Cytomegalovirus infections in seropositive patients after transfusion. The effect of red cell storage and volume

Antibody responses to cytomegalovirus (CMV) after red cell (RBC) transfusion were studied in 84 seropositive surgery patients and 82 seropositive oncology patients. The surgery patients were randomized to receive RBCs stored either 3 to 8 or 20 to 42 days after donation. Of 38 patients receiving RBCs stored 8 days or less, 3 developed a rise in titer (4-fold increase) of IgG antibody to CMV 8 to 12 weeks after transfusion. This rate of response (8%) did not differ significantly (p = 0.23) from that (16%) in the 46 patients receiving RBCs stored 20 to 42 days. Seropositive oncology patients were randomized to receive RBCs from seronegative or random donors. Five (19%) of 27 oncology patients receiving seronegative RBCs and 13 (23%) of 55 patients receiving random RBCs (mean, 2 seropositive RBC units/patient) developed a rise in titer of antibody to CMV. No CMV morbidity occurred in either patient group. For both patient groups, a rise in titer of antibody to CMV was associated with the number of transfused RBC units. These results confirm that CMV-seronegative RBCs are unnecessary for infrequently transfused seropositive patients. They also suggest that multiple transfusions of stored RBCs are as immunosuppressive as multiple transfusions of RBCs used within a few days after donation.     

Class switching is differentially regulated in RBC alloimmunization and vaccination

Background

Studies of human patients have shown that most anti-RBC alloantibodies are IgG1 or IgG3 subclasses, although it is unclear why transfused RBCs preferentially drive these subclasses over others. Though mouse models allow for the mechanistic exploration of class-switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination, and determined the role of STAT6 in their generation.

Study Design and Methods

WT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA.

Results

When compared with antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b, and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination.

Discussion

Our results show that anti-RBC class-switching occurs via alternate mechanisms when compared with the well-studied immunogen alum vaccination.     

Prevention of primary cytomegalovirus infection in patients with hematologic malignancies by intensive white cell depletion of blood products

The effect of white cell depletion of red cells and platelet concentrates on the transmission of cytomegalovirus (CMV) was studied retrospectively in 150 patients treated intensively for acute leukemia or non-Hodgkin's lymphoma. CMV infection was diagnosed on the basis of IgM and IgG antibody responses to CMV late antigen (CMV-LA). Before cytoreductive therapy for their underlying disease, 59 patients were CMV seronegative and 91 were CMV seropositive. None of the 59 CMV-seronegative patients showed persistent seroconversion 2 months after the cytoreductive treatment. The comparison group, consisting of 312 cardiac surgery patients, showed a significantly higher incidence of primary CMV infections: 10 of 86 (11.6%, p = 0.004). Twenty-five percent of the CMV-seronegative patients and controls had transient IgG antibodies to CMV-LA without IgM antibodies, which is indicative of antibodies passively acquired via blood products. These results indicate that white cell-poor blood products carry a very low risk, if any, of CMV transmission. The policy of transfusing white cell-poor blood products provides a useful alternative to the selection of CMV-seronegative donors.     

Quantitation of IgG subclass antibodies to pneumococcal capsular polysaccharides by ELISA, using Pneumovax-specific antibodies as a reference

A quantitative enzyme-linked immunosorbent assay (ELISA) method has been developed to assay the levels of IgG subclasses to pneumococcal capsular polysaccharides (PCP) by using a reference standard. This standard solution containing specific antibodies to a polyvalent pneumococcal vaccine (Pneumovax) was purified from the serum of an immunized healthy adult by affinity chromatography. In order to determine the predominant response to Pneumovax in the four IgG subclasses, specific IgG subclasses in preimmune and postimmune sera from six healthy adults were assessed quantitatively by the ELISA. With regard to peak concentrations after immunization, there was a marked increase in the IgG2 subclass, compared with those of IgG1 and IgG3. Such a quantitative assay of Pneumovax-specific IgG subclass antibodies is useful for the direct evaluation of immune responses to immunization with a polyvalent pneumococcal vaccine, and at the same time, for estimating the IgG2 response to PCP antigens in individuals.     

