anti-B Hemagglutinins Research Articles

Technological Aspects of Ensuring the Specific Safety of Human Immunoglobulin and Albumin Preparations

Modern technological approaches to ensuring the specific safety of human immunoglobulin and albumin preparations are analyzed. Introduction into human immunoglobulin preparation technology of donor screening for group- and rhesus-affiliations and chromatographic purification steps to reduce the contents of anti-A and anti-B hemagglutinins and anti-D antibodies are shown to be promising. Technological capabilities for reducing the anticomplementary activity and thrombogenic potential of immunoglobulin preparations and decreasing the contents of prekallikrein activator in human i.v. immunoglobulin and albumin preparations are discussed.

Standardization of the Method for Determining Hemagglutinin Content in Human Immunoglobulin Preparations in Russia

Documentation for standardization of the method for determining hemagglutinins (HAs) in human immunoglobulin preparations used in Russia is presented. The proposed general pharmacopoeial monograph “Determination of anti-A and anti-B hemagglutinins in medicinal preparations of human immunoglobulin” allows quality control of human immunoglobulin preparations for HAcontent using indirect hemagglutination under standard conditions. The positive and negative components of the domestic standard sample (SS) of HAs contain 1:32 anti-A and 1:16 anti-B, respectively; less than 1:2 anti-A and -B; and 1:64 components of the HA content limit. Introduction of the SS for contents of anti-A and anti-B HAs into pharmacopoeial practice would ensure that results obtained in various ranges are accurate and enable the quantitative contents of the corresponding HAs in human immunoglobulin preparations to be determined.

Стандартизация метода определения содержания гемагглютининов в препаратах иммуноглобулинов человека в России

Представлены материалы по стандартизации метода определения содержания гемагглютининов в препаратах иммуноглобулинов человека в России. Показано, что разработанная общая фармакопейная статья «Определение анти-A и анти-B гемагглютининов в лекарственных препаратах иммуноглобулинов» позволяет контролировать качество препаратов иммуноглобулинов человека по содержанию гемагглютининов методом непрямой гемагглютинации в стандартных условиях. Положительный и отрицательный компоненты отечественного стандартного образца содержания гемагглютининов содержат, соответственно, 1:32 анти-A гемагглютининов, 1:16 анти-B гемагглютининов и менее 1:2 анти-A и анти-B гемагглютининов, а компоненты лимита содержания — 1:64. Внедрение стандартного образца содержания анти-A и анти-B гемагглютининов в фармацевтический анализ позволит обеспечить достоверность полученных результатов в различных диапазонах значений, а также определить количественное содержание соответствующих гемагглютининов в препаратах иммуноглобулинов человека.

Reduction of Isoagglutinin in Intravenous Immunoglobulin (IVIG) Using Blood Group A- and B-Specific Immunoaffinity Chromatography: Industry-Scale Assessment.

BackgroundHemolysis, a rare but potentially serious complication of intravenous immunoglobulin (IVIG) therapy, is associated with the presence of antibodies to blood groups A and B (isoagglutinins) in the IVIG product. An immunoaffinity chromatography (IAC) step in the production process could decrease isoagglutinin levels in IVIG.ObjectivesOur objectives were to compare isoagglutinin levels in a large number of IVIG (Privigen®) batches produced with or without IAC and to assess the feasibility of the production process with an IAC step on an industrial scale.MethodsThe IAC column comprised a blend of anti-A and anti-B resins formed by coupling synthetic blood group antigens (A/B-trisaccharides) to a base bead matrix, and was introduced towards the end of the industrial-scale IVIG manufacturing process. Isoagglutinin levels in IVIG were determined by anti-A and anti-B hemagglutinin direct and indirect methods according to the European Pharmacopoeia (Ph. Eur.) and an isoagglutinin flow cytometry assay. IVIG product quality was assessed with respect to the retention of immunoglobulin G (IgG) subclasses, specific antibodies, and removal of IgM using standardized procedures.ResultsThe IAC step reduced isoagglutinins in IVIG by two to three titer steps compared with lots produced without IAC. The median anti-A and anti-B titers with IAC were 1:8 and 1:4, respectively, when measured by the Ph. Eur. direct method, and 1:2 and <1, respectively, when measured by the Ph. Eur. indirect method. The isoagglutinin flow cytometry assay showed an 87–90 % reduction in isoagglutinins in post-IAC versus pre-IAC fractions. IAC alone reduced anti-A and anti-B of the IgMs isotype by 92.5–97.8 % and 95.4–99.2 %, respectively. Other product quality characteristics were similar with and without IAC.ConclusionsIAC is an effective method for reducing isoagglutinin levels in IVIG, and it is feasible on an industrial scale.

