Capillary versus venous haemoglobin determination in the assessment of healthy blood donors.

BACKGROUND AND OBJECTIVES: To determine the accuracy of fingerstick haemoglobin assessment in blood donors, the performance of a portable haemoglobinometer (HemoCue Hb 201+) was prospectively compared with that of an automated haematology analyzer (Cell-Dyn 4000). Haemoglobin values obtained by the latter were used as the 'true' result. MATERIAL AND METHODS: Capillary fingerstick samples were assayed by HemoCue in 150 donors. Fingerstick samples from two sites, one on each hand, were obtained from a subset of 50 subjects. Concurrent venous samples were tested using both HemoCue and Cell-Dyn devices. RESULTS: Capillary haemoglobin values (HemoCue) were significantly greater than venous haemoglobin values (HemoCue), which in turn were significantly greater than venous haemoglobin values by Cell-Dyn (mean ± SD: 14.05 ± 1.51, 13.89 ± 1.31, 13.62 ± 1.23, respectively; P < 0.01 for all comparisons among groups). Nine donors (6%) passed haemoglobin screening criteria (≥ 12.5 g/dl) by capillary HemoCue, but were deferred by Cell-Dyn values (false-pass). Five donors (3%) were deferred by capillary sampling, but passed by Cell-Dyn (false-fail). Substantial variability in repeated fingerstick HemoCue results was seen (mean haemoglobin 13.72 vs. 13.70 g/dl, absolute mean difference between paired samples 0.76 g/dl). Hand dominance was not a factor. CONCLUSIONS: Capillary samples assessed via a portable device yielded higher haemoglobin values than venous samples assessed on an automated analyzer. False-pass and false-fail rates were low and acceptable in the donor screening setting, with 'true' values not differing by a clinically significant degree from threshold values used to assess acceptability for blood donation.

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.

Peripheral expansion of pre-existing mature T cells is an important means of CD4+ T-cell regeneration HIV-infected adults.

The CD4+ T-cell pool in HIV-infected patients is in a constant state of flux as CD4+ T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+ T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+ T-cell population relative to the CD4/CD45RO+ population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+ T cells persist in vivo for weeks to months and that the CD4+ T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.

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.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Chemokine RANTES promoter polymorphism affects risk of both HIV infection and disease progression in the Multicenter AIDS Cohort Study.

OBJECTIVE: To examine whether polymorphism in the RANTES gene is associated with HIV disease outcome. DESIGN: RANTES, a ligand of the major HIV co-receptor, CCR5, is known to block HIV-CCR5 interactions. Recently, two single nucleotide polymorphisms in the RANTES gene promoter region, designated -403G/A and -28C/G, have been described. Both polymorphisms can affect in-vitro promoter activity, and the RANTES -403A, -28G haplotype has been associated with a slower CD4 cell count decline rate in a Japanese cohort. METHODS: We compared RANTES compound genotype frequencies between HIV-positive and exposed-uninfected participants of the Multicenter AIDS Cohort Study (MACS) and rates of progression to AIDS for MACS seroconverters. RESULTS: We found that the two most common RANTES promoter compound genotypes, G1 (-403G/G, -28C/C) found in 67% of Caucasians, and G4 (-403G/A, -28C/C) found in 23% of Caucasians, were associated with altered risk of HIV transmission and progression, particularly in individuals who lacked the protective CCR5 mutation, CCR5delta32. In this study, individuals with a G4 compound genotype were more likely to acquire HIV than individuals with a G1 compound genotype (OR 1.72, P = 0.016) and the risk increased when individuals possessing CCR5delta32 were omitted from consideration (OR 2.13, P = 0.005). Among seroconverters lacking CCR5delta32, those who had the G4 compound genotype progressed significantly slower to AIDS-1993 than those with the G1 compound genotype (median time to AIDS 7.6 versus 5.4 years; RH 0.65; P = 0.007). CONCLUSIONS: These data implicate the RANTES-403A allele as a risk factor for HIV transmission and as a protective factor for HIV progression.

