods for patients with sickle cell disease.

Extended red blood cell (RBC) antigen matching is recommended to limit alloimmunization in patients with sickle cell disease (SCD). DNA-based testing to predict blood group phenotypes has enhanced availability of antigen-negative donor units and improved typing of transfused patients, but replacement of routine serologic typing for non-ABO antigens with molecular typing for patients has not been reported. This study compared the historical RBC antigen phenotypes obtained by hemagglutination methods with genotype predictions in 494 patients with SCD. For discrepant results, repeat serologic testing was performed and/or investigated by gene sequencing for silent or variant alleles. Seventy-one typing discrepancies were identified among 6360 antigen comparisons (1.1%). New specimens for repeat serologic testing were obtained for 66 discrepancies and retyping agreed with the genotype in 64 cases. One repeat Jk(b-) serologic phenotype, predicted Jk(b+) by genotype, was found by direct sequencing of JK to be a silenced allele, and one N typing discrepancy remains under investigation. Fifteen false-negative serologic results were associated with alleles encoding weak antigens or single-dose Fy(b) expression. DNA-based RBC typing provided improved accuracy and expanded information on RBC antigens compared to hemagglutination methods, leading to its implementation as the primary method for extended RBC typing for patients with SCD at our institution.

Volumetric measurement of human red blood cells by MOSFET-based microfluidic gate.

In this paper, we present a MOSFET-based (metal oxide semiconductor field-effect transistor) microfluidic gate to characterize the translocation of red blood cells (RBCs) through a gate. In the microfluidic system, the bias voltage modulated by the particles or biological cells is connected to the gate of MOSFET. The particles or cells can be detected by monitoring the MOSFET drain current instead of DC/AC-gating method across the electronic gate. Polystyrene particles with various standard sizes are utilized to calibrate the proposed device. Furthermore, RBCs from both adults and newborn blood sample are used to characterize the performance of the device in distinguishing the two types of RBCs. As compared to conventional DC/AC current modulation method, the proposed device demonstrates a higher sensitivity and is capable of being a promising platform for bioassay analysis.

Еритрометрія у вивченні адаптивних та реактивних станів спортсменів впродовж тренувального процесу

Bring the analysis of the individual characteristics of changes in the index variation of erythrocytes (IVE) and the ratio of different types of cells in the examined eritrona of the basketball players during training. A total of 7 athletes surveyed during weeks 13-16 of the training process. The obtained unstained blood smears calculates the variability of erythrocytes (IVE) by counting the 15 types of red blood cells, classified in the group. On the basis of types for each athlete during these periods of training plotted the percentage of different types of erythrocytes in erythron. In the study of specific features of the definitive components erythron athletes-basketball players found the obvious individual characteristics change as a IVE, and the distribution of the eritron components. In the surveyed athletes club team NTU "KPI" on the basis of selected morphometric study of the eritron funds set individual characteristics manifestations of reactive, adaptive changes of the whole organism during different time periods of the training process.

Molecular Characterization of a Haemagglutinating and Non Haemagglutinating Rabbit Haemorrhagic Disease Viruses From Egypt

Rabbit viral haemorrhagic disease (RVHD) is a highly contagious, peracute and acute viral disease of both wild and domestic rabbits caused by rabbit haemorrhagic disease virus (RHDV). RHDV is considered as a haemagglutinating virus; however, there is a growing evidence for presence of non haemagglutinating RHDV strains. In this study, two strains of RHDV have been characterized. The two strains were selected from ten different infected rabbit flocks. One of them had haemagglutination (HA) activity and the other one lacked HA activity. HA test was carried out on both isolates at 4°C using human red blood cells (RBC) type ‘O’. RT-PCR was performed on the liver samples of the ten infected rabbit flocks using specific primers targeting VP60 gene. The obtained partial VP60 gene sequences of the two selected strains were aligned with other haemagglutinating and non haemagglutinating strains of RHDV in the world, including ‘‘RHDVa’’ strains as France 99-05, France-Reu-00, Germany-Triptis and China TP. Phylogenetic analysis showed that the two strains were genetically different. Haemagglutinating strain was similar to Rainham strain which is low haemagglutinating virus while non haemagglutinating strain was similar to whn-1 strain which is non haeagglutinating virus. The study confirmed presence of non haemagglutinating RHDV in rabbits reared in Egypt and suggested that VP60 might be related to HA activity.

Activity of cytochrome P450 1A2 in relation to hepatic iron accumulation in transfusion-dependent β-thalassaemia major patients.