IgG subclass responses to Bordetella pertussis filamentous haemagglutinin and pertussis toxin in whooping cough

The IgG subclass response pattern to two major protective antigens of Bordetella pertussis—filamentous haemagglutinin (FHA) and pertussis toxin (PT)—was measured by enzyme-linked immunosorbent assay (ELISA) in paired sera from 46 patients with culture-confirmed whooping cough. Variations in specific subclasses related to antigen and to previous immunization status were noted. The dominant response to both antigens was of the IgG1 subclass and closely followed the total IgG response for both unimmunized children (n = 18) and previously DTP-immunized children and adults (n = 28). Neither IgG2 nor IgG4 to FHA were seen in the unimmunized children but were found in immunized children and adults. An IgG2 response to PT was noted in 70–80% of all patients irrespective of previous immunization status. An observed correlation between IgG2 and neutralizing antibodies to PT was further investigated. It was found that 10 patients without neutralizing antibodies also lacked IgG2 to PT. The study thus revealed differences in IgG subclass responses to two bacterial protein antigens, with an indication that the IgG2 response to PT reflects or correlates to the development of neutralizing antibodies to this toxin. The finding could explain discrepancies between ELISAs and neutralization tests for antibodies to bacterial toxins and have implications for vaccine research.     

Post-transfusion purpura: a survey of 12 Danish cases with special reference to immunoglobulin G subclasses of the platelet antibodies

SUMMARY. The clinical and immunological data of 12 Danish cases of post-transfusion purpura (PTP) are summarized. All patients but one were women. All except one had thrombocytopenic purpura which occurred 4–11 days after transfusion, usually with a nadir of the platelet count below 10times10⁹ 1^-1. The typical haemorrhagic symptoms were cutaneous bleedings, melena and haematuria lasting from 3 to 12 days. The time until normalization of platelet count varied from 3 to 50 days after onset. One patient had recurrence of PTP and one patient died due to intracranial haemorrhage.
Ten of the HPA-la negative patients (83%) had platelet-specific HPA-la antibodies and two HPA-lapositive individuals had anti-HPA-lbantibodies. In 10 patients, HLA antibodies were also detectable and in one patient a delayed haemolytic transfusion reaction due to anti-E was seen.
All of seven patients investigated had anti-HPA antibodies of both IgGl and IgG3 subclasses during the thrombocytopenic period while all of 10 patients had anti-HPA of only the IgGl subclass after recovery from PTP Thus, the destruction of autologous platelets in PTP seems to be associated with the presence of anti-HPA of the IgG3 subclass which may be of importance in the pathogenesis of PTP.     

The impact of blood transfusion on the occurrence of pneumonitis in primary cytomegalovirus infection after liver transplantation

The influence of blood transfusion, and particularly the transfusion of blood from cytomegalovirus (CMV)-seropositive donors, on the incidence of primary CMV infection following liver transplantation was studied prospectively in 29 consecutive CMV-seronegative adult liver transplant recipients. Fourteen patients received a liver graft from a CMV- seropositive donor (Group A), whereas for 15 patients, the donor was CMV seronegative (Group B). Eleven patients (79%) in Group A but only two (13%) in Group B developed CMV infection (odds ratio, 23.8; 95% confidence interval, 3.5-169.4). In five Group A patients, primary CMV infection resulted in pneumonitis. There was no statistical difference in the total number of blood units and the number of units of CMV- seropositive blood given to patients who did or did not develop CMV infection in both Groups A and B (odds ratio for unit of CMV- seropositive blood transfused, 1.07; 95% confidence interval, 0.96- 1.19). However, Group A patients who developed CMV pneumonitis received a higher total number of blood units (median, 44) and of CMV- seropositive blood units (median, 18) than did patients who developed other CMV infections (asymptomatic CMV, CMV syndrome, or CMV hepatitis), who received a median of 9 total units of blood and 5 units of CMV-seropositive blood (p = 0.004). It is concluded that the total number of blood units and of CMV-seropositive blood units transfused does not have an effect on the incidence of primary CMV infection after liver transplantation, but it does have an impact on the severity of the infection in recipients of a CMV-seropositive allograft.     