A study of the safety and survival of the adoptive transfer of genetically marked syngeneic lymphocytes in HIV-infected identical twins.

This phase I/II pilot project will evaluate the survival, tolerance, safety, and efficacy of infusions of activated, gene marked, syngeneic T lymphocytes obtained from HIV seronegative identical twins on the functional immune status of HIV infected twin recipients. T cells from each seronegative twin will be obtained by periodic apheresis, separated into CD4 and CD8 enriched populations by monoclonal antibody affinity binding techniques, induced to polyclonal proliferation with anti-CD3 and rIL-2 stimulation, transduced with distinctive neoR retroviral vectors, and expanded 10-1,000 fold in numbers during approximately 2 weeks of culture. These marked T cell fractions will then be infused into the seropositive twins and the survival of the uniquely marked T cell populations will be monitored by vector-specific PCR, while the recipients' functional immune status is monitored by standard in vitro and in vivo testing protocols. A total of 3 cycles of treatment will be given at intervals of 6 weeks between infusions.

Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC).

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.

High prevalence of antibodies against HERV-K10 in patients with testicular cancer but not with AIDS.

Human endogenous retrovirus K10 (HERV-K10) env and gag expression has been detected in placenta, embryonic tissue, and cell lines. By transfection, these sequences have been expressed in insect cells and developed into serological assays, revealing HERV-K10 antibodies in patients with testicular cancer. Patients with AIDS are at an increased risk for testicular cancer and frequently reactivate latent infections. We postulated that HERV-K10 seroprevalence might be increased with HIV infection or AIDS. Stored, frozen serum samples from 52 patients with testicular cancer (8 patients with HIV and 30 patients with samples near the time of diagnosis) and 84 controls (40 patients with HIV) were diluted 1:40 and tested by immunofluorescence against SF158 cells transfected with HERV-K10 env [ENV1.9(+)] or gag (pACGAG). Seroprevalence rates were compared cross-sectionally in cases and controls, excluding those with indeterminate results (3 of 30 cases and 7 of 84 controls), and also were examined longitudinally in the cases before or after diagnosis of testicular cancer. Seroprevalence to HERV-K10 Env or Gag was 17 of 27 testicular cancer patients (63%) around the time of diagnosis, compared to 4 of 77 controls (5%; P < 0.0001). Seroprevalence was similar (50% to 60%) with seminoma, teratocarcinoma, or embryonal carcinoma, and it was not increased with HIV infection in either cases (33%) or controls (3%). HERV-K10 antibodies were detected in 12 of 19 cases (63%) more than 6 months before seminoma diagnosis, as well as in four cases with residual or recurrent malignancy more than 1 month after initial diagnosis. Thus, HERV-K10 antibodies are detected frequently with testicular cancer and seem to resolve rapidly with effective therapy of the malignancy. Antibody reactivity also occurs in approximately 5% of controls, perhaps because of nonspecific or cross-reactive epitopes. HIV and AIDS were not associated with HERV-K10 antibodies, thus, leaving their higher risk of testicular cancer unexplained.

A simplified automated procedure for generation of human lymphokine-activated killer cells for use in clinical trials.

The administration of lymphokine-activated killer (LAK) cells along with interleukin-2 (IL-2) can mediate regression of tumors in selected patients. A closed automated system utilizing commercial blood cell separators has been developed for washing and Ficoll-Hypaque (FH) separation of lymphocytes, for lymphocyte culture in polyolefin bags, and for concentration of LAK cells out of culture prior to infusion. We now demonstrate that preparation of LAK cells can be simplified by elimination of the FH sedimentation step. Patient leukapheresis was performed using Fenwal CS-3000 blood cell separators, with a mean cellular yield per procedure of 54 X 10(9) WBC (95% lymphocytes), 184 X 10(9) RBC, and 306 X 10(9) platelets (n = 22). These cells were then washed in the same apheresis kit with a counter-current flow of saline, thereby eliminating 85% of platelets while retaining 88% of WBC. Aliquots of the washed cells were separated on FH gradients in 50 ml centrifuge tubes, and both FH-separated and washed-only cells were cultured at 3 X 10(6)/ml with 1500 U/ml IL-2 in polyolefin bags. Cytotoxicities of 22 preparations of LAK cells from 14 patients were evaluated in 4 h 51Cr release assays. Cells that were washed-only averaged 47, 35, and 9 lytic units/10(6) cells against K562, Daudi, and fresh tumor, while FH separated cells averaged 46, 33, and 6 lytic units/10(6) cells respectively. Cellular recoveries using the wash-only technique were 25% greater than when using FH sedimentation. Omission of FH separation saves time and expense in preparation and provides greater numbers of LAK cells for use in adoptive immunotherapy.

