The relationship between alloimmunization and posttransfusion granulocyte survival: experience in a chronic granulomatous disease cohort.

BACKGROUND: The efficacy of granulocyte transfusions in patients with HLA alloimmunization is uncertain. A flow cytometric assay using dihydrorhodamine 123 (DHR), a marker for cellular NADPH oxidase activity, was used to monitor the differential survival of transfused oxidase-positive granulocytes in alloimmunized patients with chronic granulomatous disease (CGD). STUDY DESIGN AND METHODS: Ten patients with CGD and serious infections were treated with daily granulocyte transfusions derived from steroid and granulocyte-colony-stimulating factor-stimulated donors. The proportion of neutrophils with intact oxidase activity was quantitated by DHR fluorescence on samples drawn before and 1 hour after transfusion. The incidence of acute transfusion reactions was correlated with the results of DHR fluorescence and biweekly HLA serologic screening assays. RESULTS: Eight of 10 patients experienced acute adverse reactions in association with granulocyte transfusions. Four had only chills and/or fever, and four experienced respiratory compromise; all eight exhibited HLA alloimmunization. Mean (± SD) oxidase-positive cell recovery was 19.7 ± 17.4% (n = 15 transfusions) versus 0.95 ± 1.59% (n = 16) in the absence and presence of HLA allosensitization, respectively (p < 0.01). Greater than 1% in vivo recovery of DHR-enhancing donor granulocytes was strongly correlated with lack of HLA alloimmunization. CONCLUSION: The ability to detect DHR-positive donor granulocytes by flow cytometry is strongly correlated with absence of HLA alloimmunization and lack of acute reactions to granulocyte transfusions in patients with CGD. If HLA antibodies are present and the survival of donor granulocytes is low by DHR analysis, transfusions should be discontinued, avoiding a therapy associated with high risk and unclear benefit.

The immunologic aspects of poxvirus oncolytic therapy.

The concept of using replicating oncolytic viruses in cancer therapy dates to the beginning of the twentieth century. However, in the last few years, an increasing number of pre-clinical and clinical trials have been carried out with promising preliminarily results. Novel, indeed, is the suggestion that viral oncolytic therapy might not operate exclusively through an oncolysis-mediated process but additionally requires the "assistance" of the host's immune system. Originally, the host's immune response was believed to play a predominant obstructive role against viral replication, hence limiting the anti-tumor efficacy of viral vectors. Recent data, however, suggest that the immune response may also play a key role in promoting tumor destruction in association with the oncolytic process. In fact, immune effector pathways activated during oncolytic virus-induced tumor rejection seem to follow a similar pattern to those observed when the broader phenomenon of immune-mediated tissue-specific rejection occurs in other immune-related pathologies. We recently formulated the "Immunologic Constant of Rejection" hypothesis, emphasizing commonalties in transcriptional patterns observed when tissue-destruction occurs: whether with a favorable outcome, such as in tumor rejection and pathogen clearance; or a destructive one, such as in allograft rejection or autoimmunity. Here, we propose that a similar mechanism induces clearance of virally infected tumors and that such a mechanism is primarily dependent on innate immune functions.

A preliminary comparison of the prevalence of transfusion reactions in recipients of platelet components from donors with and without human leucocyte antigen antibodies.

BACKGROUND: Human leucocyte antigen (HLA) antibodies have been implicated in transfusion-related acute lung injury, but the probability that the transfusion of a blood component containing HLA antibodies will cause a reaction is not known. This study compared the prevalence of reactions associated with the transfusion of platelet components with and without HLA antibodies. STUDY DESIGN AND METHODS: This retrospective study tested 96 consecutive apheresis platelet donors for HLA class I and II antibodies. Matched control donors without HLA antibodies were selected and records were reviewed to determine the proportion of components from each group that caused reactions. In addition, all apheresis platelet donors involved with two or more reactions were identified and tested for HLA class I antibodies. RESULTS: Five of the 96 donors had antibodies to class I or class II antigens and, of these, four had components transfused. The prevalence of reactions to components from these four donors with HLA antibodies and the 12 matched control donors without antibodies was similar (three reactions to 167 transfusions or 1.8% vs. three to 295 or 1.0%, respectively, P = 0.32). A retrospective review of the transfusion records from all platelet donors found that components from 22 caused two or more reactions and three (13.6%) had antibodies to HLA class I compared to 4.2% of the consecutively selected donors (P = 0.12). None of the patients experienced transfusion-related acute lung injury. CONCLUSION: Reactions associated with transfusion of apheresis platelets containing HLA antibodies are unusual.