Cytochrome P450 1A2 (CYP1A2) is a cytochrome enzyme with a pivotal role in hepatic drug metabolism. Data from CYP1A2((-/-)) mouse suggest that CYP1A2 plays a role in aspects of hepatic iron toxicity. The aim of this study was to assess the activity of CYP1A2 in relation to hepatic iron load in patients with transfusion-dependent β-thalassaemia major. The (13) C-methacetin continuous breath test was performed on 30 consecutive patients with transfusion-dependent β-thalassaemia major. CYP1A2 activity was measured by the rate at which the (13) C substrate is metabolized and exhaled expressed as percentage dose recovery (PDR) per hour. CYP1A2 activity was correlated with clinical and laboratory parameters and hepatic iron accumulation by T2* magnetic resonance imaging (T2*MRI). Cytochrome P450 1A2 activity in patients with transfusion-dependent β- thalassaemia major was positivity correlated with plasma ferritin levels. No correlation was found with age, duration and amount of red blood cell transfusion and type of iron chelation therapy. Low CYP1A2 activity was negatively associated with hepatic iron accumulation (T2*MRI ≤ 6.3 ms); adjusted odds ratio (OR; 95% CI) for hepatic iron accumulation in patients with low CYP1A2 activity was 0.047 (0.003-0.72; P = 0.021). Of the six patients with decreased activity of CYP1A2, five had no hepatic iron accumulation and one had mild hepatic iron accumulation by T2*MRI. Activity of CYP1A2 is associated with hepatic iron accumulation in patients with transfusion-depended β-thalassaemia major. Further studies are needed to assess the exact role of CYP1A2 in iron metabolism in human.

Blood matching and transfusion for 12 acute autoimmune hemolytic anemia patients by extracorporal hemolysis test

In order to screen the compatible red cells by using extracorporal hemolysis test for acute autoimmune hemolytic anemia (AIHA) patients who were difficult to be matched by automatic microcolumn gel indirect antiglobulin test. Twenty-six cases of AIHA were chosen as control group, to whom the same type of donor red blood cells were infused with the weakest blood agglutination; 12 cases of acute AIHA patients were chosen as test group, these patients were difficult to be matched by automatic microcolumn gel indirect antiglobulin test, and the donor red cells without hemolysis by extracoral hemolysis test were transfused for them. The results showed that compared with the control group,the effect of transfusion was better in test group (P < 0.01), with 2.26 U leukocyte-depleted erythrocyte suspension in average, whose hemoglobin, reticulocyte and total bilirubin levels were changed significantly compared with those before blood transfusion (P < 0.01) . It is concluded that the compatible red blood cells for the acute AIHA patients can be screened by the extracorporal hemolysis test, when it is difficult to screen by the automatic microcolumn gel indirect antiglobulin test.

Noninvasive Buccal Swab Antigen Sample and Molecular Testing Provides Extended Antigen Typing for Patients with Hemoglobinopathies

To demonstrate the feasibility of performing a noninvasive, molecular-based red blood cell (RBC) antigen test on infants and very young children with sickle cell disease as part of a statewide newborn screening follow-up program. A prospective pilot project was conducted using a noninvasive buccal swab and test kit to perform DNA-based, extended RBC phenotyping in 92 children participating in a newborn hemoglobinopathy screening follow-up program. Reported data include the extended panel of antigens detected by molecular analysis compared with unaffected population estimates. Molecular-based RBC antigen testing was successful, with extended RBC typing generated for all subjects. Molecular testing detected several rare negative or rare positive phenotypes, demonstrating the utility of obtaining an extended antigen panel. This study demonstrates the feasibility of performing antigen testing on buccal swab specimens from children with sickle cell disease as part of a newborn screening follow-up program with the aim of allowing specific unit matching to prevent alloimmunization with RBC transfusions. The general applicability of testing may be limited by a lack of uniform insurance coverage for buccal swab testing, however.

Two Novel α1,2-Fucosyltransferase Alleles in an H-Deficient Phenotype Individual

Background: Abnormal α1,2-fucosyltransferase activity due to gene mutation results in decreased synthesis of H antigen and leads to an H-deficient phenotype. Here we studied the underlying molecular mechanisms in 7 Chinese blood donors with the H-deficient phenotype. Methods: Red blood cell typing was performed using standard serologic tests, and ABO genotype was analyzed using ABO polymerase chain reaction with sequence-specific primer tests. The coding sequence of the FUT1 gene was amplified using the specific primers. The FUT1 alleles were identified by a pCRII-TOPO carrier for TOPO TA cloning sequencing. Results: The H-deficient phenotype frequency was estimated to be approximately 1/30,000 (6/159,515) in the Chinese Han population. The FUT1 gene mutation was demonstrated in 6 Chinese blood donors with the H-deficient phenotype. In only 1 case, no mutation was detected. Novel FUT1 alleles were found in 1 donor. One of these novel FUT1 alleles showed nucleotide 35C>T and 748C>T site mutations that resulted in amino acid substitution of Ala to Val and Trp to Arg at positions 11 and 250, respectively. Another novel FUT1 allele had a nucleotide 655G>C site mutation, causing amino acid substitution of Leu to Val at position 219. Conclusions: Two novel FUT1 alleles, 35T+748T and 655C, were identified that may greatly diminish the activity of α1,2-fucosyltransferase and result in the H-deficient phenotype.