Absence of activation of CMV by blood transfusion to HIV-infected, CMV-seropositive patients

BACKGROUND: The Viral Activation Transfusion Study (VATS) afforded an opportunity to determine whether blood transfusions, and in particular exogenous WBCs, "activate" CMV replication in HIV-infected, CMV-seropositive patients, and whether such patients can be superinfected by additional strains of CMV transmitted via blood transfusions. STUDY DESIGN AND METHODS: A total of 531 patients were randomized to receive either WBC-reduced (WBCR) or non-WBC-reduced (NWBCR) RBC units. Plasma CMV PCR assays were performed before transfusion and weekly after transfusion for 4 weeks. NWBCR cases with evidence of possible reactivation and/or superinfection were further studied for donor viremia by DNA PCR of frozen retention segments and new genotype acquisition using gB envelope sequence analysis of pre- and posttransfusion recipient specimens. RESULTS: VATS patients received a median of two RBC units during their initial transfusion. Whether positive or negative for CMV DNA at baseline, there were no significant treatment-arm differences in the percentage of patients who had positive qualitative CMV PCR or increases in CMV viral load at follow-up. Of 50 recipients randomized to NWBCR RBC and meeting criteria for possible CMV superinfection, 25 had sufficient CMV DNA load in a baseline and one or more viremic follow-up sample to permit comparison of gB genotypes. Only two recipients showed genotype shifts. Of 125 WBC pellets prepared from the seropositive units transfused into these 50 cases, only 1 tested weakly PCR positive for CMV DNA (insufficient copy number for genotyping). CONCLUSION: There was no evidence of "activation" of CMV by blood transfusion. Among the NWBCR RBC recipients, there was little evidence of possible transmission of new CMV strains. Hence, the current policy for transfusion support of HIV-infected patients, which allows transfusion of CMV-antibody-positive blood to CMV-seropositive patients, is appropriate.     

Diagnostic value of anti-arc 5 IgG antibody and analysis of the IgG subclasses in sera of patients with cystic hydatid disease

Serodiagnosis of cystic hydatid disease (CHD) due to the metacestode of Echinococcus granulosus depends on detecting antibodies specific to hydatid antigen, but cross-reactivity with other parasites is one of the major draw-backs. We used a commercially-available antigen that elicits the arc 5 in immunoelectrophoresis and analysed the immunoglobulin class and the IgG subclass response by an ELISA. We tested sera from patients with confirmed CHD, cystic mass/lesions (CML) of non-hydatid origin, cysticercosis and healthy controls. High levels of antibodies to the hydatid antigen in all three classes (IgG, IgM and IgA) were observed only in the CHD patients. Significantly, only IgG antibody levels were discriminative and of diagnostic value. Also discussed is the validity of reading the significant cut-off point in IgG-ELISA in relation to a clinically important group of patients such as those with a CML of non-hydatid origin rather than healthy controls. Analysis of the anti-arc 5 IgG subclass responses demonstrated high antibody responses in all subclasses among the hydatid patients, with IgG3 the most discriminatory. The significance of the elevation of all four subclasses and more specifically of IgG4 in CHD is discussed in relation to certain biological activities of these immunoglobulin molecules.     

IgG subclass distribution and relative functional affinity of thyroid microsomal antibodies in postpartum thyroiditis

An association between the development of postpartum hypothyroidism and high levels of IgG1 subclass microsomal (M) antibodies has been reported. Using an assay designed to detect reasonable levels of all the four IgG subclasses, we found no differences in the proportion of each IgG subclass in M antibodies of patients with postpartum hyperthyroidism or hypothyroidism compared with control postpartum patients with M antibodies but no thyroid dysfunction. However the total amount of M antibody of each IgG subclass was elevated above the controls in the patients with thyroid dysfunction. The relative functional affinity of M antibodies did not differ between controls and patients with hypothyroidism but declined 5 and 10-12 months after delivery compared to values at 2 months. These results do not support the suggestion that the amount of IgG1 subclass M antibodies particularly determines the course of postpartum thyroiditis. Rather, the total M antibody level, in all four subclasses, is associated with clinical outcome. Resolution of the disease, despite persisting M antibodies, may occur in part because the relative functional affinity of these antibodies declines after delivery.     

A lack of serologic evidence of transmission of Chlamydia pneumoniae by transfusion of buffy coat-depleted RBCs

BACKGROUND: Recent studies have shown that a high percentage of blood donors harbor Chlamydia pneumoniae DNA and antigens within their PBMNCs. The aim of the present study was to investigate whether recipients of RBC transfusions who were seronegative for C. pneumoniae before transfusion showed any evidence of seroconversion after transfusion. STUDY DESIGN AND METHODS: Patients who were possible recipients of RBC transfusion and negative in a screen test for IgG antibodies against C. pneumoniae at the time of blood group determination were candidates to be included in the study. The patients were contacted 3.0 to 3.5 months after the blood group determination, and those who accepted to participate agreed that another venous blood sample could be taken. RESULTS: Among the patients who participated, 53 had become recipients of RBC transfusion (transfused group), and 51 later did not receive any RBC transfusion (control group). No significant change was found in IgG titer against C. pneumoniae between the first and the second sample from the same patient, in either the transfused group or the control group. CONCLUSION: In our study, which was limited to 53 seronegative recipients of RBC units from seropositive donors, we found no serologic evidence that C. pneumoniae could be transmitted by RBC transfusion.     

Human IgG subclass measurements in the clinical laboratory

Complement activation, cell surface-receptor binding, blocking activity, and possibly placental transfer are among the biologically important functional differences that have been detected between the four human IgG subclasses by use of polyclonal antisera. In 1985, a IUIS/WHO panel of immunologists, using eight immunological methods, documented the specificity of select monoclonal antibodies for the IgG subclasses. Clinical assays have been developed involving these monoclonal antibodies that allow quantification of the concentration of IgG subclass protein and distribution of the IgG subclass antibodies in human immune responses. This review addresses issues of concern to investigators who are evaluating and (or) developing quantitative human IgG subclass assays in the clinical laboratory. Unique physical (structural) and biological (functional) properties of human IgG subclasses are summarized, with a focus on aspects pertinent to their clinical importance and in vitro quantification. The HP-series monoclonal antibodies with documented specificity are examined within the context of their application to several immunological methods. I describe unique technical aspects of total and antigen-specific IgG-subclass immunoassays involving these monoclonal antibodies. Finally, this report outlines clinical applications and indications for IgG-subclass measurements in the study of human health and disease.     

IgG heavy-chain subclass restriction of thyrotropin-binding inhibitory immunoglobulins in Graves'' disease

The IgG subclass composition of antibodies is an important determinant of their function. Thyrotropin receptor antibodies cause the hyperthyroidism of Graves' disease but their subclass distribution has been incompletely investigated. We have therefore purified IgG subclasses from Graves' sera by passage over affinity columns designed to deplete all but a single subclass, and then assayed those pure subclass fractions for their ability to displace radiolabelled thyrotropin from its solubilized receptor as a measure of thyrotropin receptor antibody activity. Sufficient activity was recovered for analysis in nine of 10 Graves' patients, in five of whom activity was almost completely (97-100%) restricted to the IgG1 subclass; in the remaining four patients the response was predominantly IgG1 and IgG4 with marked under-representation of the IgG2 subclass. This contrasts with the unrestricted subclass response, in the same fractions, for autoantibodies against thyroglobulin and microsomes. These results suggest that there may be a primary defect at the B-cell level in Graves' disease.     

High prevalence of cytomegalovirus DNA in plasma samples of blood donors in connection with seroconversion

BACKGROUND: Human cytomegalovirus (CMV) is considered to latently infect blood cells. Transfusion-transmitted infection (TT-CMV) of immunocompromised patients occurs despite the use of CMV-seronegative or leukoreduced units. STUDY DESIGN AND METHODS: The prevalence of CMV DNA in plasma was investigated in 82 blood donors who had previously been seronegative for CMV and showed anti-CMV immunoglobulin G for the first time, 598 blood donors who were seropositive for at least 1 year, and 150 seronegative blood donors. In a second part of the study, the overall prevalence of CMV DNA in blood donations was assessed based on 31,745 donations. RESULTS: CMV DNA was repeatedly detected in plasma samples of 44 percent of newly seropositive donors (12%-62%, depending on the interval to the last seronegative donation). All steadily seropositive or seronegative donors were negative for the presence of CMV DNA. Detection of CMV DNA in connection with seroconversion was accompanied by significantly increased neopterin, increased alanine aminotransferase, and reduced white blood cell counts, but the sensitivity of these surrogate markers was only 71 percent. The overall prevalence of CMV DNA in blood products due to primary CMV infection of donors was at least 0.13 percent. CONCLUSION: Viremia of newly seropositive donors may be an important reason for the residual risk of TT-CMV despite leukoreduction. Furthermore, transfusion of WBC-reduced blood components from seronegative donors could imply a greater risk of TT-CMV than transfusion of WBC-reduced blood from donors who have been seropositive for at least 1 year, because window-phase donations but no reactivation could be detected in this study.     

Immunoglobulin G subclasses in immunodeficiency

Immunodeficiencies have been described ranging from stem cell dysfunction to low levels of isolated IgG subclasses. Subclass deficiencies seem in most cases to depend on regulatory defects rather than absence of genes encoding Ig heavy chains. It is, thus, not surprising that low levels of IgG subclasses can be seen in various immunodeficiency syndromes, where regulatory defects of the immune response may be present. Isolated IgG subclass deficiencies, however, are often found among patients with increased susceptibility to e.g. infections. The frequent B and/or T cell abnormalities observed in IgG subclass deficient individuals may explain the increased susceptibility to infections, suggesting that low subclass levels may function as an indicator of a clinically important immunodeficiency.     

Transfusion and cytomegalovirus-associated AIDS-defining disease in hemophiliacs. Hemophilia Malignancy Study

BACKGROUND: Whether transfusion increases the risk of AIDS-defining cytomegalovirus (CMV) infection (CMV AIDS) in immunosuppressed patients is not known. Because of concerns about the risk of transfusion transmission of CMV and potential exposure to multiple strains of CMV through transfusion, the National Hemophilia Foundation recently recommended that CMV-negative blood be used in human immunodeficiency virus-positive hemophiliacs, regardless of their CMV serologic status. Although the multiple strains of CMV cause different CMV disease manifestations in transplant recipients, there are no data on CMV disease in human immunodeficiency virus-positive hemophiliacs. STUDY DESIGN AND METHODS: It was hypothesized that if the transmission of CMV through transfusion causes CMV disease in human immunodeficiency virus- positive hemophiliacs, then hemophiliacs with CMV AIDS would be more likely to have received transfusions than those with AIDS-defining disease not caused by CMV (non-CMV AIDS). The number and type of transfusions were evaluated in 334 hemophiliacs with AIDS (35 with CMV AIDS and 299 with non-CMV AIDS) enrolled in the multicenter Hemophilia Malignancy Study. RESULTS: There were no differences between hemophiliacs with CMV AIDS and those with non-CMV AIDS in age, type, and severity of hemophilia; the proportion receiving transfusions; or the mean number of units transfused. These findings persisted after correction for transfusion practice, (i.e., CMV-unscreened blood vs. CMV-negative and/or white cell-reduced blood). There was no difference between the groups in CMV lgG titers or in the proportion who were CMV seropositive, and there was no difference between these parameters in those who had received transfusion(s) and those who had not. CONCLUSION: Transfusion appears to have little, if any, effect on the development of CMV AIDS or CMV lgG seroprevalence in patients with hemophilia.     