Development of an automated closed system for generation of human lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) can mediate tumor regression in some patients with advanced cancer. The activation of large numbers of LAK cells was performed in roller bottles in a research laboratory setting and required meticulous aseptic technique, at least one skilled technician per patient and one laminar flow hood per patient. To reduce the complexity and expense of LAK cell generation for human immunotherapy trials we have developed a closed-system automated procedure using a continuous flow blood cell separator. PBL were obtained by standard apheresis techniques. Platelets and plasma were elutriated using countercentrifugal flow of saline in the cell separator machine. The washed PBL were underlaid with Ficoll-Hypaque (FH) in the original separation bag. Lymphocytes were then flushed into a collection bag where they were concentrated and washed with 2 liters of saline. Mean recovery from the automated FH technique was 54.6 +/- 4.3% compared to 62.3 +/- 4.0% using manual methods in 50 ml tubes (P greater than 0.05). Cells were diluted in the collection bag with RPMI 1640 +/- 2% human AB serum and could be dispensed in an automated fashion to polyolefin bags via a sample port with 1000-1500 U/ml IL-2. After 3-4 days of culture in 5% CO2 at 37 degrees C, activated cells from the bags were harvested and washed in a closed system using the continuous flow cell separator. Cell yield from the harvest was 79.2 +/- 5.4% in the automated system compared to 64.9 +/- 5.0% in the standard procedure using manual harvest of roller bottles (P less than 0.01). Lytic capacity of the cells against fresh human tumor in a 4 h 51Cr release assay was equivalent in cells processed either by the automated or the conventional manual method. The advantages of a closed system include decreased potential for microbial contamination and reduced labor and capital equipment costs. This technique may be easily adapted for use with other cell collection and culture systems.

Treatment of refractory familial hypercholesterolemia by low-density lipoprotein apheresis using an automated dextran sulfate cellulose adsorption system. The Liposorber Study Group.

A subgroup of patients with familial hypercholesterolemia (FH) respond inadequately to standard diet and drug therapy, and are therefore at high risk for the premature development or progression of coronary artery disease. This study evaluated low-density lipoprotein (LDL) cholesterol and lipoprotein (a) removal in a multicenter, controlled trial with a new LDL apheresis procedure (Liposorber LA-15 System). The study comprised patients with FH who had not responded adequately to diet and maximal drug therapy. There were 54 patients with heterozygous FH (45 randomized to treatment and 9 control subjects) and 10 with homozygous FH (all of whom received LDL apheresis). The study included three 6-week treatment phases and a 4-week rebound phase. Treatments were administered at 7- to 14-day intervals. Mean acute reductions in LDL cholesterol were 76% in heterozygous FH patients and 81% in homozygous ones. Time-averaged levels of LDL cholesterol were reduced 41% (243 to 143 mg/dl) in heterozygous FH patients and 53% (447 to 210 mg/dl) in homozygous ones. The substantial acute reduction of lipoprotein (a) (means: 65%, heterozygous FH; 68%, homozygous FH) has not been reported with other therapies. The Liposorber LA-15 System represents an important therapeutic option in FH patients who respond inadequately to diet and drug therapy.

Long-term effects of low-density lipoprotein apheresis using an automated dextran sulfate cellulose adsorption system. Liposorber Study Group.

The short-term effectiveness of low-density lipoprotein (LDL) apheresis using a dextran sulfate cellulose adsorption column technique was previously examined in a 9-center, 22-week controlled trial in 64 patients with familial hypercholesterolemia (FH) who did not adequately respond to diet and drug therapy. Forty-nine patients (40 treatment, 9 controls) subsequently received LDL apheresis procedures as part of an optional follow-up phase. This study reports on the long-term safety, lipid lowering, and clinical efficacy of LDL apheresis for the 5-year period that includes both the initial controlled study and follow-up phase. During this time, patients received a total of 3,902 treatments of which 3,314 treatments were given during the follow-up phase. Adverse events were infrequent, occurring in 142 procedures (3.6%). Immediate reduction in LDL cholesterol was 76% both in homozygotes and in heterozygotes. Patients with homozygous FH had a progressive decrease in pretreatment LDL cholesterol level along with an increase in high-density lipoprotein (HDL) cholesterol level. There was no appreciable change in pretreatment lipoprotein level over time in heterozygotes. The rate of cardiovascular events during therapy with LDL apheresis and lipid-lowering drugs was 3.5 events per 1,000 patient-months of treatment compared with 6.3 events per 1,000 patient-months for the 5 years before LDL apheresis therapy. These findings support the long-term safety and clinical efficacy of LDL apheresis in patients with heterozygous and homozygous FH who are inadequately controlled with drug therapy.

Cyclosporine is required to prevent severe acute GVHD following T-cell-depleted peripheral blood stem cell transplantation.

Reduced immunosuppression may improve immune recovery and increase the graft-versus-leukemia effect after allogeneic stem cell transplantation. Furthermore, the requirement for post-transplant immunosuppression following extensive T-cell depletion remains unclear. We therefore evaluated the role of cyclosporine (CSA) in recipients of HLA-identical T-cell-depleted peripheral blood stem cell transplants (PBSCT), followed by donor lymphocyte infusions (DLIs) scheduled on days +45 and +100. Before day+45, successive cohorts of patients received decreasing amounts of CSA: standard-dose (SD) CSA, low-dose (LD) CSA, or no CSA until day+45. LD CSA was as effective as SD CSA in preventing acute graft-versus-host disease (GVHD). However, moderate-to-severe acute GVHD was significantly more frequent before the day +45 DLI in patients receiving no CSA (33.3 vs 12.7%, P=0.036, including the only four grade III-IV cases). As a result of higher rates of early acute GVHD, more patients in the 'no CSA' group failed to receive any DLI (30.7 vs 7.1%, P=0.01). Overall, there was no difference in the incidence of acute GVHD, as patients receiving CSA developed more GVHD after DLI. Similarly, no significant differences were found in chronic GVHD, transplant-related mortality, or survival. These results define a role for CSA in preventing GVHD at low T-cell doses following PBSCT.

Utilization patterns of frozen autologous red blood cells. Experience in a referral center and a community hospital.

The risks of homologous blood transfusion have motivated some blood centers and private industry to consider providing long-term storage of frozen, autologous red blood cells as a service. The usefulness of this practice is unknown. We performed a retrospective analysis of frozen autologous red blood cell use in two hospitals. Records were available for 21- and 9-year intervals, respectively. A total of 104 autologous units were cryopreserved for 41 patients. Fifteen (37%) of 41 patients received one or more of their stored units of red blood cells. Twenty-two patients had autologous units frozen in anticipation of elective surgery; 11 (50%) of these 22 patients received some or all of their stored units. Sixteen patients had autologous units stored because of potential transfusion problems related to rare blood types or to the presence of multiple blood cell alloantibodies, and another 3 patients had units frozen simply at their personal request. Only 4 (21%) of these latter 19 patients who donated without a specific planned use eventually received their frozen autologous red blood cells. Long-term autologous frozen red blood cell storage can improve medical management of some patients with anticipated surgical procedures or unusual requirements for transfusion. However, our study suggests that most autologous units frozen without specific planned use will not be transfused.

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.