Patient-specific transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-associated mortality. The inadvertent transfusion of neutrophil antibodies can cause pulmonary transfusion reactions and TRALI. However, not all patients transfused with neutrophil antibodies experience transfusion reactions. A 22-year-old man with severe aplastic anaemia (SAA) experienced TRALI after a platelet transfusion. The donor was found to be alloimmunized to human neutrophil antigen (HNA)-3a, an antigen expressed by neutrophils from approximately 90% of Caucasians. Eleven other platelet components from this donor were transfused prior to this event and two caused reactions: one chills and one TRALI. Both episodes of TRALI occurred in the same male patient with SAA. The fact that one patient experienced TRALI following both exposures to anti-HNA-3a from the same donor whereas nine other recipients did not adds evidence to the observation that patient factors make a significant contribution to neutrophil antibody-mediated transfusion reactions.

Cryopreserving and deglycerolizing sickle cell trait red blood cell components using an automated cell-processing system.

RBC components with rare phenotypes are sometimes required for patients with sickle cell disease, and these rare components can often be found among donors with sickle cell trait. Cryopreserving RBC components from sickle cell trait donors requires a modified deglycerolization method to preserve the integrity of the RBCs. This study evaluated the feasibility of using an automated cell-processing system to cryopreserve and deglycerolize sickle cell trait donor RBC components. CP2D/AS-3 RBC components were collected from three donors with sickle cell trait. Each component was processed with an automated cell-processing system (ACP 215, Haemonetics Corp., Braintree, MA) and cryopreserved within 6 days of collection. The components were stored at -65 degrees C or less for at least 2 days and were deglycerolized using the automated cell-processing system's standard procedure. Before cryopreservation and after deglycerolization, several variables were measured. Deglycerolization resulted in recovery of 43.0, 76.5, and 67.5 percent of RBCs from the three sickle-cell-trait donor components compared with 80 percent or greater for all six control components. A small, dark red, jelly-like mass was noted in the bowl of the disposable set after deglycerolization of each of the three RBC sickle cell trait components. The osmolalities of all three sickle cell trait components were less than 400 mOsm/kg, but only one of the three was acceptable for a 14-day outdate. Freezing and deglycerolization of sickle cell trait donor RBC components with the automated cell-processing system resulted in recovery of some RBCs, but a decrease in RBC recovery was problematic. Modifications of the procedure are needed for processing sickle cell trait donor RBC components.

Rapid, single-subject genotyping to predict red blood cell antigen expression.

Genotyping is useful to predict the expression of those RBC antigens for which antisera are difficult to obtain and to determine the probable phenotype of highly transfused patients, and it can be used to test stored DNA when a blood sample is not available. This study assessed a sequence-specific primer (SSP)-based genotyping system for blood group alleles suitable for the rapid testing of a small number of samples and assessed the use of stored whole blood. Genomic DNA was isolated from fresh and 1- and 2-week-old stored blood from 20 donors with known ABO and Rh phenotypes and was used for ABO, RHD, and RHCE genotyping using SSPs. The amplicons were analyzed using gel electrophoresis and a novel microfluidic on-chip electrophoresis system. Analysis of DNA from fresh and 1- and 2-week-old blood by SSP and gel electrophoresis yielded the correct ABO, RHD, and RHCE type in all samples, but with DNA from 2-week-old stored blood the amplicons were more difficult to visualize. Analysis of the same samples with the SSP on-chip electrophoresis assay correctly typed all samples except for one RHCE typing discrepancy of a fresh sample and one RHCE typing discrepancy of a 2-week-old sample. Analysis of amplicons by on-chip electrophoresis required one tenth the DNA that gel electrophoresis did and could be completed within 30 minutes compared with 2 hours with gel electrophoresis. Amplicons were also more readily visualized with on-chip electrophoresis. Fresh and 1- and 2-week-old samples could be ABO and RH genotyped with SSP. Analysis using on-chip electrophoresis was easier and more rapid than that using gel electrophoresis, but test reliability was slightly more variable.

Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation.

