Metabolomic profile in patients with primary warm autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is a rare clinical condition with immunoglobulin fixation on the surface of erythrocytes, with or without complement activation. The pathophysiology of AIHA is complex and multifactorial, presenting functional abnormalities of T and B lymphocytes that generate an imbalance between lymphocyte activation, immunotolerance and cytokine production that culminates in autoimmune haemolysis. In AIHA, further laboratory data are needed to predict relapse and refractoriness of therapy, and thus, prevent adverse side-effects and treatment-induced toxicity. The metabolomic profile of AIHA has not yet been described. Our group developed a cross-sectional study with follow-up to assess the metabolomic profile in these patients, as well as to compare the metabolites found depending on the activity and intensity of haemolysis. We analysed the plasma of 26 patients with primary warm AIHA compared to 150 healthy individuals by mass spectrometry. Of the 95 metabolites found in the patients with AIHA, four acylcarnitines, two phosphatidylcholines (PC), asymmetric dimethylarginine (ADMA) and three sphingomyelins were significantly increased. There was an increase in PC, spermine and spermidine in the AIHA group with haemolytic activity. The PC ae 34:3/PC ae 40:2 ratio, seen only in the 12-month relapse group, was a predictor of relapse with 81% specificity and 100% sensitivity. Increased sphingomyelin, ADMA, PC and polyamines in patients with warm AIHA can interfere in autoantigen and autoimmune recognition mechanisms in a number of ways (deficient action of regulatory T lymphocytes on erythrocyte recognition as self, negative regulation of macrophage nuclear factor kappa beta activity, perpetuation of effector T lymphocyte and antibody production against erythrocyte antigens). The presence of PC ae 34:3/PC ae 40:2 ratio as a relapse predictor can help in identifying cases that require more frequent follow-up or early second-line therapies.

Serologic and molecular studies to identify neonatal alloimmune neutropenia in a cohort of 10,000 neonates.

Neonatal alloimmune neutropenia (NAIN) is caused by maternal alloimmunisation to fetal human neutrophil antigens (HNAs). This study investigated maternal HNA/HLA alloantibodies involved with NAIN and identified the frequency of NAIN in Brazilian neonates. Neonatal neutropenia (neutrophil count < 1.5 × 109 /L) was investigated in samples from 10,000 unselected neonates, resulting in 88 neutropenic newborns (NBs) and their 83 mothers. Genotyping was performed by PCR-SSP (HNA-1/-4) and PCR-RFLP (HNA-3/-5). Serologic studies were performed by GAT (granulocyte agglutination test), Flow-WIFT (white blood cells immunofluorescence test) and LABScreen-Multi-HNA-Kit (OneLambda®) (LSM). Neonatal neutropenia was identified in 88/10,000 (0·9%) NBs. Genotyping revealed 60·2% maternal-fetal HNA incompatibilities (31·8% for HNA-1; 14·8% for HNA-3; 15·9% for HNA-4; 21·6% for HNA-5). Serologic studies revealed 37·3% of mothers with positive results with at least one technique. The detected anti-HNA specificities were confirmed in eight positive cases related to HNA-1/-3 systems. In cases with maternal-fetal HNA-4/-5 incompatibility, no specific neutrophil alloantibodies were found but anti-HLA I/II were present. Anti-HNA-2 was not identified. This is a large Brazilian study which involved the investigation of antibodies against all five HNA systems in neutropenia cases and showed a frequency of NAIN in 8/10,000 neonates. Among the HNA antibodies identified, we highlight the anti-HNA-1d and anti-HNA-3b, antibodies unusual in alloimmunised women, and rarely related to NAIN cases.

Antibodies against human neutrophil antigens in non-transfused women with red blood cell alloimmunisation induced by pregnancy.

BACKGROUND: Alloantibodies against human neutrophil antigens (HNA) resulting from allogeneic exposure may be associated with transfusion-related acute lung injury and immune neutropenia. Understanding the risk factors for the formation of such antibodies could have a great impact on the adoption of measures to prevent potentially fatal transfusion reactions. The aim of the study was to determine the prevalence of anti-HNA alloantibodies in non-transfused pregnant women with and without red blood cell (RBC) alloantibodies. MATERIALS AND METHODS: HNA alloantibodies were investigated in blood samples from 147 pregnant women with RBC alloimmunisation induced by pregnancy as the only allogeneic stimulus (group 1). The control group (group 2) consisted of 563 women with at least one pregnancy without RBC alloimmunisation. Both groups were investigated for the presence and identity of HNA alloantibodies using granulocyte agglutination tests, white blood cell immunofluorescence testing, and the bead-based LABScreen Multi Kit. Genotyping was performed to confirm the specificity of the HNA alloantibodies. RESULTS: Group 1 women had a statistically higher number of HNA alloantibodies compared to group 2 women (9/147 [6.1%] vs 9/563 [1.6%]; p=0.005, OR=4.01; 95% CI 1.5-10.3). Considering only multiparous women, there was a higher statistical significance for the difference in the presence of HNA alloantibodies between the two groups (7/82 [8.5%] vs 9/493 [1.8%]; p=0.002, OR=5.02; 95% CI 1.8-13.9). DISCUSSION: Our data show that RBC alloimmunisation is significantly associated with the development of anti-HNA alloantibodies, corroborating the hypothesis that some individuals are better immune responders and react strongly to allogeneic exposure. The presence of RBC alloantibodies can, therefore, facilitate the identification of individuals with a higher risk of alloimmunisation to antigens from other cells, also acting as a tool to avoid potentially fatal transfusion reactions.

RHD and RHCE molecular analysis in weak D blood donors and in patients with Rh antibodies against their own corresponding Rh antigen.

BACKGROUND: The Rh system is the largest and most polymorphic blood group system. The existence of a large number of RH alleles results in variant phenotypes that often complicate blood donor phenotyping and the distinction between auto- and allo-antibodies in recipients who have anti-Rh antibodies in the presence of their own corresponding Rh antigen. Knowledge of these variants is necessary in order to make blood transfusion safer. MATERIALS AND METHODS: Samples from 48 blood donors with serological weak D and from 29 patients who had anti-Rh antibody in the presence of their own corresponding Rh antigen were evaluated molecularly for RHD and RHCE alleles using a blood-multiplex ligation-dependent probe amplification assay and Sanger sequencing. RESULTS: Rh variants were found in 45 of the 48 blood donors: 24/45 (53%) were weak D, 2/45 (4%) partial D and 19/45 (42%) were weak and partial D. The remaining three donors (6%) did not show a mutation in the RHD allele. Among the 29 patients, 13/29 had anti-e, of whom 4/13 had genotypes that predicted a partial e antigen; 11/29 had anti-D, with 6/11 being identified as partial D; 2/29 had anti-c, of whom 1/2 was predicted to express partial c antigen; 4/29 who had anti-E and 4/29 who had anti-C did not show mutations in RHCE*C or RHCE*E. DISCUSSION: It was possible to find individuals with clinically significant Rh phenotypes due to the weak reactivity of the D antigen, detected through serological tests in blood donors. In patients, when found with the anti-Rh antibody in the presence of the same Rh antigen, it is difficult to distinguish an auto-antibody from an allo-antibody by serological tests; in these cases, molecular methods (genotyping) can help us to determine whether there are changes in the RH alleles and to discover the nature of the antibody (allo or auto).

Study on the Change in Powder Properties of Rice Flour by Different Milling Processes.

The aim of this study was to clarify the change in the powder properties of rice flour depending on the milling process. Rice flour samples, which have gradual mechanical shock properties, were prepared using different milling methods. Furthermore, the correlation between the starch damage, owing to mechanical shock, and powder properties of rice flour was investigated. The particle size was changed gradually through each milling process; however, the change did not clearly correlate with starch damage. The results of the X-ray diffraction (XRD) pattern of nongelatinized samples showed the typical A-type structure of starch. The crystal structure of starch in rice flour may change to a disorder state with the progress of milling; thus, in this study, instead of crystallinity, we considered the disorder index (DI) calculated from the XRD intensity of samples. Relationship between DI and starch damage was confirmed with R 2 = 0.923. Therefore, the mechanical shock caused by the milling process contributes to the crystal state of starch. The parameter q m calculated from the Guggenheim-Anderson-de Boer (GAB) equation of each sample corresponded to the DI. This result suggested that the sorption site of rice flour decreased, and a positive correlation was observed between the parameter K and DI. Thus, the interaction between the rice flour and water molecules weakened because of the mechanical shock. In addition, the use of a SEM image supports the insight that the change in parameter K may reflect the structural change in the solid phase. These results demonstrated that the change in powder properties of rice flour caused by mechanical shock of the milling could evaluate quantitatively.

RHD gene polymorphisms in alloimmunized RhD-negative individuals with high rate of racial admixture.

BACKGROUND: The D-negative phenotype is the result of the total RHD gene deletion in almost all Caucasians, but it accounts for only about 20% in Africans and 70% in Asians. In Africans the RHDΨ that is one of the most important causes of the D-negative phenotype. We investigated the RHD polymorphisms in D-negative phenotype mixed Brazilians who have anti-D alloantibody. STUDY DESIGN AND METHODS: Blood samples from 130 individuals previously typed as D-negative were phenotyped again using: (a) two tube reagents (Anti-D blend reagent, Cellular line TH-28, MS-26; and Anti-D polyclonal); (b) one gel test ID-Card for Rh subgroups including C(w) and K antigen; and (c) ABO/Rh (Anti-D blend reagent, Cellular line 175-2, LDM3). The method used for RHD screening detected the presence of RHD exon 10 and intron 4. Sequence analysis was performed on PCR products amplified from genomic DNA for all 10 exons RHD gene. RESULTS: We found that 118/130 (90.8%) of D-negative tested individuals had total RHD gene deletion, while 12/130 (9.2%) showed RHD gene polymorphisms. The RHDΨ was found in 10 (7.7%) individuals, one sample (0.77%) hybrid RHD-CE-D(s) /RHDΨ, and another (0.77%) weak D type 4.2. CONCLUSIONS: The results showed that the RHD gene was present in 9.2% of racially mixed Brazilians who produced usually clinically significant anti-D alloantibodies. Therefore, the data showed that careful attention is necessary for clinicians in applying RhD genotyping to transfusion medicine in populations with high rate of racial admixture.

FCGR3B*03 allele inheritance pattern in Brazilian families and some new variants of gene FCGR3B.

BACKGROUND: The FCGR3B gene encoding the FcyRIIIb receptor for immunoglobulin G has three polymorphic forms known as HNA-1a, HNA-1b, and HNA-1c, encoded by the alleles FCGR3B*01, FCGR3B*02, and FCGR3B*03, respectively. It is not clear whether the inheritance of the FCGR3B*03 allele, which encodes the HNA-1c, is linked or not to the other two alleles. The objective of this study was to identify the inheritance pattern of the FCGR3B*03 allele in Brazilians. STUDY DESIGN AND METHODS: Blood samples from nine families with at least one FCGR3B*03(+) member, totalizing 47 individuals, were studied. The presence of the FCGR3B*01, FCGR3B*02, and FCGR3B*03 alleles was detected by the polymerase chain reaction with sequence-specific priming method, and all DNA samples were sequenced. RESULTS: In three of the nine studied families, the FCGR3B*03 was passed down with the FCGR3B*02, while in one family the FCGR3B*03 was inherited in linkage with FCGR3B*01. The other families were not informative regarding FCGR3B*03 inheritance. Sequencing showed for the first time one single-nucleotide polymorphism at Position 264 resulting from a simple substitution C→T; three other different substitutions at Position 230, T→A, T→G; and the presence of three nucleotides at Position 230 (T, G, and A). The previously reported variants FCGR3B*01A227G and FCGR3BG330T were also found. CONCLUSION: In this Brazilian FCGR3B*03(+) group we found that the inheritance of FCGR3B*03 took place by a linkage to FCGR3B*02 or to FCGR3B*01. Linkage of FCGR3B*03 to FCGR3B*02 was the most common. Additionally, we report SNPs that have not been described, suggesting that they might be more common than previously thought.

Hemolytic anemia after kidney transplantation: a prospective analysis.

BACKGROUND: Hemolysis may occur in 9% to 40% of patients after solid organ transplantation and be caused by the passenger lymphocyte syndrome (PLS). STUDY DESIGN AND METHODS: We have prospectively examined 217 kidney transplant recipients before (Day -1) and after (up to Days +10, +20, and +30) surgery. ABO-identical transplant was performed in 180 (82.9%) patients, while 37 (17.1%) individuals received ABO-compatible nonidentical grafts. Direct antiglobulin tests (DATs) were performed by tube technique (polyspecific anti-human globulin [IgG + C3d]), positive DAT samples were further tested by gel agglutination (monospecific anti-IgG, -IgM, -IgA, or -C3), and eluates were prepared from DAT-positive red blood cells (RBCs) by the dichloromethane elution test. RESULTS: We observed that 34 of 217 (15.7%) patients developed a positive DAT up to Day +30. The percentage of patients with positive DATs was significantly higher in those having ABO-compatible nonidentical transplants compared to those that received ABO-identical grafts (10/37 = 27.0% vs. 24/180 = 13.3%; p = 0.037). Specific RBC antibodies (anti-A or anti-B) were found in only 5 of 37 (13.5%) patients having ABO-compatible nonidentical transplants who presented with clinical hemolysis. We found only three reactive eluates from 24 patients with positive DATs who received ABO-identical transplants but had no hemolysis. CONCLUSIONS: Our data collected prospectively demonstrated that: 1) positive DATs occurred in 15.7% of all patients up to Day +30 after a kidney transplant; 2) the DAT positivity occurred up to Day +10 in 9.7% of all transplanted patients; 3) the majority of the transplant recipients with a positive DAT had a nonreactive RBC eluate; and 4) PLS was the cause of a positive DAT in 13.5% of patients submitted to ABO-compatible nonidentical kidney transplants.

Impact of using different laboratory assays to detect human leukocyte antigen antibodies in female blood donors.

BACKGROUND: HLA antibodies passively transferred to transfused recipients may cause transfusion reactions such as transfusion-related acute lung injury (TRALI), but in many of the reported TRALI incidents, no white blood cell antibodies have been identified. We investigated whether a higher number of anti-HLA would be detected in donor's plasma by using a method with potential higher sensitivity rate. STUDY DESIGN AND METHODS: Sera from 300 previously pregnant female blood donors were screened for anti-HLA using a solid-phase mixed-antigen assay (enzyme-linked immunosorbent assay [ELISA]). Samples from 60 women with three or more pregnancies with a negative ELISA were further tested using microbead-flow assays (LABScreen mixed, panel-reactive antibodies [PRA], and single antigen). RESULTS: Anti-HLA Class I and/or Class II were detected by ELISA in 26.7% (80/300) of all women and in 37.0% (37/100) of women with three or more pregnancies. The LABScreen assays detected additional anti-HLA specificities (44 Class I and 17 Class II) in 28.3% (17/60) of ELISA-negative donors with three or more pregnancies. HLA antibodies were detected in 8.3% (5/60), 18.3% (11/60), and 21.7% (13/60) of ELISA-negative women by LABScreen mixed, PRA, or single antigen, respectively. CONCLUSION: Our data showed that the microbead-flow detected more HLA antibodies than ELISA, but the clinical significance of these antibodies is currently unknown. Detecting anti-HLA is useful for donor management and could contribute to the decision to definitively defer blood donors involved in TRALI incidents. However, further studies are necessary to better determinate the relative risk of TRALI induced by anti-HLA detected only by techniques with higher sensitivity rate.

Human neutrophil alloantigens systems.

Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcgamma receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the alphaM (CD11b) and alphaL (CD11a) subunits of the leucocyte adhesion molecules (beta2 integrins). Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.

Human neutrophil alloantigens systems

Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcγ receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the αM (CD11b) and αL (CD11a) subunits of the leucocyte adhesion molecules (β2 integrins). Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.

Os aloantígenos de neutrófilos estão associados a várias condições clínicas como neutropenias imunes, insuficiência pulmonar relacionada à transfusão (TRALI), refratariedade à transfusão de granulócitos, e reações transfusionais febris. Na última década, foi observado considerável progresso na caracterização dos aloantígenos envolvidos nestas condições clínicas. Atualmente sete antígenos estão incluídos em cinco sistemas de antígenos de neutrófilo humano (HNA). Os antígenos HNA-1a, HNA-1b e HNA-1c foram identificados como formas polimórficas do receptor Fcγ RIIIb (CD16b), codificados por três alelos. Recentemente, a estrutura primária do antígeno HNA-2a foi elucidada e a glicoproteína carreadora do antígeno foi identificada como um membro da superfamília Ly-6/uPARe designada como CD177. O antígeno HNA-3a está localizadoem uma glicoproteína de 70-90 kDa, entretanto sua base molecular ainda é desconhecida. Finalmente, os antígenos HNA-4ae HNA-5a são resultantes de mutações de um único nucleotídeo nas subunidades αM (CD11b) and αL (CD11a) das moléculas de adesão de leucócitos (β2 integrinas). A caracterização molecular e bioquímica dos antígenos neutrofílicos permitiu a expansão das ferramentas diagnósticas pela introdução de técnicas de genotipagem e imunoensaios para a identificação de anticorpos. Novos estudos envolvendo a imunologia de granulócitos serão de grande valor para a prevenção e tratamento de reações transfusionais e doenças imunes causadas por aloanticorpos de neutrófilos.

Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy.

The movement of cellular water accompanies changes in growth within dormant buds. To further understand this process, accumulation of tonoplast deltaTIP1 and plasma membrane PIP2 aquaporin transcripts was measured by quantitative reverse transcriptase-polymerase chain reaction and the water dynamics in dormant peach (Prunus persica L.) flower buds was studied by magnetic resonance imaging. Proton density (PD), spin-spin relaxation time (T(2)) and apparent diffusion coefficient (ADC) were used to observe water dynamics during dormancy. The expression of deltaTIP1 and PIP2 aquaporins, PD and T(2) in the upper part of the bud including primordia, in the basal part of the bud and the bud trace increased earlier in the low-chill cultivar 'Coral' than in the high-chill cultivar 'Kansuke Hakuto,' reflecting the difference in timing for the end of endodormancy in the two cultivars. deltaTIP1 mRNA accumulated mainly in the basal part of the bud, whereas PIP2 mRNA was detected mainly in the upper part. These findings may reflect the activation of inter- and intracell communication through membrane transport properties of aquaporins resulting in a gradual increase in water content to that required for bud activity at the end of endodormancy. An apparent decrease in the expression of deltaTIP1 and PIP2 mRNAs was, however, observed in late winter in some portions of the buds of both cultivars just before sprouting.

Anti-N-like and anti-Form red cell antibodies in chronic hemodialysis patients.

Allogeneic red blood cell (RBC) transfusions and the use of reusable dialyzers sterilized with formaldehyde can lead to RBC alloimmunization in chronic hemodialysis patients. The formed RBC alloantibodies have been implicated in immediate kidney allograft failure and decreased RBC survival observed in these patients. Using indirect antiglobulin test, direct antiglobulin test (DAT), and direct Polibrene test (DPT), we detected an RBC alloimmunization rate of 17.2% (11/64) in transfused hemodialysis patients, and found the presence of anti-N-like and anti-Form antibodies in 5 (5.7%) and 53 (60.9%) individuals, respectively. The sensitivity rate of the DPT was significantly higher than that of the DAT in detecting anti-Form, but the DAT showed a higher specificity rate compared with the DPT. We conclude that patients treated with reusable dialyzers sterilized with formaldehyde may develop specific RBC alloantibodies that could increase the potential risk of hemolysis, decrease survival of RBCs, and increase the need of blood supply.

Application of noninvasive phagocytic cellular assays using autologous monocytes to assess red cell alloantibodies in sickle cell patients.

We investigated red cell (RBC) alloantibodies in 125 sickle cell anemia (SCA) patients using tube indirect antiglobulin test (PEG, LISS or enzyme) and gel centrifugation test (LISS or enzyme). Prediction of clinical significance of alloantibodies was evaluated by the monocyte monolayer assay (MMA) and the chemiluminescence test (CLT) using autologous monocytes. The alloimmunization rate was 20.8% and the gel test detected a higher number of alloantibodies than the tube test (26 v 21, p = 0.02). We observed 58.3% and 69.2% positive MMA and CLT results, respectively. Eighteen (69.2%) antibodies exhibited clinical relevance, 14 (58.3%) antibodies reacted by both MMA and CLT, while 4 (15.4%) antibodies reacted only by CLT. In conclusion, the application of phagocytic cellular assays using autologous monocytes defined clinical significance of about 70% of RBC alloantibodies detected in SCA patients. The data also suggest that the CLT may be more valuable than the MMA as a noninvasive test for predicting hemolysis after transfusion of incompatible blood in SCA patients.

RBC-associated IgG in patients with visceral leishmaniasis (kala-azar): a prospective analysis.

BACKGROUND: Despite the fact that anemia is one of the most striking clinical features of visceral leishmaniasis (kala-azar), the factors involved in its pathogenesis are not fully understood. Although the cause of the anemia seen in these patients is often multifactorial, sequestration and destruction of the RBCs in the enlarged spleen, immune mechanisms, and alterations in RBC membrane permeability have been implicated. STUDY DESIGN AND METHODS: To investigate whether RBCs of patients with kala-azar were coated with IgG, blood samples of 67 patients were tested, prospectively, before (Day 1), during (Day 30), and after (Day 90) antimonial therapy, to examine the presence of RBC-associated IgG, circulating immune complexes (CICs), and rheumatoid factor (RF). RESULTS: The prevalence of a positive DAT on Day 1 was significantly greater than the prevalence of a positive DAT performed on Day 30 and on Day 90 (32.8 vs. 4 vs. 0%, p < 0.001). With an enzyme-linked antiglobulin test (ELAT) to measure the number of IgG molecules per RBC more accurately, it was found that the amount of IgG molecules per RBC was increased (mean, 298 molecules of IgG per RBC) in the group of patients with kala-azar tested before antimonial therapy, but was considered normal (<50 molecules of IgG per RBC) in all patients tested 90 days after treatment. The prevalence of a positive eluate test was low (15.0%) in DAT (anti-IgG)-positive patients and the positivity of DATs and ELATs correlated with the presence of either RF or CICs, respectively. CONCLUSIONS: These data suggest that a nonspecific adsorption of CICs on the RBC surface is probably the most important factor involved in the increased amount of RBC-associated IgG in patients with untreated visceral leishmaniasis; however, further prospective studies are required to establish the exact role of the RBC-associated antibodies, CICs, and RF as contributing factors of the anemia seen in these patients.