Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

ABO genotyping in leukemia patients reveals new ABO variant alleles.

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

Hyperhomocystinemia in patients with coronary artery disease.

Hyperhomocystinemia has been related to an increased risk of cardiovascular disease in several studies. The C677T polymorphism for the gene that encodes the methylenetetrahydrofolate reductase enzyme (MTHFR) and low plasma folate levels are common causes of hyperhomocystinemia. Due to differences in nutritional patterns and genetic background among different countries, we evaluated the role of hyperhomocystinemia as a coronary artery disease (CAD) risk factor in a Brazilian population. The relation between homocysteine (Hcy) and the extent of CAD, measured by an angiographic score, was determined. A total of 236 patients referred for coronary angiography for clinical reasons were included. CAD was found in 148 (62.7%) patients and 88 subjects had normal or near normal arteries. Patients with CAD had higher Hcy levels [mean (SD)] than those without disease (14 (6.8) vs 12.5 (4.0) microM; P = 0.04). Hyperhomocystinemia (Hcy >17.8 microM) prevalence was higher in the CAD group: 31.1 vs 12.2% (P = 0.01). After adjustment for major risk factors, we found an independent association between hyperhomocystinemia and CAD (OR = 2.48; 95% CI = 1.02-6.14). Patients with a more advanced coronary score had a higher frequency of hyperhomocystinemia and tended to have higher mean Hcy levels. An inverse relation between plasma folate and Hcy levels was found (r = -0.14; P = 0.04). Individuals with the MTHFR C677T polymorphism had a higher prevalence of hyperhomocystinemia than those without the mutated allele. We conclude that hyperhomocystinemia is independently associated with CAD, with a positive association between Hcy level and disease severity.

Results of CHOP chemotherapy for diffuse large B-cell lymphoma.

Patients with diffuse large B-cell lymphoma treated in a University Hospital were studied from 1990 to 2001. Two treatment regimens were used: ProMACE-CytaBOM and then, from November 1996 on, the CHOP regimen. Complete remission (CR), disease-free survival (DFS), and overall survival (OS) rates were determined. Primary refractory patients and relapsed patients were also assessed. A total of 111 patients under 60 years of age were assessed and ranked according to the international prognostic index adjusted to age. Twenty (18%) of them were classified as low risk, 40 (36%) as intermediate risk, 33 (29.7%) as high intermediate risk, and 18 (16.3%) as high risk. Over a five-year period, OS and DFS rates were 71 and 59%, respectively, for all patients. For the same time period, OS and DFS rates were 72.8 and 61.3%, respectively, for 77 patients treated with CHOP chemotherapy and 71.3 and 60% for patients treated with the ProMACE-CytaBOM protocol. There was no significant difference in OS or DFS between the two groups. Eleven of 50 refractory and relapsed patients were consolidated with high doses of chemotherapy. Three received allogenic and 8 autologous bone marrow transplantation. For the latter, CR was 62.5% and mean OS was 41.1 months. The clinical behavior, CR, DFS, and OS of the present patients were similar to those reported in the literature. We conclude that both the CHOP and ProMACE-CytaBOM protocols can be used to treat diffuse large B-cell lymphoma patients, although the CHOP protocol is preferable because of its lower cost and lower toxicity.

Plasma kinetics and biodistribution of a lipid emulsion resembling low-density lipoprotein in patients with acute leukemia.

Low-density lipoprotein (LDL) could be used as a carrier of chemotherapeutic agents to neoplastic cells that overexpress LDL receptors (rLDL), but LDL is difficult to obtain and handle. Recently, it was observed that a protein-free emulsion resembling the lipid portion of LDL (LDE) behave like native LDL when injected into the bloodstream. In this study, the evidence that LDE is taken up by rLDL was expanded by comparing LDL and LDE plasma decay curves in rabbits and by competition experiments with lymphocytes. To verify whether LDE could be removed from the plasma by neoplastic cells with increased rLDL, LDE labeled with 14Ccholesteryl ester was injected into 14 patients with acute myeloid leukemia (AML) and into 7 with acute lymphocytic leukemia (ALL). In AML rLDL expression is increased but in ALL it is normal. LDE plasma fractional clearance rate (FCR, in h-1) was calculated from the remaining radioactivity measured in plasma samples collected during 24 h following injection. LDE FCR was 3-fold greater in AML than in ALL patients 0.192 +/- 0.210 (SD) and 0.066 +/- 0.033 h-1, respectively, P < 0.035. When LDE injection was repeated in 9 AML patients in hematological remission, LDE FCR diminished 66% compared to the pretreatment values (from 0.192 +/- 0.210 to 0.065 +/- 0.038 h-1, P < 0.02), so that it could be estimated that nearly 66% of the emulsion was taken up by AML cells and only 34% by the normal tissues. As expected, LDE FCR was unchanged in 4 patients with ALL in hematological remission (0.069 +/- 0.044 h-1). Gamma camera images obtained 6 h after the injection of 99mTc-label LDE into one patient with ALL showed biodistribution similar to that of LDL. In one AML patient LDE was comparatively more concentrated over the areas corresponding to the bone marrow infiltrated by AML cells. Our results indicate that LDE FCR is increased in a disease known to contain malignant cells that overexpress rLDL, suggesting that LDE is taken up by malignant cells with increased rLDL.