Dangerous universal donors: the reality of the Hemocentro in Belo Horizonte, Minas Gerais.

BackgroundThe term dangerous universal blood donor refers to potential agglutination of the erythrocytes of non-O recipients due to plasma of an O blood group donor, which contains high titers of anti-A and/or anti-B hemagglutinins. Thus, prior titration of anti-A and anti-B hemagglutinins is recommended to prevent transfusion reactions.ObjectiveThe aim of this study was to estimate the frequency of dangerous universal donors in the blood bank of Belo Horizonte (Fundação Central de Imuno-Hematologia – Fundação Hemominas – Minas Gerais) by determining the titers of anti-A and anti-B hemagglutinins in O blood group donors.MethodA total of 400 O blood group donors were randomly selected, from March 2014 to January 2015. The titers of anti-A and anti-B hemagglutinins (IgM and IgG classes) were obtained using the tube titration technique. Dangerous donors were those whose titers of anti-A or anti-B IgM were ≥128 and/or the titers of anti-A or anti-B IgG were ≥256. Donors were characterized according to gender, age and ethnicity. The hemagglutinins were characterized by specificity (anti-A and anti-B) and antibody class (IgG and IgM).ResultsAlmost one-third (30.5%) of the O blood group donors were universal dangerous. The frequency among women was higher than that of men (p-value = 0.019; odds ratio: 1.66; 95% confidence interval: 1.08–2.56) and among young donors (18–29 years old) it was higher than for donors between 49 and 59 years old (p-value = 0.015; odds ratio: 3.05; 95% confidence interval: 1.22–7.69). There was no significant association between dangerous universal donors and ethnicity, agglutinin specificity or antibody class.ConclusionEspecially platelet concentrates obtained by apheresis (that contain a substantial volume of plasma), coming from dangerous universal donors should be transfused in isogroup recipients whenever possible in order to prevent the occurrence of transfusion reactions.

Anti-A and anti-B hemagglutinin depletion during Cohn purification process of 5% immunoglobulin.

Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are widely used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. For high-quality products and to minimize adverse events related to the use of intravenous IgG (IVIG) it is very important to perform detailed analyses of their components. One of these components, that in rare cases can cause severe hemolytic conditions, is the amount of hemagglutinins, natural antibodies that bind A and/or B (anti-A or -B) antigens present in red blood cells (RBCs). To characterize different IgG batches and to monitor the efficacy of the production procedure in the hemagglutinin reduction, a direct agglutination test (DAT) and a new flow cytometry (FC)-based assay were used for measuring the activity and the content of hemagglutinins in IgG samples obtained at different stages of the purification process. A total of 113 batches of 5% IVIG, produced in 2013 by Kedrion Biopharma, were analyzed for the ability to agglutinate RBCs by DAT. All batches tested were within the limits set by the European Pharmacopoeia. Three batches of 5% IVIG were analyzed for their hemagglutinin levels. The finished products and the production intermediates were evaluated by the DAT and the FC assay. A significant decrease of anti-A and anti-B titer after the Fraction (F)III precipitation was observed in all batches tested and an evaluation of the results obtained by the two methods was performed. This study shows that the hemagglutinin titer, accurately measured in a high number of 5% IVIG batches, is within the allowed limits for the DAT method. The specific production process employed, in particular the FIII precipitation step, successfully removes IgM and significantly reduces IgG class hemagglutinins.