Delayed donor erythropoiesis and pure red-cell aplasia (PRCA) complicate major-ABO mismatched non-myeloablative allogeneic stem-cell transplantation. To characterize these events, we analysed red-cell serology and chimaerism in lymphohaematopoietic lineages, including plasma cells and B cells, in 12 consecutive major-ABO incompatible transplants following cyclophosphamide/fludarabine-based conditioning. Donor erythropoiesis was delayed to more than 100 days in nine (75%) patients including six (50%) who developed PRCA. During PRCA, all patients had persistent anti-donor isohaemagglutinins and recipient plasma cells (5-42%), while myeloid and T cells were completely donor in origin. In contrast, B-cell chimaerism was frequently full-donor when significant anti-donor isohaemagglutinins persisted. Four patients with early mixed haematopoietic chimaerism and the prolonged presence of anti-donor isohaemagglutinins and recipient plasma cells developed delayed-onset (>100 days post-transplant) red cell transfusion dependence and PRCA after myeloid chimaerism converted from mixed to full donor. These findings confirm that donor-erythropoiesis is impacted by temporal disparities in donor immune-mediated eradication of recipient lymphohaematopoietic cells during major-ABO incompatibility and suggest that plasma cells are relatively resistant to graft-versus-host haematopoietic effects.

Neutrophil and platelet antibodies in autoimmune lymphoproliferative syndrome.

BACKGROUND AND OBJECTIVES: Autoimmune lymphoproliferative syndrome (ALPS), is an inherited disorder characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly, accumulation of T-cell receptor (TCR)-alphabeta+ CD4- CD8- T cells (double-negative T cells) and autoimmunity. We investigated the incidence and nature of neutrophil and platelet antibodies in patients with ALPS. MATERIALS AND METHODS: Sera from 26 patients with ALPS were tested for neutrophil antibodies by granulocyte immunofluorescence, granulocyte agglutination and monoclonal antibody immobilization assays of granulocyte antigens, and for platelet antibodies using a solid-phase antibody-detection system. RESULTS: Neutrophil antibodies were detected in 46% of patients with ALPS. Antibody specificity could be defined in eight of the 12 patients with neutrophil antibodies. Among these eight patients, four had antibodies directed against more than one antigen. Overall, 14 antibodies directed to specific antigens were identified: three were directed to the HNA-1a antigen of FcgammaRIIIb; two to the HNA-1b antigen of Fcgamma-RIIIb; two to epitopes common to all FcgammaRIIIb molecules; four to the HNA-2a antigen of the NB1 glycoprotein; and three to neutrophil beta2 integrins. Platelet antibodies were detected in 35% of patients with ALPS. No antibody specificities were identified among the platelet antibodies. There was no association between the detection of neutrophil antibodies and a history of clinical neutropenia, or between the detection of platelet antibodies and a history of clinical thromobocytopenia. CONCLUSIONS: Neutrophil and platelet antibodies are important markers of ALPS, but do not always cause clinical cytopenias. The specificities of neutrophil antibody were similar to those found in children with autoimmune neutropenia but without ALPS.

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d.

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.

Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures.

BACKGROUND: Detection of immunoglobulin or complement bound to RBCs by using the DAT is valuable in the diagnosis of immune-mediated hemolytic anemia. Traditionally, the DAT has been performed by tube agglutination using anti-IgG or anti-C3d. The purpose of this study was to compare the tube agglutination DAT to gel microcolumn, affinity microcolumn, and flow cytometric DATs on RBCs coated in vitro and on patient RBC samples. STUDY DESIGN AND METHODS: RBCs from 84 patients were assessed by tube agglutination DAT, one gel microcolumn DAT, and two affinity microcolumn DATs. One affinity microcolumn assay was unmodified and one was modified by the addition of polyspecific antiglobulin or anti-IgG as a secondary antibody. RBCs from 15 of the 84 patients underwent analysis by flow cytometry with fluorescence-labeled anti-IgG. The assays were also compared by using D+ RBCs sensitized with serially adjusted concentrations of anti-D. RESULTS: Both tube agglutination and gel microcolumn DATs were positive in 49 patient samples; both assays were negative in 20 samples, and the results were discordant in 15. Gel microcolumn DATs were more likely than were tube agglutination DATs to detect IgG on RBCs. Affinity microcolumn DATs were less likely than gel microcolumn or tube agglutination DATs to detect IgG on RBCs. Flow cytometry results were the same as gel microcolumn results in 12 of 15 patient samples and the same as tube agglutination results in 13 of 15. Tube agglutination and both affinity microcolumn assays reacted with RBCs coated with anti-D that was diluted 1-in-100. The gel microcolumn and flow cytometry assays reacted with RBCs coated with anti-D diluted 1-in-400. There was no correlation between tube agglutination and gel microcolumn DATs in detecting bound C3d. CONCLUSION: Detection of IgG bound to RBCs was not consistent with the methods described. Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs. Given the varied results of these assays, reference laboratories should not rely on a single method for DATs. More comprehensive testing should be performed when the tube agglutination DAT is negative in a patient with suspected immune-mediated hemolytic anemia. Further comparisons are necessary to determine the proficiency of flow cytometric assays.

Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation.

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.

Administration of G--CSF plus dexamethasone produces greater granulocyte concentrate yields while causing no more donor toxicity than G--CSF alone.

BACKGROUND: G-CSF with or without dexamethasone is becoming the standard agent for mobilizing granulocytes for transfusion. The purpose of this study was to determine if the toxicities of G--CSF with or without dexamethasone are offset by greater collection yields and to define the minimum interval that should separate sequential collections. STUDY DESIGN AND METHODS: Twenty donors were studied on three occasions. They were given either dexamethasone (8 mg, by mouth) plus a placebo injection, G--CSF (5 microg/kg, given subcutaneously) plus placebo capsules, or G--CSF plus dexamethasone. Granulocytes were collected by apheresis. A donor symptom survey was administered, and cell counts and blood chemistries were assessed before collection and 1, 2, 7, 14, 21, 28, and 35 days after collection. RESULTS: More granulocytes were collected when G--CSF was given than when dexamethasone was given (41.1 +/- 20.4 x 10(9) vs. 21.0 +/- 10.0 x 10(9); p<0.001), but the use of G--CSF plus dexamethasone produced the greatest yields (67.1 +/- 22.0 x 10(9); p<0.002). When the donors were given dexamethasone alone, 58 percent experienced at least one symptom, compared to 85 percent of those given G--CSF and 75 percent of those given G--CSF plus dexamethasone. In all three regimens, platelet counts fell 19 percent to 24 percent after collection and remained below baseline for 7 to 14 days. Granulocyte counts returned to baseline within 3 to 7 days, but, in all three regimens, a mild granulocytopenia occurred 21 days after collection. With each of the regimens, blood chemistries changed, but the changes were mild and most returned to baseline within 7 days; however, changes in albumin, bilirubin, and AST persisted until 28 days after collection. CONCLUSION: These results support the use of G--CSF plus dexamethasone in granulocyte donors. G--CSF plus dexamethasone resulted in greater granulocyte yields than either agent alone and was associated with donor symptoms and changes in blood cell counts and chemistries similar to those seen with G--CSF alone or dexamethasone alone. Granulocytes can be safely collected a second time after a 7-day interval; however, for regular donors, it may be best to separate collections by 4 weeks.

The expression of NA antigens in people with unusual Fcgamma receptor III genotypes.

BACKGROUND: The Fcgamma receptor IIIb (FcgammaRIIIB) genes that encode neutrophil-specific antigens NA1 and NA2 differ at 5 nucleotides (nts); in 4, the result is an amino acid (AA) difference between the two alleles. The role of each of these differences in antigen expression is not known. Persons with FcgammaRIIIB genes that differ from NA1-FcgammaRIIIB and NA2-FcgammaRIIIB by 1 nt have been described. This study compared NA1 and NA2 expression on granulocytes in persons with variant FcgammaRIIIB genes and in healthy blood donors. STUDY DESIGN AND METHODS: Reactions of NA1- and NA2-specific MoAbs and alloantibodies with granulocytes were assessed by flow cytometry in 74 healthy blood donors and 6 persons with known variant FcgammaRIIIB genes. The granulocytes were tested with 1 NA1-specific MoAb, 1 NA2-specific MoAb, 4 NA1-specific alloantibodies, and 4 NA2-specific alloantibodies. RESULTS: Analysis of granulocytes from persons with variant NA genotypes found that single-base substitutions in FcgammaRIIIB at 141 and at 349 are important in NA1 expression and those at 227 and 277 are important in NA2 expression. Among blood donors, neither age, sex, nor race affected the expression of NA1 or NA2. The NA2-specific MoAb reacted more intensely with granulocytes from NA2-double-dose cells than with those from NA-single-dose cells, but this was not true for the NA2-specific alloantibodies. There was no difference in the reactions of the NA1-specific MoAbs and alloantibodies with donor samples of known NA1-double-dose or NA-single-dose cells. The intensity of reactions of both the NA1- and NA2-specific MoAbs and alloantibodies were strongly correlated on double-dose cells but not on single-dose cells. In fact, granulocytes from 7 healthy blood donors, phenotyped as NA-single-dose with the MoAbs, were phenotyped as NA2-double-dose with the alloantibodies. Variations in FcgammaRIIIB are common in blacks, but 5 of the 6 donors were white. These results suggest that FcgammaRIIIB variations may be common in both whites and blacks. CONCLUSIONS: NA2 expression is affected by polymorphisms in FcgammaRIIIB 227 and FcgammaRIIIB 277, both of which are involved in an FcgammaRIIIb N-glycosylation site. Polymorphisms in FcgammaRIIIB at 141 and 349 appear more important to NA1 expression.

Evaluation of solutions for the storage of granulocyte colony-stimulating factor-mobilized granulocyte concentrates.

BACKGROUND: High cell counts in granulocyte colony-stimulating factor (G-CSF)-mobilized granulocytes are detrimental to concentrate storage. An eightfold dilution with autologous plasma improves storage, but this method is impractical. The purpose of this study was to identify an infusible solution that could be used in place of autologous plasma to dilute and store granulocytes. MATERIALS AND METHODS: Granulocytes collected from donors given dexamethasone (8 mg per os) and/or G-CSF (5 micrograms/kg subcutaneously [SQ]) were diluted eightfold in the following cell culture media: X-Vivo 10, Dulbecco's modified Eagle's minimal essential medium (DMEM) or Iscoves modified Dulbecco's medium (IMDM); or in the following infusible solutions: Plasma-Lyte A; Normosol R; lactated ringers, supplemented with 1% human serum albumin and 50 mM histidine (LRAH); or Plasma-Lyte A supplemented with 50 mM histidine buffer or 25 mM HEPES buffer plus 1% human serum albumin. The granulocytes were stored for 48 h at room temperature. White blood cell (WBC) counts, WBC viability and pH were measured after approximately 2 h, 24 h and 48 h of storage. RESULTS: Cell counts, viability and pH were maintained after 2 h, 24 h and 48 h in cells stored in the three cell culture media. The pH fell slightly after 48 h to 6.86 +/- 0.10 in granulocyte concentrates diluted in LRAH, but fell to a greater extent after 24 h and 48 h, to 6.36 +/- 0.23 (48-h value) in granulocyte concentrates diluted in Plasma-Lyte A and to 6.40 +/- 0.19 (48-h value) in granulocyte concentrates diluted in Normosol R. The cell counts of concentrates diluted in LRAH were stable for 48 h, but fell in granulocyte concentrates stored in Plasma-Lyte A and Normosol R. Plasma-Lyte A supplemented with histidine maintained the pH of diluted granulocyte concentrates better than Plasma-Lyte A supplemented with HEPES; 6.91 +/- 0.10 and 6.65 +/- 0.11, respectively, after 24 h. Cell counts were maintained best in granulocyte concentrates diluted in Plasma-Lyte A supplemented with albumin and one or both of the buffers. CONCLUSIONS: Culture media were best for granulocyte storage, but they are not approved for in vivo use. Infusible solutions are not buffered adequately and lack sufficient protein, but infusible solutions, such as lactated Ringer's solution or Plasma-Lyte A supplemented with buffers and albumin, hold promise as effective and licensable solutions for granulocyte storage.

RBC autoantibodies in autoimmune lymphoproliferative syndrome.

BACKGROUND: Patients with autoimmune lymphoproliferative syndrome (ALPS) have an autosomal dominant genetic defect that affects lymphocyte apoptosis and is associated with chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity, particularly affecting RBCs, WBCs, and platelets. STUDY DESIGN AND METHODS: DATs were performed on 34 consecutive patients with ALPS and 37 of their clinically unaffected relatives. The effects of age, sex, race, and immunoglobulin levels on the incidence of autoantibodies and clinical hemolysis were assessed. RESULTS: The DAT was positive in 21 (62%) of ALPS patients but in only 1 (3%) of their relatives (p = 0.001). The DAT reacted because of IgG alone in 43 percent, complement alone in 5 percent, and IgG plus complement in 19 percent; 33 percent of the patients' cells had a positive reaction with polyspecific reagent only. All 10 ALPS patients with a history of hemolytic anemia had a positive DAT. Sixty percent of them had only IgG on their cells, 30 percent had IgG and complement, and 10 percent reacted only with polyspecific reagent. Of the 11 patients with a positive DAT and no history of hemolytic anemia, IgG alone was present in 27 percent, complement alone in 9%, and IgG plus complement in 9 percent; 55 percent had positive DATs only with polyspecific reagent. Among ALPS patients, those with a positive DAT had greater quantities of cells with increased alpha and ss T-cell receptors that phenotyped as CD4-CD8- and higher IgG levels. CONCLUSIONS: The DAT results in ALPS patients are most similar to those found in warm autoimmune hemolytic anemia. The DAT is useful to distinguish affected and unaffected persons within an ALPS family.

Detection of granulocyte antibodies by flow cytometry without the use of pure granulocyte isolates.

Established methods used to detect serum antibodies to granulocytes require the isolation of granulocytes. Flow cytometric analysis of granulocytes with monoclonal antibodies eliminates the need for granulocyte isolation. The purpose of this study was to develop a method to evaluate reactions of antibodies to granulocytes without separating granulocytes from other leukocytes. Three screening cell samples for granulocyte antibody detection were prepared from whole-blood samples in which the red blood cells (RBCs) were lysed and remaining leukocytes tested against sera at 4 degrees C. Binding of human alloantibodies to the screening cells was determined by flow cytometric analysis using phycoerythrin-conjugated antibody to human immunoglobulin. Forward and side scatter were used to analyze granulocytes separately from other leukocytes. The assay was validated by testing granulocytes with reference alloantibodies directed to NA1, NA2, 5b, and Mart antigens. Samples from 32 patients were tested, and the results of the assays were compared with the results of testing the samples in a granulocyte immunofluorescence (GIF) assay performed by a reference laboratory. In the whole-blood flow cytometric (WBFC) assay the mean fluorescence intensities of reference antisera with antigen-positive cells, expressed in arbitrary units, were anti-NA1 = 48 to 221, anti-NA2 = 24 to 69, anti-5b = 13 to 57, and anti-Mart = 42 to 72. In contrast, the mean fluorescence intensity of type AB-negative control sera ranged from 3 to 11. Of the 32 patient sera tested, 23 were positive (range = 12 to 56) and 9 were negative (range = 3 to 10). When compared with the results obtained by the reference laboratory, 27 sera were concordant between the WBFC and the GIF assays. Four of the samples were positive in WBFC (range = 11 to 31) and negative in GIF and one sample was negative in WBFC (range = 5 to 6) and positive in GIF. Leukocytes prepared from whole blood after lysis of RBCs can be used in flow cytometric analysis to detect granulocyte alloantibodies. The results of testing for granulocyte antibodies with this assay were similar to results of testing sera in GIF. Further comparative studies are indicated to confirm findings and explain the discordant results.

Intravenous Rh immune globulin prevents alloimmunization in D- granulocyte recipients but obscures the detection of an allo-anti-K.

Rh immune globulin (RhIG) has been used to prevent alloimmunization in D(-) recipients of apheresis platelet transfusions from D(+) donors that may contain up to 5 mL of D(+) red blood cells (RBCs). Granulocyte concentrates contain approximately 30 mL of RBCs and it has been necessary to give D(-) recipients granulocyte transfusions from D(+) donors. Intravenous RhIG has not yet been demonstrated to be effective in preventing D alloimmunization with granulocyte transfusions. Four D(-) recipients received multiple D(+) granulocyte transfusions from D(+) donors and multiple injections of intravenous RhIG at a standard dose of 600 microg for each D(+) transfusion. Two D(-) males with chronic granulomatous disease were given 32 and 13 daily granulocyte transfusions, 18 and 2 of which, respectively, were D(+). After the first dose of intravenous RhIG, both patients exhibited circulating anti-D that was undetectable 3 to 4 years later. Two patients with severe aplastic anemia were given 5 and 14 granulocyte transfusions, 4 and 7 of which, respectively, were D(+). Both patients died before the effectiveness of RhIG could be assessed. In one of these patients the indirect and direct antiglobulin tests became positive after the first dose of intravenous RhIG, which required that subsequent granulocyte transfusions from D(+) donors be crossmatched by immediate spin (IS) testing only. A delayed hemolytic reaction attributed to allo-anti-K occurred after granulocytes from a K(+) donor were given to this patient. These results suggest that intravenous RhIG can be used to prevent alloimmunization to D in D(-) patients receiving large quantities of RBCs from D(+) granulocyte transfusions. However, anti-D and other passive antibodies from RhIG prohibit the use of the antiglobulin crossmatch with antigen-positive granulocyte donor samples. It may be important to frequently collect new samples to screen for newly formed allo-antibodies when IS crossmatches are used in place of the antiglobulin crossmatch.

Storage of G-CSF-mobilized granulocyte concentrates.

BACKGROUND: Current standards limit granulocyte storage to 24 hours. Since G-CSF inhibits granulocyte apoptosis, it may be possible to store G-CSF-mobilized granulocytes for longer periods while maintaining cell viability and function. However, G-CSF mobilization increases the yield of granulocytes several times, and the resulting higher cell concentrations may diminish viability during storage and significant levels of pyrogenic cytokines may be produced. STUDY DESIGN: Ten granulocyte donors were given dexamethasone (8 mg PO), G-CSF (5 microg/kg SQ), or both and on the next day granulocyte concentrates were collected using a blood cell separator. Component cell counts, cell viablilities, pH, and IL-1beta, IL-6, IL-8 and TNF levels were measured at 2 to 4 (2), 20 to 28 (24), and 44 to 52 hours (48 hours). RESULTS: Significantly more granulocytes were collected when donors were given G-CSF (4.2 +/- 2.3 x 10(10)) or G-CSF plus dexamethasone (6.4 +/- 2.5 x 10(10)) compared with that collected with dexamethasone alone (2.2 +/- 1.2 x 10(10)); p = 0.03 and p = 0.002, respectively. Storage had little effect on WBC count. Slight but significant increases in IL-1beta and IL-8 occurred after 24 and 48 hours as compared to the levels at 2 hours' storage. Levels of IL-6 and TNF did not change. The pH dropped significantly with time in granulocytes mobilized with each regimen. Granulocytes mobilized with G-CSF plus dexamethasone were acidic immediately after collection, and pH was below 6.0 after 24 hours. To assess the effect of cell concentrations on pH, serial dilutions were performed on 13 granulocyte concentrates in autologous plasma prior to storage. The pH remained above 7.0 only when dexamethasone-mobilized granulocytes were diluted 1-in-8 and when the G-CSF plus dexamethasone-mobilized granulocytes were diluted 1-in-16. CONCLUSIONS: To optimize storage pH, mobilized granulocyte concentrates require a 1-in-8 to 1-in-16 dilution, which is operationally impractical. Clinical-grade granulocyte preservative solutions are needed to maintain pH during storage.

Detection of HLA antibodies by using flow cytometry and latex beads coated with HLA antigens.

BACKGROUND: Detection of HLA class I antibodies in sera is needed in various clinical situations. The standard method for detecting HLA class I antibodies is the complement-dependent lymphocytotoxicity (CDC) assay, but solid-phase assays are now available. STUDY DESIGN AND METHODS: This study assessed the ability of a flow cytometric assay using latex beads coated with HLA class I antigens to detect HLA class I-specific antibodies. The CDC assay was compared with the pooled-bead assay for the detection of HLA class I antibodies. Thirty-one randomly selected serum samples previously tested by CDC assay were tested with pooled beads and analyzed by flow cytometry. Twenty-seven additional serum samples, chosen by clinical criteria and CDC assay results, were tested against the pooled beads. Next, samples from six patients from whom three or more serum samples were drawn on consecutive days were tested with both methods. Finally, serum samples that were proved positive by both methods were tested with selected beads coated with antigens from a single person. RESULTS: Among the randomly selected serum samples, there was 90-percent agreement between the two assays. There was 96-percent agreement between the two assays of the 27 samples that were selected by clinical criteria and CDC assay results. Testing the sera with individual beads suggested that the HLA class I antibodies react with beads expressing the corresponding HLA antigen and beads expressing antigens in the same cross-reactive group. CONCLUSION: The pooled-bead assay can be used as an alternative method for detecting HLA class I antibodies. However, if the specificity of the HLA class I antibody is required, another assay must be used.