Possible benefit of consolidation therapy with high-dose cytarabine on overall survival of adults with non-promyelocytic acute myeloid leukemia.

In adults with non-promyelocytic acute myeloid leukemia (AML), high-dose cytarabine consolidation therapy has been shown to influence survival in selected patients, although the appropriate doses and schemes have not been defined. We evaluated survival after calculating the actual dose of cytarabine that patients received for consolidation therapy and divided them into 3 groups according to dose. We conducted a single-center, retrospective study involving 311 non-promyelocytic AML patients with a median age of 36 years (16-79 years) who received curative treatment between 1978 and 2007. The 131 patients who received cytarabine consolidation were assigned to study groups by their cytarabine dose protocol. Group 1 (n=69) received <1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles. The remaining patients received high-dose cytarabine (≥1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles). The actual dose received during the entire consolidation period in these patients was calculated, allowing us to divide these patients into 2 additional groups. Group 2 (n=27) received an intermediate-high-dose (<27 g/m2), and group 3 (n=35) received a very-high-dose (≥27 g/m2). Among the 311 patients receiving curative treatment, the 5-year survival rate was 20.2% (63 patients). The cytarabine consolidation dose was an independent determinant of survival in multivariate analysis; age, karyotype, induction protocol, French-American-British classification, and de novo leukemia were not. Comparisons showed that the risk of death was higher in the intermediate-high-dose group 2 (hazard ratio [HR]=4.51; 95% confidence interval [CI]: 1.81-11.21) and the low-dose group 1 (HR=4.43; 95% CI: 1.97-9.96) than in the very-high-dose group 3, with no significant difference between those two groups. Our findings indicated that very-high-dose cytarabine during consolidation in adults with non-promyelocytic AML may improve survival.

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis.

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.

Addition of low-dose busulfan to cyclophosphamide in aplastic anemia patients prior to allogeneic bone marrow transplantation to reduce rejection.

Busulfan was added at the dose of 4 mg/kg to 200 mg/kg cyclophosphamide in 81 patients (3-53 years, median 24) with aplastic anemia to reduce graft rejection. Graft-versus-host disease (GVHD) prophylaxis comprised cyclosporine-methotrexate. The number of prior transfusions was 0-276 (median 26), and 48% had received prior immunosuppressive therapy. Two patients experienced primary graft failure, and 10 secondary rejection at 28-1001 days (median 317 days). The cumulative incidence of rejection was 22%; for heavily transfused patients (>/=50 U) it was 43% compared to 16% for the rest (P=0.06). Overall survival rate at 8 years was 56%; patients who received 15 transfusions was 78 and 50%, respectively (P=0.01), whereas it was 67 and 28% for 50 transfusions, respectively (P=0.002). In multivariate analysis, higher number of prior transfusions, shorter period of immunosuppression with cyclosporine and GVHD were associated with inferior survival; moreover, a higher risk of graft rejection were associated with a higher number of prior transfusions and a trend was observed for a shorter cyclosporine administration. Low-dose busulfan is feasible and may be helpful in patients exposed to <50 transfusions. However, rejection remains a significant problem, mainly in heavily transfused patients.

Unexpected low prevalence of delta antibodies in the east Amazon region and São Paulo: evidence for regional differences in the epidemiology of delta hepatitis virus within Brazil.

Antibodies (anti-HD) to hepatitis delta virus (HDV) were tested by radioimmunoassay in 207 human serum samples from the eastern Amazon (states of Pará and Amapá) and São Paulo, Brazil. 42 Amazon HBsAg asymptomatic carriers were negative for anti-HD. 84 São Paulo HBsAg asymptomatic carriers were also negative. Among the 81 HBsAg patients from São Paulo with different liver diseases, only one had anti-HD. Liver biopsy of this chronic active hepatitis case was positive for HBsAg, HBcAg and HDAg in liver, by an immunoperoxidase technique. The low prevalence of HDV infections in São Paulo and eastern Amazon was unexpected and contrasts with the recent reports of high prevalence in the western Amazon region. Such regional differences emphasize the need for extensive and precise worldwide epidemiological studies of HDV.

Aggregation of platelets in whole blood from children with pulmonary hypertension.