A study on salivary hemagglutinins in a Central Indian population

PurposeTo study the presence of salivary hemagglutinins in a Central Indian Population. MethodA drop of supernatant of centrifuged saliva samples was placed in cavity tiles. A drop of 0.2% cell suspensions of twice washed cells of known A and B blood groups was added into the respectively marked cavities. After mixing, the tiles were kept in a moist chamber for 30min and examined microscopically. Clumps formed of at least 3–4 cells were taken as positive. ResultA sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has been developed. Presence of salivary hemagglutinins in Maharashtrians was quite high, i.e., 69.41% as compared with Gonds 17% (Chatterjee and Badiye, 2012) and the Punjabis 35.98% (Chattopadhyay and Ganeson, 1983). ConclusionSaliva in the form of stains is encountered as physical evidence in many cases, such as anonymous letters, secret writing, sexual assault, rape, murder, disputed paternity, cigarette butt ends, etc. If analyzed properly saliva can not only help in the elimination of the innocents, but also in the actual identification of a specific individual. Like blood grouping, this can also be used for forensic purposes.

Efficacy and Accuracy of ABO Blood Group Determination from Saliva

Objectives: The saliva was first analyzed for the presence of anti-A and anti-B hemagglutinins in 1928. However, it was not been utilized as evidence in criminal investigations because of insufficient techniques available at that time. Hence, the present study was designed to evaluate feasibility of ABO group in medicolegal cases by determining the efficacy and accuracy of ABO blood group determination from saliva and to determine the pattern of antigen secretion in saliva in family members. Methods: A total of 200 subjects (101 males and 99 females) studied. Out of these. 100 subjects were taken at random (sample I) and 100 subjects were taken from 25 families (sample I0. The blood group of each subject was determined from saliva by absorption-inhibition method. Results: The present study showed that 83% subjects are secretors of antigen in saliva. If the subject was secretor, one of his/her parents was essentially a secretor. If both parents were secretor both the children in the family were secretors. Conclusion Results can be utilized for identification of subjects, ruling out identification, determination of inheritance in medicolegal cases.

Freeze dried plasma: a French army specialty

We read with interest the article by Nascimento and colleagues [1] on fresh frozen plasma (FFP) in massive bleedings. We fully agree with their conclusion when they say that we need immediately available universal donor AB plasma. Hemorrhagic shock is the primary cause of avoidable mortality in combatants. Data from the recent US army war experience show clearly that in cases of massive transfusion the transfusion policy strongly impacts mortality. Namely, for massive bleeding the ratio between red blood cells and clotting factors should be close to the composition of whole blood [2]. Plasma is thus essential for massive transfusions. But FFP use is impractical in uncertain environments such as a battlefi eld. During military operations, refrigerated transportation and storage are logistical problems. Th awing of FFP takes a long time, with an important loss of plasma in austere environments. For example, Mabry and colleagues [3] report that, during the Mogadishu urban battle, the available FFP was stored in bags that fractured one-third of the time upon thawing. For these reasons, the French army has used freezedried and secured plasma (FDSP) since 1994. Plasma separated from fresh blood of at least ten donors is lyophilized to produce FSDP. Blood type selection allows the dilution and neutralization of natural anti-A and antiB hemagglutinins. Th is FDSP is thus compatible with any blood type. In addition, FDSP is shelf-stable in ambient temperatures for 2 years and easily rehydrated with 200 ml of water for injections in less than 3 minutes, allowing immediate provision with the fi rst packed red blood cells [4]. FDSP contains all clotting factors and proteins. After more than 2 years storage at ambient temperature, the fi brinogen and clotting factor levels of FDSP are equivalent to FFP [4]. Th e securization process is quarantine (i.e. the plasma is held until the donor returns and is retested after a period that is longer than the window period of known viruses). Plasma as FDSP is thus a logistically superior product, without compromising hemostatic properties, quickly available in cases of emergency for any blood type.

Thrombotic Microangiopathy After ABO-Incompatible Living Donor Liver Transplantation: A Case Report