Nanostructure of Red Blood Cell Membranes in Premature Neonates with Respiratory Distress Syndrome

Objective: to study the nanostructure of red blood cell membranes in premature babies with neonatal respiratory distress syndrome (NRDS), by applying atomic force microscopy. Subjects and methods. The investigation included 27 newborn infants, of them 13 premature babies with NRDS formed a study group. The mean gestational age was 33.1±2.3 weeks; their birth weight was 1800±299.3 g. A comparison group consisted of 14 full-term babies with favorable pregnancy and term labor. The mean gestational age of the babies was 39.4±0.5 weeks; their birth weight was 3131.7±588.8 g; the infants had a one minute Apgar score of 8±0.4. Their red blood cells were examined using an atomic force microscope. The objects to be examined were residual umbilical cord blood (RUCB) from the premature infants; central venous blood after 7 hours of birth and neonatal venous blood taken on day 7 of life. Results. RUCB from full-term babies contained planocytes that were a major morphological type of red blood cells. In physiological pregnancy and acute fetal hypoxia, the morphological composition of red blood cells in premature neonates with NRDS was close to that in full-term babies. The planocytes are also a major morphological type of red blood cells in the premature infants; the frequency of their occurrence varies. Stomatocytes are typical of all the neonates in the NRDS group; their frequency levels vary greatly: from 8 to 65% of the total number of erythrocytes. The examination revealed that the premature infants of 31—36 weeks gestation were characterized by abnormal erythrocyte shapes that showed a high variability. At birth, the premature babies were found to have changes in the nanostructure of red blood cell membranes, which were influenced by intrauterine hypoxia. The first-order value reflecting flickering in the red blood cell membrane varies to the most extent. Conclusion. Atomic force microscopy showed that the greatest changes in the structure of red blood cell membranes were found in RUCB. The premature babies with NRDS had intrauterine poikilocytosis caused by unfavorable factors, as confirmed by the presence of multiple correlations. Analysis of the nanostructure of red blood cell membranes revealed that the first-order value reflecting flickering in the red blood cell membrane was most sensitive; this indicator showed a slow normalization. Correspondence to: Key words: red blood cell membrane, nanostructure, planocytes, stomatocytes, premature newborn infants, respiratory distress syndrome.

Applying molecular immunohaematology to regularly transfused thalassaemic patients in Thailand.

Red blood cell transfusion is the principal therapy in patients with severe thalassaemias and haemoglobinopathies, which are prevalent in Thailand. Serological red blood cell typing is confounded by chronic transfusion, because of circulating donor red blood cells. We evaluated the concordance of serological phenotypes between a routine and a reference laboratory and with red cell genotyping. Ten consecutive Thai patients with β-thalassemia major who received regular transfusions were enrolled in Thailand. Phenotypes were tested serologically at Songklanagarind Hospital and at the National Institutes of Health. Red blood cell genotyping was performed with commercially available kits and a platform. In only three patients was the red cell genotyping concordant with the serological phenotypes for five antithetical antigen pairs in four blood group systems at the two institutions. At the National Institutes of Health, 32 of the 100 serological tests yielded invalid or discrepant results. The positive predictive value of serology did not reach 1 for any blood group system at either of the two institutions in this set of ten patients. Within this small study, numerous discrepancies were observed between serological phenotypes at the two institutes; red cell genotyping enabled determination of the blood group when serology failed due to transfused red blood cells. We question the utility of serological tests in regularly transfused paediatric patients and propose relying solely on red cell genotyping, which requires training for laboratory personnel and physicians. Red cell genotyping outperformed red cell serology by an order of magnitude in regularly transfused patients.

