Erythrocyte Blood Group Antigens Research Articles

Comparative analysis of asexual and sexual stage Plasmodium falciparum development in different red blood cell types

BackgroundRed blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development.MethodsApproximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy.ResultsMerozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05).ConclusionsParasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

Detection of red cell alloantibodies in thalassaemia patients

Background: β-thalassaemia patients receive regular blood transfusion to thrive. Due to antigen disparity between the blood donors and these patients they develop red cell alloantibodies due to alloimmunization. The objective of this study is to predict the frequency of red cell alloimmunization amongst β-thalassaemia major patients receiving regular blood transfusion.Methods: This study including 106 patients with β-thalassaemia was conducted in the department of Transfusion Medicine, S. C. B. Medical College, Cuttack for a period of 12 months. Alloantibodies to different red cell blood group antigens in multi-transfused thalassaemia patients were detected using the glass bead technology for blood group serology in the present study.Results: Out of 106 β-thalassaemia major patients included in the study, 7.5% of patients developed alloantibodies, all being clinically significant. The alloantibodies were anti-E, anti c, anti e and anti-D. The rate of incidence of these alloantibodies was 3.8%, 1.9%, 0.9% and 0.9% respectively. There was a significant association between alloantibody formation with number of transfused packed red cells (Mann-Whitney Test: p value = 0.035) and age at first transfusion (p value = 0.001). The factors having no association with alloimmunization to red cell antigens are age and gender.Conclusions: Alloimmunization to various erythrocyte blood group antigens is a common problem in multi-transfused β-thalassaemia patients. There is an association between number of transfused packed red cells and age at first transfusion with alloantibody formation in the study.

Specific Combinations of Erythrocyte Group Antigens in Blood Donors.

Blood groups are inherited biological characteristics that do not change throughout life only in healthy people. Blood group antigens determine the adaptation of a human, as a biological species, to the surrounding environment. Our goal was to study the combination and frequency characteristics of three group systems (ABO, RH, KELL) in blood donors. The blood of 743 donors (aged ≥ 18years) has been investigated on erythrocyte blood group antigens (521 male and 222 female). The sample has been provided from diagnostic laboratory of "Health Centre Medina Ltd" of Batumi. Research was agreed with hematology department of the clinic. Blood typing of the sample has been carried out on the basis of immunogenetic laboratory of Batumi Shota Rustaveli State University. The express method with universal mono-clone antibodies was used in order to reveal blood group system antigens. ABO blood group are distributed the following order: O > A > B > AB. We allocated 16 phenotypic groups. O, Rh+, K- (38.62 ± 1.7%) are represented with the highest percentage among donors and a rather large number of the investigated donors (287 donors) is the carrier of this phenotype. A frequency distribution of the A, Rh+, K- phenotype group 10% (28.93 ± 1.6) was less. 215 donors carry A, Rh+, K- phenotype combination. O, Rh+, K+ and A, Rh+, K+ variations of the phenotype are represented with an equal percentage (3.23 ± 0.4%). 7.94 ± 0.9% donors are characterized by features of the O, Rh-, K- Erythrocyte group. A frequency distribution of B, Rh+, K- phenotype is slightly less - 6.86 ± 0.9%. A, Rh-, K combination of phenotype occurs with a frequency of 4.97 ± 0.7%, and AB, Rh+, K- occurs with a frequency of 2.28 ± 0.5%. I n the donor population there were only single cases of B, Rh-, K+ (0.13 ± 0.01); AB, Rh+, K+ (0.13 ± 0.01); AB, Rh-, K- (0.40 ± 0.2); B, Rh+, K+ (0.26 ± 0.1); O, Rh-, K+ (0.40 ± 0.2); A, Rh-, K+ (0.67 ± 0.2) combination of phenotype. The phenotype AB, Rh-, K+ was not identified. The existence of this database will increase the safety level of transfusion, widen donors database and allow the clinics to find quickly the rare combination of blood group for transfusion and decrease of the risk of post-transfusion complications.

Prevalence of Blood Groups at the Blood Transfusion Center at the Military Hospital Avicenna of Marrakech

The ABO and RHD systems represent the most immunogenic systems and the most sought after in blood transfusion. The objective of this work was to present new statistics of phenotypic prevalence of ABO and RHD systems in Morocco using a new sample. Our study was a retrospective, descriptive and epidemiological one carried out at the Blood Transfusion Center at the Avicenne Military Hospital in Marrakech on a sample of 10,000 donors collected between the year 2009 and the year 2017. The study of the expression Erythrocyte blood group antigens by the serological phenotyping technique was based on haemagglutination. Double determination was necessary to avoid the risk of grouping errors. This technique was simple and inexpensive, and today remains a gold standard for immunohematology. At the end of this study, the frequency of the antigens of the red blood cell systems ABO and RHD was as follows: ABO system: A (32.7%), B (16.33%), AB (5.26%), O (45.71%). RHD system: RHD positive (92.29%), RHD negative (7.71%). Our results were compared to other previous Moroccan and other foreign countries studies; this allowed us to locate hemotypologically the transfusion center of the Avicenne military hospital of Marrakech in the world. At the end of this study, we have concluded that our results were consistent with previous studies in Morocco. These results were identical to those found in the Mediterranean countries and showed that Morocco was in an intermediate position between the countries of Europe and those of Black Africa.

Molecular basis for an individual with rare p phenotype in P1Pk blood group system

To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system. Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing. The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position. The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.

Identification of two new blood group systems, Langereis (LAN) and Junior (JR)

Background The high‐prevalence erythrocyte blood group antigens Lan and Jra were first reported in 1962 and 1970 respectively. While both anti‐Lan and anti‐Jra can cause acute transfusion reactions, anti‐Jra is of particular concern in obstetrics as it can cause fatal hemolytic disease of the fetus and the newborn (HDFN). The biochemical and genetic bases of Jra and Lan have not yet been elucidated despite the isolation of two hybridoma secreting human monoclonal antibodies specific for Jra (HMR0921) and Lan (OSK43).Results Using the OSK43 antibody, we were able to immunoprecipitate a single protein from the erythrocytes of a random (Lan+) blood donor. This protein was identified by mass spectrometry as ABCB6, a member of the ATP binding cassette (ABC) transporter family. In order to validate ABCB6 as a novel genetic locus encoding blood group antigens, we first confirmed by immunoblot analysis that ABCB6 was present in the membrane of Lan+ erythrocytes. In contrast, we detected no ABCB6 products in Lan− erythrocytes, suggesting that null alleles of ABCB6 may be responsible for the Lan− phenotype. We then established K‐562 cells stably transfected with a construct encoding ABCB6 and showed that exogenous expression of ABCB6 correlated with cell surface expression of the Lan antigen. Targeted sequencing of ABCB6 in 12 unrelated Lan− individuals identified 10 different null mutations. All these Lan− individuals were either homozygous for a single null mutation or heterozygous for two null mutations in ABCB6, confirming that null alleles of ABCB6 are responsible for the Lan− phenotype. Using the HMR0921 antibody, we similarly purified and identified the ABC transporter ABCG2 as the carrier of the Jra antigen. Analysis of Jr(a+) or Jr(a−) erythrocytes, as well as K‐562 cells transfected with ABCG2 cDNA, showed that the Jra antigen is encoded by ABCG2 and that Jr(a−) erythrocytes lack ABCG2. Targeted sequencing of ABCG2 in 18 unrelated Jr(a−) individuals identified eight different null mutations, which were responsible for their Jr(a−) phenotype, and revealed that the prevalence of the Jr(a−) phenotype in the Japanese and European Gypsy populations is related to the mutations p.Q126X and p.R236X in ABCG2, respectively.Conclusions We demonstrated that the related transporters ABCB6 and ABCG2 specify two new blood group systems called Langereis (LAN) and Junior (JR), respectively. The elucidation of their molecular bases will facilitate the detection of individuals with the rare Lan− and Jr(a−) phenotypes and hence improve their transfusion security and/or their pregnancy follow‐up.

ABCG2 null alleles define the Jr(a−) blood group phenotype

The high-incidence erythrocyte blood group antigen Jr(a) has been known in transfusion medicine for over 40 years. To identify the gene encoding Jr(a), we performed SNP analysis of genomic DNA from six Jr(a-) individuals. All individuals shared a homozygous region of 397,000 bp at chromosome 4q22.1 that contained the gene ABCG2, and DNA sequence analysis showed that ABCG2 null alleles define the Jr(a-) phenotype.

Funções biológicas dos antígenos eritrocitários

Os antigenos de grupos sanguineos eritrocitarios sao estruturas macromoleculares localizadas na superficie extracelular da membrana eritrocitaria. Com o desenvolvimento de estudos moleculares, mais de 250 antigenos sao conhecidos e estao organizados em 29 sistemas de grupos sanguineos reconhecidos pela Sociedade Internacional de Transfusao Sanguinea (ISBT). Estudos tem revelado que os antigenos de grupo sanguineo estao expressos na membrana eritrocitaria com ampla diversidade estrutural, incluindo epitopos de carboidratos em glicoproteinas e/ou glicolipidios e em proteinas inseridas na membrana via um dominio, via dominios de multipassagem ou ligados a glicosilfosfatidinositol. Alem das diversidades estruturais, muitas funcoes importantes tem sido associadas aos antigenos eritrocitarios recentemente identificadas, podendo ser esquematicamente divididas em: estruturais, transportadores, receptores e moleculas de adesao, enzimas, proteinas controladoras do complemento e outras. Esta revisao tem como foco as funcoes potenciais das moleculas que expressam os antigenos eritrocitarios.

Impact of Erythrocyte Duffy Antigen Genetic Polymorphism on the Distribution of GroB-T, a Novel Human CXC Chemokine

Grobeta-T, a human CXC chemokine, has been studied for its potential to mobilize stem cells. Chemokines bind specifically to receptors on target immune cells but also to a homologous erythrocyte blood group antigen, the Duffy Antigen/Receptor for Chemokines (DARC)that is subject to genetic polymorphism in humans.A mutation in the DARC gene is common among African Americans and results in lack of expression of the erythrocyte antigen. We used a combination of in vitro studies of Grobeta-DARC interaction and pharmacokinetic simulation to anticipate the potential impact of this polymorphism on the pharmacokinetics of Grobeta-T. [125I]Grobeta-T was incubated in Caucasian blood to characterize the concentration dependence of the blood to plasma concentration ratio (B/P). Affinity and capacity of binding was estimated by Scatchard analysis; specificity was investigated by competitive displacement with a CC chemokine. The B/P value (7 nM) was then determined in blood from 8 African American subjects. Duffy antigen expression was determined by antibody agglutination. A pharmacokinetic model was developed which accounted for blood-cell binding. Simulations were performed to explore effects of dose regimen and DARC expression on the Grobeta-T plasma concentration-time profile. Grobeta-T affinity and capacity for DARC (Caucasian blood)were 23.0 +/- 1.2 and 37.7 +/- 0.6 nM, respectively;excess CC chemokine fully displaced [125I]Grobeta-T. Chemokine binding was highly correlated with the presence or absence of the Duffy antigen (p<0.01) in African American blood; the proportion of subjects for which binding was observed (3/8), was consistent with the reported frequency of DARC expression in this population. Counter to intuition,in the terminal disposition phase at low doses,concentrations of free Grobeta-T in the presence of DARC may be substantially higher than in the absence of DARC. Dissociation from the erythrocyte antigen may lead to greater persistence, at low doses, of free Grobetabeta-T in the blood of individuals expressing the chemokine sink.

Decay-Accelerating Factor Ameliorates Systemic Autoimmune Disease in MRL/lpr Mice via Both Complement-Dependent and -Independent Mechanisms

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. Previous studies have established a significant protective activity of DAF in the MRL/lpr murine model of human systemic lupus erythematosus. To dissect the mechanism of protection by DAF in this disease model, we evaluated the effect of C3 gene ablation on disease development in MRL/lpr-Daf-1(-/-) mice. We found no significant difference in lymphadenopathy, splenomegaly, or anti-chromatin autoantibody titer between complement-sufficient and complement-deficient MRL/lpr-Daf-1(-/-) mice. On the other hand, complement deficiency strikingly reduced the incidence and severity of dermatitis in MRL/lpr-Daf-1(-/-) mice. To assess the contribution of DAF expression on lymphocytes versus local tissues in suppressing dermatitis, we generated BM chimeric mice between MRL/lpr-Daf-1(-/-) and MRL/lpr-Daf-1(+/+) mice. Compared with MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(-/-) controls, MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(+/+) chimeras developed significantly attenuated dermatitis, suggesting that the protective effect of DAF in suppressing dermatitis is primarily attributable to its local expression. We conclude that DAF works as a complement regulator in the skin to protect MRL/lpr mice from skin inflammation, whereas its inhibitory role in the induction phase of MRL/lpr autoimmunity is complement-independent. Together, these results reveal multiple mechanisms of action for DAF in ameliorating systemic autoimmunity.

Erythrocyte Adhesion Receptors: Blood Group Antigens and Related Molecules

During the second half of the 20th century, blood bankers quickly expanded our knowledge of human erythrocyte blood group antigens. By the dawn of the 21st century, several hundred blood group antigen polymorphisms had been identified. Hot on the heels of the serologists, membrane biochemists and molecular geneticists defined both the biochemical and genetic bases of most of these antigens. Perhaps to their surprise, this work has led to the discovery of functionally diverse and important membrane proteins expressed on the surface of red cells, including numerous adhesion molecules. Red cells express an unexpected number of such adhesion receptors, some of which contribute to human disease, as well as to normal red cell development. And perhaps most interestingly, study of these molecules has elucidated ways in which even mature red cells respond to external stimuli, such as adrenergic hormones.

Distribution of ABO, Rh, MNSs, Kell and Duffy blood-group antigens in population of Vojvodina

Analysis of erythrocyte blood group antigen polymorphisms and genetic variability in population of Vojvodina was performed by investigating gene and genotype frequencies which determine antigens of ABO, Rh, MNSs, Kell and Duffy blood-group systems. We investigated 350 unrelated persons from Vojvodina in regard to appurtenance of ABO, Rh, MNSs, Kell and Duffy blood-group systems. We calculated gene, genotype, phenotype frequencies and proportion significance test. Results of investigation revealed that gene and genotype frequencies of investigated blood-group systems are similar to corresponding data for majority of European populations, while statistically significant differences were established in inhabitants of geographically distant regions. Values of proportion significance test revealed statistically significant differences of genotype frequencies for ABO and MNSs blood-group antigens in populations of: Australian Aborigines, Chinese population, Arabians, Blacks, Eskimos, American Indians (Navaho and Pueblo) and population of Papua New Guinea. Statistically significant differences of genotype frequencies were established in inhabitants of narrow geographical areas of Europe such as: Finland, Germany, Sweden, Albania, England and Netherlands. Our results point to the fact that erythrocyte blood-groups have different frequencies in some parts of the world, and that there are great differences in frequencies of some blood-groups among inhabitants of various continents and races. Genetical peculiarity of the population of Vojvodina points to the fact that differences in blood-group frequencies are also present among inhabitants of narrow geographical areas.

Molecular Basis of Erythrocyte Blood Group Antigens and Applications in Transfusion Medicine

Molecular Basis of Erythrocyte Blood Group Antigens and Applications in Transfusion Medicine

Terminologie numérique des antigènes de groupes sanguins érythrocytaires

Numeric terminology of erythrocyte blood group antigens Since the discovery of the first human blood groups, the terminology for erythrocyte blood group antigens has evolved and become inconsistent. In some cases, a single letter is used (eg, A / B, E / c), in some others a symbol with a superscript to denote allelic products is used (eg, Fy a / Fy b, Jk a / Jk b) while in still others, a numerical notation is used (eg, Fy3, Lu4, K11). Even within the same blood group system, antigens have been named by different terminology (eg, K/k, Kp a/Kp b, K11/K17) and the same antigen was given different names in different laboratories. Therefore a necessary alternative ‘popular’ terminology was suggested by the ISBT (International Society of Blood Transfusion) Working Party; In its last monograph, published in 1995 and reviewed in 1996, erythrocyte blood group antigens are classified into 23 systems, 5 collections and 2 series. The Working Party also suggested guidelines for establishing new blood group systems and including new specificities into the nomenclature.

Biologic functions of blood group antigens.

In the past few years, we have learned a great deal about the biologic function of structures bearing blood group antigens. Some blood group antigen-bearing proteins function as major transport channels within the erythrocyte membrane; these include the anion transporter (band 3: Diego and Wright antigens), the water channel (aquaporin: Colton antigens), and the urea transporter (Kidd antigens). At least two erythrocyte blood group antigen proteins have complement regulatory functions: the complement receptor type 1 (CR1, CD35: Knops antigens) and decay accelerating factor (DAF, CD55: Cromer antigens). Some blood group antigens reside on proteins with known receptor functions, such as the chemokine receptor (Duffy) and the hyaluronan receptor (Indian). The Cartwright antigens reside on an enzyme, acetylcholinesterase, and the Kell antigens reside on a protein that belongs to the CALLA-related family of neutral metalloproteinases. Finally, some blood group antigens reside on proteins that serve crucial structural functions necessary to normal erythrocyte lifespan and morphology. These proteins include band 3, glycophorins C/D (bearing the Gerbich antigens), and the Rh proteins. Both oligosaccharide and protein blood group antigens may act as receptors for bacterial, viral, and parasitic infectious agents.

Isolation of the JMH Antigen on a Novel Phosphatidylinositol-Linked Human Membrane Protein

AbstractJMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospho-lipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI-linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.

Isolation of the JMH antigen on a novel phosphatidylinositol-linked human membrane protein

JMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospholipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI- linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.

Phosphatidylinositol-linked red blood cell membrane proteins and blood group antigens

Abstract A new class of membrane proteins has recently been described. Unlike integral membrane proteins, which traverse the membrane with one or more hydrophobic peptide domains, the peptide domains of these more newly described proteins are entirely extracellular and are anchored to the cell membrane via a phosphatidylinositol-glycan (GPI) anchor. Erythrocyte membrane proteins of this class include proteins with diverse functions; several, however, are complement regulatory proteins. Moreover, it is the lack of expression of GPI-anchored proteins that is responsible for manifestations of the acquired hematologic disease paroxysmal nocturnal hemoglobinuria. Recently, several investigators have also demonstrated that a number of erythrocyte blood group antigens reside on this class of proteins. These antigens include those of the Cromer blood group, JMH, Holley/Gregory, Cartwright, and Dombrock. The biochemical basis for the Cromer, JMH, and Holley/Gregory antigens have so far been partly delineated. Immunohematology 1991;7:65–72.

Relationship of Inb Antigen to Other Antigens on In(Lu)‐related p801

The Ina and Inb antigens have recently been added to the list of diverse erythrocyte blood group antigens whose expression is down-regulated by the In(Lu) gene. Evidence has been provided that these antigens constitute polymorphisms of an erythrocyte glycoprotein previously defined by its reactivity with murine monoclonal antibodies, such as A3D8, and designated In(Lu)-related p80. This study has now confirmed the assignment of the Inb antigen to p80 and has further studied the relationship of the Inb epitope to other p80 epitopes recognized by both monoclonal antibodies as well as polyclonal antiserum produced against purified erythrocyte p80.