Thrombotic microangiopathy (TMA) has rarely been reported in the setting of liver transplantation. Herein we have reported a successful case of TMA after ABO-incompatible living donor liver transplantation (LDLT) treated with plasma exchange and high-dose intravenous gamma-globulin infusion. A 50-year-old woman was diagnosed with hepatitis C virus-related cirrhosis. We performed an ABO-incompatible LDLT (group B to O) with preoperative plasma exchange to reduce the anti-B hemagglutinin titers to 1:8. The immunosuppressants consisted of tacrolimus, mycophenolate mofetil, and steroid. On postoperative day (POD) 8, her anti-B hemagglutinin titer suddenly increased to 1:64. The serum lactate dehydrogenase (LDH) level was grossly elevated (1518 IU/L). On POD 13, we suspected infection of an intra-abdominal hematoma ( Serratia marcescens) which was drained surgically. On day 5 after the reoperation, thrombocytopenia developed with a platelet count of 3×10 4/mm 3. A peripheral blood film showed severe red blood cell (RBC) fragmentation. Thus, we made a clinical diagnosis of TMA and reduced the tacrolimus dose. We started intensive daily plasma exchange (4 L/d) with fresh frozen plasma and high-dose intravenous gamma-globulin infusions. One week thereafter, thrombocytopenia improved with reduced transfusion requirements. The peripheral blood film showed normal RBC morphology. The serum LDH returned to baseline levels. Four factors were considered to have caused TMA in this case: the prescription of tacrolimus, ABO-incompatible liver transplantation, bacterial infection, and surgical stress. These factors may have all contributed by causing significant endothelial injury and TMA.

Detection and Use of Salivary Hemagglutinins for Forensic Blood Grouping

A sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has been developed. The method utilizes a bromelin treated erythrocyte suspension as indicator cells and includes a simple procedure to concentrate these hemagglutinins. Antiserum directed against immunoglobulin A enhances the hemagglutination assay. We find that these salivary hemagglutinins are present in over 90% of the population and that their titer remains stable over a period of two months. These hemagglutinins can be used to blood type the donor of a saliva sample and can be used in a confirmatory test that complements the commonly used absorption-inhibition test which is used to detect salivary blood group agglutinogens. In preliminary studies we have determined that hemagglutinins can be successfully isolated and analyzed from dried saliva stains.

Purification and properties of an anti-B hemagglutinin produced by Streptomyces species

An anti-B hemagglutinin was purified to homogeneity from the culture filtrate of a strain of Streptomyces sp. by affinity chromatography. The Streptomyces hemagglutinin was adsorbed to insolubilized gum arabic and eluted with 1 M NaCl containing 1 M D-galactose. The purified hemagglutinin is thought to be homogeneous judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 7.2, disc gel electrophoresis at pH 4.3, isoelectric focusing, and ultracentrifugation. The molecular weight was estimated to be 11,000 from results of gel filtration in 6 M guanidine hydrochloride (Gdn-HCl), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sedimentation equilibrium analysis. The amino acid analyses revealed that the hemagglutinin contained large amounts of alanine, glycine, and valine, 47% of the total amino acid residues, and no phenylalanine. Carbohydrate analysis demonstrated that the hemagglutinin might not be a glycoprotein. The circular dichroic (CD) spectrum of the protein is quite different from those of usual proteins in having a large positive peak at 226 nm (theta = 10,000) and a negative band at 212 nm (theta =-2600). The hemagglutinin showed a typical precipitation curve with gum arabic, and agglutinated human blood group B erythrocytes 256 times as strongly as A or O erythrocytes. These activities were not affected by pH (from 4 to 12). The anti-B activity was further confirmed by serological tests. The hemagglutination-inhibition studies indicated that D-galactose was inhibitory, but alpha-D-galactosides were not necessarily better inhibitors than beta-D-galactosides. L-Rhamnose was the best inhibitor among the monosaccharides tested, and L-arabinose and D-fucose were also inhibitory.

Sugar specificity of anti-B hemagglutinin produced by [formula omitted] sp

A hemagglutinin specific for blood group B antigen has been purified to 190-fold from the culture fluid of a strain of Streptomyces sp. by conventional procedure involving ammonium sulfate fractionation and column chromatography. The molecular weight of the partially purified preparation was estimated to be approximately 5000±1000; this value is extremely small as compared with those of hemagglutinins which have been so far isolated from various sources. Hemagglutination-inhibition tests revealed that the Streptomyces agglutinin has a specificity to combine with D-galactose and several saccharides having D-galactose residues at the non-reducing terminal, and that the special configuration of the hydroxyl groups at C-2 and C-4, particularly the hydroxyl group at C-2, is essential for binding of the sugars to the hemagglutinin.

A STUDY ON THE OCCURRENCE OF THE ANTI-A AND ANTI-B HEMAGGLUTININS.

A STUDY ON THE OCCURRENCE OF THE ANTI-A AND ANTI-B HEMAGGLUTININS.