Direct quantitation of IgG subclasses 1, 2, and 3 bound to red cells by Rh1 (D) antibodies

The authors developed quantitative radioimmunoassays to allow direct measurement of total human IgG and individual IgG subclasses among antibodies bound to cell surfaces. The assays use four mouse monoclonal radioiodinated antibodies, one that reacts equally well with all four human IgG subclasses and three that are specific for human IgG subclasses 1, 2, or 3. The assays were used to analyze IgG subclass composition in 21 high-titer anti-D samples from Rh-negative volunteers immunized for Rh immunoglobulin production. Anti-D activity was restricted primarily to the IgG1 and IgG3 subclasses. Eleven of 21 sera demonstrated red cell antibodies with a marked predominance of IgG1 (87 +/- 3.6% of total IgG antibody, +/- SEM) and low levels of IgG3 (1.4 +/- 0.73%). In the remaining 10 sera, IgG3 made up a greater proportion of total IgG antibody (32 +/- 3.8%), although IgG1 was still predominant (61 +/- 4.1%). This observed dichotomy in the IgG subclass profiles of different anti-D sera may be a consideration in the selection of anti-D sera for the production of the immunoglobulin used in the prophylaxis of Rh-incompatible pregnancies.     

IgG subclasses in varicella-zoster virus infections

Sera from cases of varicella and zoster together with sera from healthy people seropositive for varicella-zoster virus (VZV) have been examined for their specific IgG subclass profile.Specific IgG₁ was present in all sera containing VZV-specific IgG antibody. Sera from healthy individuals seropositive for VZV also commonly contained specific IgG₂, IgG₃ and IgG₄. Specific IgG₁ and IgG₃ were the main subclasses detected after varicella in sera taken up to 2 months after the onset of symptoms. Specific IgG₃ was also detected soon after zoster, but did not persist for as long as after varicella. Specific IgG₂ and IgG₄ can also be detected frequently after zoster, but rarely in the 2 months after varicella.Thus the IgG subclass profiles can be of some help in serologically distinguishing between varicella and zoster but cannot be used as the sole discriminatory factor.     

Acquisition of cytomegalovirus infection in infants following exchange transfusion: a prospective study

A prospective study of newborn infants who required exchange transfusion was undertaken to evaluate the risk of transmission of cytomegalovirus (CMV). Buffy coat-, urine- and saliva-saturated throat swabs for viral cultures and serum specimens for CMV complement-fixing (CF) antibody were obtained from 45 infant-mother pairs. Buffy coat from the donor blood was cultured and CMV CF titers measured. Viral studies were repeated on infants and mothers at six and 12 weeks after exchange transfusion. Fifteen infants received CMV seropositive blood and 14 infants received CMV seronegative blood. Sixteen infants who did not receive blood or blood products served as controls. Three of 12 antibody-positive newborns developed infection after getting seropositive blood. One of three antibody-negative newborns developed infection after getting seropositive blood. The presence of transplacental antibody does not appear to protect the infants. None of the control infants developed CMV infection. None of the infected infants were symptomatic. Although CMV infection in infancy can be acquired by routes other than blood, exchange transfusion with seropositive blood enhances the likelihood of acquiring infection.     

Immunochemical characterization of the anti-B response in an ABO-incompatible blood transfusion: presence of antibodies recognizing glucosylceramide

Summary. A male patient of blood group A was accidentally given two units of group B blood when operated upon because of injury. This resulted in an immediate haemolytic transfusion reaction. The patient was treated with exchange transfusions in order to remove the incompatible red cells and thus prevent renal damage. Two weeks after the transfusion, an anti-B humoral immune response appeared. The antibodies were mainly IgG and IgA types, of IgG3 and IgA1 subclasses. The anti-B antibodies seemed specific for the B trisaccharide [Galα1–3(Fucα1–2)Gal] as tested by radioimmunoassay and chromatogram binding assay. No antibodies recognizing part of the core saccharide chain could be detected, i.e. antibodies capable of differentiating between B type 1 and B type 2 chains did not occur. An unsuspected finding was that the patient had IgG antibodies recognizing glucosylceramide (and weakly also galactosylceramide) before and after the transfusion. These antibodies were still present 9 years after the ABO-incompatible blood transfusion.