Expansion of the Kell blood group system: two new high‐prevalence antigens and two novel K0 (Kellnull) phenotypes

The number of KEL alleles associated with new antigens or loss of expression of high-prevalence antigens continues to increase. We investigated KEL in five samples: two with K0 (null) phenotypes and three with normal Kell expression and antibodies to high-prevalence antigens. Red blood cell (RBC) typing and antibody identification were by standard methods. Genomic DNA was isolated from white blood cells and DNA array testing and sequencing of KEL exons was performed by standard methods. Proband 1, an Asian woman with Kp(b+) RBCs, presented with alloanti-Kp(b) . Four years later, the antibody was reactive with all RBCs except K0 . She was homozygous for KEL c.877C>T change (p.Arg293Trp), and the high-prevalence antigen absent from her RBCs was named KHUL. Probands 2 and 3, both Japanese and homozygous for KEL c.875G>A (p.Arg292Gln), presented with an antibody reactive with all except K0 RBCs. The antibody, named KYOR, recognizes an antigen antithetical to KYO (KEL31). Proband 4, a pregnant Middle Eastern woman, presented with alloanti-Kp(b) , but her RBCs did not express Kell antigens. She was homozygous for KEL c.230G>T (p.Cys77Phe). Proband 5, a multiply transfused Caucasian female with an antibody reactive with all RBCs except K0 and lacking Kell antigens, was a compound heterozygote carrying a silenced allele c.574C>T (p.Arg192Stop) in trans to c.1664G>A (p.Gly555Glu). We describe two new high-prevalence Kell antigens, KHUL (ISBT 006037; KEL37) and KYOR (ISBT 006038; KEL38), and two novel alleles encoding K0 phenotypes. We caution that antibodies produced by individuals with K0 RBCs or lacking high-prevalence antigens can present as anti-Kp(b) .

Morphological Features of Red Blood Cells in Patients with Severe Concomitant Injury

Objective: to reveal changes in the structure and shape of red blood cells depending on blood loss (BL) volume in patients with severe concomitant injury (SCI). Subjects and methods. Eighteen patients (9 men, 9 women) aged 48.6±16.1 years who had sustained severe concomitant mechanical injury (CMI) with different BL volumes and hemodynamic disorders were examined. According to the volume of BL, the patients were divided into two groups: 1) 7 victims with a BL volume of &lt; 750 ml (5.7±1.9 ml/kg), grade 1 BL; 2) 11 victims with a BL volume of &gt; 2000 ml (37.5±5.1 ml/kg), grade 4 BL. A comparison group consisted of 5 apparently healthy volunteers whose mean age was 26.4±2.7 years. The shapes and sizes of red blood cells were examined by light optical and atomic force microscopy (AFM). To study the composition of red blood cells, ten microliters of whole blood were applied to the slides and red blood cell monolayers were prepared using a V-sampler. The membrane surface was scanned by semicontact resonance AFM. The investigators used NSGL 01-A cantilevers with a resonance frequency in the range of 80200 kHz, a probe radius of 10 nm, 512 and 1024 scanning points, and 100X100 ^m and 10X10 ^m scanning fields. Planar and 3D images were obtained. Results. Calculation of 1000 cells by light optical microscopy and AFM showed significantly different counts of macro- and microcytes in the comparison group. The 100X100-^m field exhibited the following types of red blood cells: discocytes (97.9±1.5% and 96±5%), echinocytes (2.1±0.9% and 3±1%), and squamous cells (0.1±0.02% and 1±0.5%). Within the first 24 hours after injury, the victim group displayed lower normocyte counts and higher counts of macrocytes and microcytes than the control group. AFM in the 100X100-^m field revealed that the victims with SMI when admitted to an intensive care unit exhibited a significant decrease in the counts of discocytes counts and increases in those of echinocytes, stomatocytes, and squamous cells, as compared to the controls. Anisocytosis and poikylocytosis was found to depended on the degree of BL. The squamous cells are most likely to be a variety of young erythrocytes. An appreciable increase in macrocytes and squamous cells on day 5 most likely reflects enhanced compensatory erythropoiesis in response to BL. Examination of red blood cell shapes within the first 24 hours after injury in the AFM field revealed profound discocytes with a protuberance in the center. On day 1 of injury changes in the nanostructure of red blood cell membranes included: 9-fold increase of first-order height (hj); 1.5-fold and 3-fold increase of second- and third-order heights (h2 and h3), respectively. The magnitude of changes in hj, h2 and h3 depended on the volume of BL and tended to decrease within the first two weeks of injury. Conclusion. The victims with SMI were observed to possess significant anisocytosis and poikylocytosis. The changes were associated with the volume of BL and included profound discocytes with a protuberance in the center as well aso changes in heights of nanostructural patterns of red blood cell membranes. Key words: severe concomitant injury, blood loss, atomic force microscopy, light optical microscopy, nanostructure of red blood cell membranes.

A new strategy to identify rare blood donors: single polymerase chain reaction multiplex SNaPshot reaction for detection of 16 blood group alleles.

As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood.