Treatment of autoimmune hemolytic anemias.

Treatment of autoimmune hemolytic anemias varies depending on whether the patient has autoimmune hemolytic anemia of warm antibody type, cold agglutinin syndrome, paroxysmal cold hemoglobinuria, or autoimmune hemolytic anemia secondary to an underlying disorder. Initial therapy for warm antibody autoimmune hemolytic anemia should be corticosteroids, such as prednisone at conventional doses of 1 to 1.5 mg/kg/d orally. Criteria must be established to determine whether the therapeutic response is adequate, because long-term therapy may lead to significant detrimental side effects. Splenectomy has the advantage over therapeutic options in that it has the potential for complete and long-term remission. The major adverse effect is the syndrome of overwhelming postsplenectomy infection. Other therapeutic options, which are less likely to have long-term benefit, are immunosuppressive drugs, danazol, intravenous immunoglobulin, and plasma exchange. Therapy of cold agglutinin syndrome often is unsatisfactory. All patients should avoid exposure to cold, and if additional therapy is necessary, the therapies used for warm antibody autoimmune hemolytic anemia may be tried with less likelihood of response. Paroxysmal cold hemoglobinuria requires aggressive supportive therapy, generally supplemented by corticosteroids. Hemolysis usually terminates spontaneously. Patients with secondary autoimmune hemolytic anemia may be treated similarly to those with idiopathic autoimmune hemolytic anemia, and additional therapy for the underlying disorder also may result in remission of the hemolysis.

In vitro studies of the impact of transfusion on the detection of alloantibodies after autoadsorption.

BACKGROUND: In a patient with warm autoantibodies who has recently received a transfusion, it is not recommended to perform adsorptions using autologous RBCs to detect alloantibodies. Although not scientifically documented, this position is based on the theory that transfused RBCs in the patient's circulation would be capable of adsorbing alloantibodies that may be present. This in vitro study was designed to determine what percentage of transfused RBCs might completely remove alloantibodies in vivo. STUDY DESIGN AND METHODS: Selected D, E, K, Fy(a), and Jk(a) antibodies were adsorbed with mixtures of antigen-positive and antigen-negative RBCs to determine the lowest concentration of antigen-positive RBCs capable of removing all alloantibody reactivity. The percentage of antigen-positive RBCs in each mixture was determined by flow cytometry. RESULTS: Small amounts of antigen-positive RBCs (2-6%, as determined by flow cytometry) completely removed anti-D, -E, and -Fy(a) reactivity. Reactivity of two examples of anti-K was removed by 11 percent and 17 percent of K+ RBCs, respectively. Anti-Jk(a) reactivity was completely removed by 4 to 5 percent Jk(a+) RBCs using a PEG adsorption; the endpoint (>11%) was estimated, but complete adsorption with ZZAP-treated RBCs was not performed. CONCLUSION: Small amounts of antigen-positive RBCs are generally capable of removing all alloantibody reactivity. Thus, waiting for 3 months after transfusion before performing autologous adsorptions is a prudent policy.

Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter.

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.

Regulation of the estrogen-responsive pS2 gene in MCF-7 human breast cancer cells.

To understand how hormones and antihormones regulate transcription of estrogen-responsive genes, in vivo footprinting was used to examine the endogenous pS2 gene in MCF-7 cells. While the consensus pS2 estrogen response element (ERE) half site was protected in the absence of hormone, both the consensus and imperfect ERE half sites were protected in the presence of estrogen. 4-Hydroxytamoxifen and ICI 182,780 elicited distinct footprinting patterns, which differed from those observed with vehicle- or with estrogen-treated cells suggesting that the partial agonist/antagonist and antagonist properties of 4-hydroxytamoxifen or ICI 182,780, respectively, may be partially explained by modulation of protein-DNA interactions. Footprinting patterns in and around the TATA and CAAT sequences were identical in the presence and in the absence of estrogen suggesting that the basal promoter is accessible and poised for transcription even in the absence of hormone. In vitro DNase I footprinting experiments demonstrated that the estrogen receptor bound to the pS2 ERE and that adjacent nucleotides were protected by MCF-7 nuclear proteins. These findings indicate that transcription of the pS2 gene is modulated by alterations in protein binding to multiple sites upstream of the basal promoter, but not by changes in protein-DNA interactions in the basal promoter.

Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity.

BACKGROUND: Patients who are refractory to platelet transfusion as a result of HLA alloimmunization are generally given HLA-matched or crossmatched platelets. However, HLA-matched platelets that are matched at HLA-A and -B loci (A-matched) or those without any mismatched or cross-reactive antigens (BU-matched) are frequently unavailable. A disadvantage of crossmatching is that crossmatched platelets have a shelf life of only 5 days, so that crossmatch tests must be performed frequently for patients requiring long-term platelet transfusions. An alternative method is the selection of platelets according to the patient's HLA antibody specificity, called the antibody specificity prediction (ASP) method. STUDY DESIGN AND METHODS: An anti-human globulin-enhanced microlymphocytotoxicity test modified by a double addition of serum and a computer program were used to determine the specificity of patients' HLA antibodies. Platelet crossmatching was performed with a solid-phase adherence assay. The percentage of platelet recovery (PPR) was determined in 1621 platelet transfusions in an observational study in 114 patients, and the PPR of platelets selected by the ASP method was compared with the PPR of those that were HLA-matched, crossmatched, or randomly selected. The numbers of potential donors in files of HLA-typed donors as identified by HLA matching vs. the ASP method were determined. RESULTS: After adjustments for covariates, the mean +/- SEM PPR was similar for HLA-matched (21 +/-4%), cross-matched (23+/-4%), and ASP-selected (24+/-3%) platelets and was significantly lower for randomly selected (15+/-1.4%) platelets. For 29 alloimmunized HLA-typed patients, the mean number of potential donors found in a file of 7247 HLA-typed donors was 6 who were an HLA-A match (median = 1), 33 who were an HLA-BU match (median = 20), and 1426 who were identified by the ASP method (median = 1365). CONCLUSION: The ASP method of donor selection for refractory alloimmunized patients appears as effective as HLA matching or crossmatching. Far more donors are identified in a file of HLA-typed donors by the ASP method than by HLA matching, and this indicates that the ASP method provides important advantages regarding the availability of compatible platelet components.

Distinguishing immunosilent AIDS from the acute retroviral syndrome in a frequent blood donor.

BACKGROUND: There are seven reports of "immunosilent AIDS" in which there was a lack of development of anti-HIV for more than 6 months. Thus, when a frequent blood donor presented with clinical findings highly suggestive of overt AIDS, there was concern that he may have had a prolonged immunosilent infection. CASE REPORT: A 24-year-old man who had donated blood six times in the previous year was diagnosed as having AIDS; he presented with fever, nausea, vomiting, diarrhea, weight loss, and oral candidiasis. The anti-HIV enzyme immunoassay was positive, the Western blot was indeterminate (gp160 only), the CD4+ count was 174 per mL, the HIV polymerase chain reaction was positive (2.8 x 10(6) copies/mL), and the HIV p24 antigen assay was positive. Twelve components from previous donations had been transfused, and 2 units of fresh-frozen plasma were still in inventory. Repeat donor testing 57 days after donation indicated seroconversion with a positive anti-HIV enzyme immunoassay, a positive Western blot, a negative HIV p24 antigen assay, and a positive test for HIV by polymerase chain reaction (89,000 copies/mL). Both units of fresh-frozen plasma tested negative for HIV by polymerase chain reaction. Four transfusion recipients had died, and the remaining eight are anti-HIV negative with >6 months' follow-up. CONCLUSION: The donor had an unusually severe acute retroviral syndrome and presented with findings that were difficult to distinguish from overt AIDS.

Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes.

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.

Effectiveness of a prospective physician self-audit transfusion-monitoring system.

BACKGROUND: The purpose of this study was to search for a more effective transfusion-monitoring system than the existing system of retrospective peer review. STUDY DESIGN AND METHODS: This research used a study-control, preintervention and postintervention design, to evaluate the effectiveness of a prospective physician self-audit transfusion-monitoring system that functioned without the direct involvement of transfusion service physicians. This research also evaluated the effectiveness of issuing to physicians a memo with transfusion guidelines. Three process indicators were used to assess physician behavior at various stages of the blood-ordering process: 1) the number of crossmatches ordered per admission, 2) the transfusion-to-crossmatch ratio, and 3) the number of blood units returned to the laboratory after physician self-auditing. The study used two outcome indicators to reflect overall blood utilization: 1) the percentage of patients who received red cell transfusions and 2) the number of blood units transfused per recipient each month. RESULTS: The prospective physician self-audit system implemented at the study hospital did not reverse physician transfusion decisions, and the process of issuing to physicians a memo with transfusion guidelines at the control hospital failed to reduce blood usage. However, a transient reduction in blood utilization was observed at the study hospital. CONCLUSION: The reduction was hypothesized to be due to a Hawthorne effect, in which observed behavior is affected by the subject's awareness of the research study.

The impact of T-cell depletion on the effects of HLA DR beta 1 and DQ beta allele matching in HLA serologically identical unrelated donor bone marrow transplantation.

Unrelated donor bone marrow transplants have been associated with relatively high rates of acute graft-vs.-host disease and treatment-related mortality. These complications reflect histo-incompatibility between donor and recipient. Molecular technology has recently been applied to HLA typing to identify alleles not distinguishable with serologic typing techniques. We report results in 92 unrelated marrow transplant recipients who were HLA seroidentical with donor HLA-A, -B, and -DR antigens and assess the effect of DR beta 1 and DQ beta compatibility using sequence specific oligonucleotide primers. Forty-eight patients received T-cell depleted marrow grafts, and 44 received unmodified grafts. Among recipients of unmodified marrow grafts, matching for both DR beta 1 and DQ beta reduced the rate of grade 3-4 acute graft-vs.-host disease to 38 +/- 20% vs. 73 +/- 20% among recipients mismatched for either allele (p = 0.02). This difference was not observed in recipients of T-cell depleted marrow grafts. Multivariate analysis confirmed matching for both DR beta 1 and DQ beta loci (p = 0.015), and receiving a T-cell depleted graft (p = 0.008) independently predicted for reduced risk of grade 3-4 acute graft-vs.-host disease. In conclusion, both DR beta 1 and DQ beta appear biologically important for development of acute graft-vs.-host disease in patients receiving unmanipulated marrow grafts for unrelated donor transplant.

The sickle cell hemolytic transfusion reaction syndrome.

BACKGROUND: Patients with sickle cell anemia may develop serious, life-threatening hemolytic transfusion reactions (HTRs). More severe anemia may develop after the HTR than was present before transfusion, which suggests the possibility of an increased rate of hemolysis of autologous red cells. STUDY DESIGN AND METHODS: The signs and symptoms occurring during eight severe HTRs that occurred in five patients with sickle cell anemia were reviewed, as were published reports by other investigators. Calculations of red cell production and destruction incorporating known correction factors for reticulocyte maturation were performed to determine the most probable mechanism for the striking drop in hematocrit observed in several instances. RESULTS: A characteristic constellation of findings was recognized in some severe HTRs in patients with sickle cell anemia. Calculations of daily red cell production and senescence indicated that a marked drop in hematocrit occurs when erythropoiesis is suppressed in a patient with a short red cell life span and that this could account for severe posttransfusion anemia when donor red cells are hemolyzed during an HTR. CONCLUSION: A sickle cell HTR syndrome was defined. A rapid increase in the severity of anemia occurs in patients with sickle cell anemia when all donor red cells are hemolyzed during an HTR and when there is suppression of erythropoiesis, as commonly occurs as a result of transfusion or concomitant illness. Although an increased rate of hemolysis of autologous red cells may also occur, more definitive data are required to document that in these patients.

DNA bending is induced by binding of the glucocorticoid receptor DNA binding domain and progesterone receptors to their response element.

Circular permutation analysis was used to determine the degree of DNA bending induced by binding of the glucocorticoid receptor (GR) DNA binding domain (DBD), the human progesterone receptor (PR) DBD, PR-A:A and PR-B:B homodimers, and PR-A:B heterodimers to the glucocorticoid response element/progesterone response element (GRE/PRE). The bending angles induced by the GR DBD and the PR DBD were approximately 28 degrees and 25 degrees, respectively. The PR-B:B and PR-A:A homodimers and the PR-A:B heterodimers all induced similar DNA bending angles of 72-77 degrees. The substantially greater DNA bend induced by full-length PR compared to the PR DBD indicates that sequences outside the classic zinc finger DNA binding domain may play an important role in the interaction of PR with the GRE/PRE. Because PR-A:A and PR-B:B homodimers and the PR-A:B heterodimers induce similar DNA bends, the different abilities of the PR-A and PR-B isoforms to activate transcription are not due to differences in their abilities to distort DNA structure.

Are retrospective peer-review transfusion monitoring systems effective in reducing red blood cell utilization?

OBJECTIVE: This research used a study-control group design and examined data collected from five hospitals to evaluate the effectiveness of retrospective peer-review systems on reducing utilization of red blood cells (RBCs). DESIGN: The effects of retrospective peer-review systems were studied in three parts: (1) trends of RBC utilization were compared by the slopes of linear regression lines that assessed the effect of time on RBC utilization among four study hospitals and one control hospital, (2) diagnosis-specific RBC utilization was compared between the control hospital and one matched study hospital, and (3) the effect of the retrospective review system of one study hospital was assessed by linear regression using data accumulated 1 year before and 2 years after implementation of the program. RESULTS: Three study hospitals showed no significant changes in RBC utilization during the 10-month study period. One study hospital and the control hospital demonstrated trends of reduced RBC use with negative slopes of regression lines; however, there was no difference in the degree of the two slopes, and the diagnosis-specific RBC utilization was not lower at the study hospital than at the control hospital. Furthermore, implementation of the retrospective peer-review system at one study hospital demonstrated no effect on RBC utilization. CONCLUSIONS: We conclude that the retrospective peer-review systems implemented at these four hospitals had no effect on reducing red blood cell utilization.

A comprehensive transfusion medicine curriculum for medical students. Transfusion Medicine Academic Award Group.

BACKGROUND: The Transfusion Medicine Academic Awards (TMAA) program, sponsored by the National Heart, Lung, and Blood Institute, has provided grants to medical schools to help them develop comprehensive curricula in transfusion medicine. In 1989, the TMAA Group published a set of comprehensive curricular goals for teaching transfusion medicine. The medical student portion of this curriculum has now been revised to reflect new developments in transfusion medicine and recent trends in medical school education. STUDY DESIGN AND METHODS: Two medical schools independently revised the 1989 curriculum for their students. Because significant similarities were noted between curricula of the two institutions, the two revisions were combined and submitted to all TMAA institutions for comment. As a result, a revised medical school curriculum was developed and approved by the TMAA Group. RESULTS: The revised curriculum consists of 28 objectives in six major areas of transfusion medicine. It is presented in its entirety in this article. CONCLUSION: The TMAA transfusion medicine curriculum should provide to medical schools a valuable resource for evaluating their teaching of transfusion medicine and should provide to medical school deans and curriculum committees an authoritative source of transfusion medicine expertise.