Papain-treated Red Blood Cells Research Articles

SARS CoV 2 infection in chronic myelogenous leukemia: Severe hematological presentation

Infection with SARS-CoV-2, the cause of coronavirus infectious disease-19 (COVID-19), has caused a pandemic. Few data are available about the risk of COVID-19 infection in persons with hematological cancer, but controversy whether these persons have the same clinical signs and outcomes. We describe a case of life‐threatening COVID-19 infection complicated by severe anemia in patients affected also by chronic myelogenous leukemia. The screening for RBC antibodies and the direct antiglobulin test (DAT) turned positive. The identification of the antibodies, showed the presence of an alloantibody with anti-Lewis b specificity, which was reactive at room temperature, in the anti-human globulin phase (AGH) and with papain-treated red blood cells. At the same time hemophagocytic lymphohistiocytosis (HLH), on the basis of major laboratory findings including hyperferritnemia, increase of triglicerides levels and according to the HLH score was suspected. Patients received antiviral therapy, steroids and intravenous immunoglobulins. Hemolysis resolved and ferritin dramatically decreased after administration of Ig and a Afull recovery was achieved after viral infection resolution.This case highlights the novel and multifaceted hematological findings during sever COVID 19 infection. COVID 19-related pneumonia is mediated by hyper activation of effector T cells and excessive production of inflammatory cytokines, such as IL-6, IL-1, interferon-gamma, and TNF. This inflammatory process called "cytokine storm" is a life-threatening complication of COVID 19 infection. In this case severe immunohematological consequences are reported for the first time and recognition of this complications are probably underestimated.

Spontaneous cold agglutination phenomenon associated with auto-anti-“N”-like antibody in a healthy blood donor

Background: The low titer cold autoagglutinins (CAs) are found in every individual; the high-titer CAs are associated with certain infections, malignancies, or autoimmune disorders. Usually, they have specificities as anti-I; occasionally, they occur as anti-i or anti-Pr. However, CAs found that as spontaneous cold autoagglutinations (SpCAs) among the healthy blood donors are predominantly anti-i or anti-Pr besides being anti-I for their specificities. Aim: The present study is aimed to ascertain the specificity of autoantibody associated with SpCA. Study Design: The antibody in the donor's plasma and the eluate augmented from his red blood cells (RBCs) was studied. Specificity was determined using commercial and in-house RBCs panel. Standard serological methods were used. Results: A 49-year-old male volunteer donor grouped as B, RhD-negative, and M+N+ and showed a positive test for antibody screening. Direct antiglobulin test was negative though the autocontrol test was strongly positive at 4°C. The antibody eluted from the donor's RBCs showed anti-“N”-like specificity when it reacted with the trypsin-treated RBCs but not with the papain-treated RBCs. Its specificity as anti-“N” was ascertained by the failure of its reactivity with the RBCs from the M+N-S-s-U-phenotype that lacks the “N” antigen. Conclusion: SpCA phenomenon observed in this case differs from the reported cases for its specificity as anti-“N”-like.

Anti‐U‐like as an alloantibody in S−s−U− and S−s−U+var black people

S, s, and U antigens belong to the MNS system. They are carried by glycophorin B (GPB), encoded by GYPB. Black people with the low-prevalence S-s- phenotype, either U- or U+(var), can make a clinically significant anti-U. Anti-U-like, a cold immunoglobulin G autoantibody quite commonly observed in S-s+U+ black persons, was previously described to be nonreactive with ficin-, α-chymotrypsin-, and pronase-treated red blood cells (RBCs); nonreactive or weakly reactive with papain-treated RBCs; and reactive with trypsin-treated RBCs. Here we describe, in S-s- people from different molecular backgrounds, an alloantibody to a high-prevalence GPB antigen, which presents the same pattern of reactivity with proteases as autoanti-U-like. Four S-s- patients with an alloantibody to a high-prevalence GPB antigen were investigated by serologic and molecular methods. An alloantibody was observed in two S-s-U-/Del GYPB, one S-s-U+(var)/GYPB(P2), and one S-s-U+(var)/GYPB(NY) patients. As this alloantibody showed the same pattern of reactivity with proteases as autoanti-U-like, we decided to name it "anti-U-like." Anti-U-like made by the two S-s-U- patients was reactive with the S-s-U+(var) RBCs of the two other patients. S-s-U-/Del GYPB, S-s-U+(var)/GYPB(P2), and S-s-U+(var)/GYPB(NY) patients can make an alloanti-U-like. Anti-U-like made by S-s-U- people appears reactive with GYPB(P2) and GYPB(NY) RBCs, which both express a weak and partial U-like reactivity. We recommend transfusing S-s-U- RBCs in S-s-U- patients showing alloanti-U-like. Our study contributes to a better understanding of alloimmunization to GPB in black people and confirms importance of genotyping in S-s- patients, especially those with sickle cell disease to be frequently transfused.

ANTI-hrs associated with the RHD-RHCE HAPLOTYPE DAR-ceAR in a patient with Sickle Cell Trait

Introduction: The RH blood group system is the most polymorphic and immunogenic among blood groups and some rare Rh phenotypes are found exclusively in the black population. The RH 18 phenotype (Hr-negative) that is one of these phenotypes is characterized by a high incidence antigen. The production of anti-RH 18 imposes to use equivalent rare antigen-negative red blood cells (RBCs), or deleted-Rh RBCs (Rhnull or D- -) for transfusion.Case Report: A 24-year-old female patient with two previous abortions and one ectopic pregnancy and miscarriage had to be transfused with a RBC unit. She was typed as blood group A, Dccee phenotype with weak D, positive RBC antibody screening, auto-control negative and TAD negative. The antibody was anti-e (title 1/64) determined by testing serum against a commercial RBC panel (Fresenius-Kabi) by IgG indirect antiglobulin test tube (PEG-IAT), and nontreated (LissCoombs) and papain-treated RBCs on a gel matrix (DiaMed, Latino America). After adsorption of serum on Rhe-negative RBC (DccEE), the retrieving antibody was an anti-e-like antibody reacting with all normal Rhe-positive RBCs. Molecular and serologic Rh typing was also performed on blood samples obtained from five relatives of the patient and her husband.Results: Patient and relatives cDNA sequence of gene RHD and RHCE transcripts were analyzed for RHCE exons 4 to 5 and exon 6; for RHD exons 4 to 5, 6 and 7. Sequencing of cDNA from the patient, her father and her sister showed in RHD the presence of the DAR allele point mutation 602 C>G in exon 4; 667 T>G and 744 C>T in exon 5; 957 G>A and 1025 T>C in exon 7; while on gene RHCE we found the ceAR allele carrying 712 A>G, 733 C>G e 787 A>G in exon 5. Hemoglobin tests revealed that the patient, her father and sister had a sickle cell trait (patient HbA1=55%; HbA2=2,8%; HbS=41%; HbF=1,2%; father HbA1=58,5%; HbA2=1,4%; HbS=37,7%; HbF=2,4%; sister HbA1=57,4%; HbA2=2,5%; HbS=38,6%; HbF=1,5%).Discussion: The family study showed that the haplotype DAR-ceAR was inherited from her father. Individuals carrying the ceAR allele may produce anti-hrs antibody that is defined as a Rhe-like which does not react with rare Rhe positive variant RBCs. The antibody may be clinically significant and represents a great risk for those patients who are not transfused with a similar rare phenotype variant. Serologically, DAR has shown weaker reactions with anti-D, it is characterized by complete loss of at least 9 of 37 RhD epitopes, and may produce anti-D. Both mutated alleles show a replacement of RHD exons by RHCE counterparts.

Analysis of enzyme‐treated red blood cell surface and haemagglutination using a theory of soft particle electrophoresis

Enzymatic treatment of red blood cells is thought to reduce the cell zeta (zeta) potential, effectively decreasing the distance between cells to less than the length of an immunoglobulin G antibody binding site, and resulting in agglutination of cells. However, the zeta potential given by Smoluchowski's formula is based on theories of the electrophoresis of hard colloidal particles. A theory has recently been developed for the electrophoresis of colloidal particles covered with polyelectrolytes, which we call 'soft particles'. The electrophoretic mobility of red blood cell treated with papain and neuraminidase was measured as the electrolyte concentration of the medium using phosphate buffer. The results were analysed via the formula for 'soft particles'. This mobility formula involved two parameters, the fixed charge density (ZN) and parameter 1/lambda characterizing the 'softness' of the cell surface layer. The best-fit curves of 0.1 units neuraminidase-treated red blood cells indicated that ZN decreased by 76% and 1/lambda decreased by 8% compared to intact red blood cells. In contrast, in 5 units of papain-treated red blood cells ZN decreased by 45% and 1/lambda decreased by 33% compared to intact red blood cells. The present study shows that the change in ZN for neuraminidase-treated cells was very large, but the cells did not become agglutinable. Papain-treated cells had changes in both ZN and 1/lambda, and the cells became agglutinable. 1/lambda is one of the important factors for agglutination.

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens.

Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.

Inhibition of Growth ofAspergillus flavusand Fungal α-Amylases by a Lectin-Like Protein fromLablab purpureus

Aspergillus flavus is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. Associated with the disease is the production of aflatoxins, which are a series of structurally related mycotoxins known to be carcinogenic. Previous research has suggested that the alpha-amylase of A. flavus promotes aflatoxin production in the endosperm of infected maize kernels. We report here the isolation and characterization of a 36-kDa alpha-amylase inhibitor from Lablab purpureus (AILP). AILP inhibited the alpha-amylases from several fungi but had little effect on those from animal and plant sources. The protein inhibited conidial germination and hyphal growth of A. flavus. The amino acid sequence indicated that AILP is similar to lectin members of a lectin-arcelin-alpha-amylase inhibitor family described in common bean and shown to be a component of plant resistance to insect pests. AILP also agglutinated papain-treated red blood cells from human and rabbit. These data indicate that AILP represents a novel variant in the lectin-arcelin-alpha-amylase inhibitor family of proteins having lectin-like and alpha-amylase inhibitory activity.

The Diego blood group system-an update

Abstract Anti-Jka was detected by solid-phase red cell adherence(SPRCA) anti-body detection and identification tests in the plasma of a 9-month-old female intant during a roatine presurgical evaluation. the pation and her nonidentical twin sister who also had anti-Jka in her plasma, were products at an uncomplicated in vitro fertilization, full -term preganancy, and vaginal delivery Neither twin had been tranfered recently infected. or treated with medication. their mother had no prior pregnancies or transfusions. Red blood cells (RBCs) from the patient and her sister typed as Jk(a-b+) by direct hemagghutination and this phenotype was confirmed by negative adsorption and elution studies. Both infants plasma samples were strongly reactive with 20 examples of Jk(a+) RBCs and nonreactive with 20 examples of Jk(a-) RBCs by SPRCA assays. Anti-Jka was not detected in either twins plasma by Indirect antiglobulin tests by tube method in lowionie-strength saline solution or polyethylene glycol, or with ficin- or papain-treated RBCs. Monocyte monolayer assays using Jk(a+) RBCs sensitized by either twins’ serum were nonreactive (0%). RBCs from both parents typed as Jk(a+b+). Both parents’ antibody detection test results by SPRCA assay were negative. The absence of a history of exposure to allogeneic RBCs or possible passive transfer of maternal or other alloantibody classifies these antibodies as natually-occurring anti-Jka. Immunohemalology 1999;15: 159-162.

Reactivity of 62 Rh MAbs with weak and variant antigens.

We characterized serologically 5 anti-C (4 IgM and 1 IgG), 3 anti-c (2 IgM and 1 IgG), 4 anti-E (1 IgM and 3 IgG), 4 anti-e (3 IgM and 1 IgG) and 46 anti-D (16 IgM and 30 IgG) monoclonal antibodies, provided by the Rh Section of the Third International Workshop and Symposium on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (1996) for their ability to detect weak and variant antigens. The agglutination patterns were established using untreated and papain-treated red blood cells in a column agglutination technology system (BioVue, Ortho). Significant differences were found between the IgM and IgG antibodies. The papain treatment seemed to be important for IgM but not for IgG antibodies. Almost all of the IgM anti-D antibodies detected untreated DIV samples and almost all of the IgG anti-D antibodies detected untreated weak D samples. Both IgM and IgG anti-D antibodies showed the highest number of negative reactions with DVI and Rh 33 red blood cells. The CwCw sample was detected by only one of the 4 anti-C IgM MAbs using enzyme-treated red blood cells. All anti-c MAbs were able to detect treated Cx samples. Because of the small number of weakly expressed E and e samples, definitive conclusions cannot be drawn on the ability of these antibodies to detect these antigens.

Misidentification of anti-Vel due to inappropriate use of prewarming and adsorption techniques

Abstract Two units of red blood cells (RBCs) were ordered for a 44-year-old Caucasian woman with renal failure and cancer. Pretransfusion testing performed at the regional reference laboratory had revealed the presence of an antibody reactive with all cells at the indirect antiglobulin test (LAT) but apparently nonreactive by a prewarmed IAT. The patient’s RBCs were direct antiglobulin test negative. Adsorption of the serum with rabbit erythrocyte stroma or with allogeneic RBCs at 4°C reduced the reactivity at the IAT. The patient was transfused with two units of washed RBCs and died 6 to 8 hours later. Retrospective testing in our laboratory detected anti-Vel in both pretransfusion and posttransfusion samples. The pretransfnsio serum was hemolytic when tested in LISS or with papain-treated RBCs. Weak reactivity (1+) was observed at the IAT. EDTA-treated serum (to prevent C’-mediated hemolysis) was strongly reactive (3+s) with Vel+ RBCs but compatible with 10 examples of Vel-RBCs. Adsorptions with rabbit RBCs did not affect reactivity. Pretransfusion RBCs were nonreactive with three examples of anti-Vel at the IAT. The posttransfusion serum was grossly hemolyzed and anti-Vel was demonstrable, although weaker than in the pretransfusion sample. The antibody was subclassed as IgG1 and IgG3. In this case, the use of a prewarming technique precluded detection of hemolysis caused by the antibody prior to the IAT. This case stresses the importance of antibody identification and the correct use of such techniques as prewarming and adsorptions. Immunohematology 1994;10:83-86.

First example of Rh:–32,–46 red cell phenotype

Abstract The red cells of a white male blood donor typed as Rh:–1, –2, –3,w4,w5,6,–17,w19,–31,–32,–34, and –46. Although the donor has no history of transfusion, his serum contains an alloantibody that is weakly reactive with most red blood cells (RBCs) tested. Only Rhnull and D–– RBCs are nonreactive. Reactivity is enhanced with ficin- or papain-treated RBCs and is unaffected by AET or DTT treatment of the RBCs. Previously described Rh:–46 RBCs have been of deletion types D––, D••, and Rhnull or Rh:32. In three multitransfused patients, the Rh46 antigen was temporarily suppressed and the phenotype eventually reverted to normal. This is the first report of RBCs of the Rh:–32,–46 phenotype that are not of a rare Rh deletion or Rhnull type. In addition, the Rh:w5,w19,–31,–34 phenotype is rarely found in whites. Immunohematology 1994; 10:130–133.

Recherche des anticorps irréguliers érythrocytaires par la méthode du gel-test. Analyse de 35 882 échantillons

The results of screening for irregular blood-group antibodies in 35,882 blood-specimens using the gel-test method were analysed. The samples came from different clinics and hospital units. 1,179 samples from 728 different individuals (3.2%) contained one or several irregular antibodies. The gel low ionic strength antiglobulin test detected Kell, Duffy, and Ss antibodies with a normal frequency compared to bibliographic data, whereas Kidd antibodies seemed to be abnormally infrequent. The best detection of Rhesus antibodies was obtained with the gel enzyme test (papain). The prevalence of cold, naturally occurring, anti-Lewis and anti-P1 antibodies was low. The gel enzyme test with papain-treated red blood cells seems to be necessary for detecting Lewis antibodies (only 10 out of 95 were detected with the gel low ionic strength antiglobulin test). As a counterpart, the gel enzyme test also detected many antibodies with doubtful clinical significance: anti-E and anti-Cw reacting only with the gel enzyme test, autoantibodies with Rhesus specificity (82 examples), antibodies mimicking Jka (4 examples) and S (1 example) specificities in individuals with the corresponding antigen.

Cryptic cold agglutinin activity of monoclonal macroglobulins

One hundred and twenty sera with monoclonal immunoglobulin M's (IgM's) were tested for cryptic cold agglutinin activity against papain- and neuraminidase-treated adult and cord red blood cells (RBC). Fifteen sera (12.5%) agglutinated papain-treated RBC. Fourteen of these 15 sera also agglutinated neuraminidase-treated RBC. Ten additional sera agglutinated neuraminidase-treated but not papain-treated cells. Three patterns were observed among agglutinators of papain-treated RBC: either predominant agglutination of adult RBC, or of cord RBC or equal reaction, thus resembling I, i and non-Ii patterns of conventional cold agglutinins. We propose to call these patterns, tentatively, Icr (cr for cryptic), icr, and non-Iicr, respectively. Purified monoclonal IgM's retained the above patterns. Eluates from enzyme-treated cells contained monoclonal IgM's. Investigation of neuraminidase-treated RBC has shown that in distinction to T-anti-T system (Thomsen-Friedenreich receptor system), the majority of sera with monoclonal IgM's agglutinated more strongly adult RBC, and some agglutinated more strongly cord RBC. There was no loss of agglutinating activity after repeat freezing and thawing of the sera. Thus, it seems that monoclonal IgM's which react with neuraminidase-treated RBC detect antigenic determinants different from the T antigen. Cryptic CA activity of IgM's should be added to the list of antibody activities of these monoclonal immunoglobulins.