The response of aggregation of platelets to adenosine diphosphate (7.5-120 microM) and collagen (1.25 micrograms/ml) was assessed in whole blood (impedance method) in 10 children with pulmonary hypertension (hematocrit range, 42 to 71%). The response to collagen was normal (9.08 +/- 3.47 vs. 10.36 +/- 1.86 ohms in controls, P = NS) while there was a decreased response to adenosine diphosphate (6.98 +/- 3.83 vs. 11.21 +/- 2.02 ohms, P less than 0.01), in spite of high concentrations of the inducer. Lowering the hematocrit in vitro to 40% with autologous platelet-rich plasma resulted in a rise in the platelet count from 171 +/- 63 to 225 +/- 84 x 10(9) platelets/1 (P less than 0.001) and a significant increase in the response to adenosine diphosphate from 6.98 +/- 3.83 to 9.89 +/- 3.66 ohms (P less than 0.02). As in the baseline condition, high concentrations of adenosine diphosphate were required. The response to collagen did not change significantly. The results indicate that aggregatory response of platelets is relatively preserved in these children. The decreased response to adenosine diphosphate may be a result of a low count and interference of red cells on the accretion of platelets on the electrodes. Because high concentrations of adenosine diphosphate were still required after hemodilution to achieve an aggregatory response close to normal, we speculate that leakage of endogenous adenosine diphosphate from red cells may have accounted for partial activation of the platelets, resulting in a relative refractory state to in vitro stimulation.

Papaverine inhibits human platelet aggregation induced by ADP.

1. The in vitro and ex vivo effect of therapeutic levels of papaverine on human platelet aggregation induced by 3-5 microM adenosine-5'-diphosphate (ADP) was evaluated in platelet-rich plasma (PRP) by photometric and impedance aggregometry, and in whole blood by impedance aggregometry. 2. Platelet aggregation induced by 3-5 microM ADP in whole blood was significantly inhibited by 5.32 and 10.64 microM papaverine in vitro. This effect was also observed in PRP enriched with erythrocytes but not in PRP alone or enriched with leukocytes. 3. Papaverine (5.32 microM) significantly enhanced the antiplatelet activity of adenosine (0.75 microM) in human whole blood, an effect that was not observed in PRP. 4. A single oral dose of 100 mg papaverine hydrochloride, given to eight healthy human volunteers 1 h before the platelet aggregation evaluation, significantly inhibited the platelet aggregation induced by 3-5 microM ADP in whole blood. This effect was not observed in PRP. 5. Oral administration of the same dose at 8-h intervals (10 times) to seven additional healthy human volunteers led to a significant negative correlation (r = -0.55, P < 0.01) between the slope of platelet aggregation in whole blood and plasma papaverine levels (0.12-0.75 microM). 6. Papaverine and adenosine, alone or together, had no in vitro effect on whole blood platelet aggregation of male Wistar rats measured by impedance aggregometry. 7. These results suggest that papaverine inhibits human platelet aggregation in whole blood by an interaction with red blood cells.

An aqueous extract of guaraná (Paullinia cupana) decreases platelet thromboxane synthesis.

The effects of an aqueous extract of guaraná (Paullinia cupana) on rabbit platelet aggregation and thromboxane synthesis were examined. The guaraná extract (100 mg/ml) and fractions separated by TLC (origin and xanthines) decreased platelet aggregation (37, 27 and 31% of control values, respectively) and platelet thromboxane formation from [14C]-arachidonic acid (78, 70 and 50% of control values, respectively). The decreased thromboxane synthesis could be responsible, at least in part, for the antiaggregatory action of guaraná.

Platelet regeneration time: a statistical approach.

The determination of platelet regeneration half-time (PRT t1/2) by measuring malondialdehyde after intake of acetylsalicylic acid is a simple nonisotopic method for the estimation of platelet survival. There is no available information concerning the populational distribution of PRT t1/2. Consequently, there is controversy about the utilization of parametric or nonparametric statistical tests in studies of PRT. In the present study, we demonstrate the closeness of the fit of log PRT t1/2 to the normal (Gaussian) distribution.

Aluminum induces lipid peroxidation and aggregation of human blood platelets.

Aluminum (Al3+) intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al(3+) -induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL) of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 microM) stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA) (100 microM) and n-propyl gallate (NPG) (100 microM), inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA) (100 microM), an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation.

Plasma anticoagulant system in patients with pulmonary hypertension.

Adults with pulmonary hypertension and polycythemia (N = 22) have low levels of plasma antithrombin III (84 +/- 18 vs 98 +/- 13% for controls, N = 35, P less than 0.005) and protein C (66 +/- 21 vs 125 +/- 30%, N = 8, P less than 0.0002) but normal levels of total protein S. Data are reported as means +/- SD and percent normal values obtained for pooled plasma from normal healthy adults. Children with the same disorder (N = 6) also had low protein C levels (66 +/- 16 vs 85 +/- 5%, P less than 0.025). Total protein S was normal for children, but free protein S was decreased (66 +/- 13 vs 91 +/- 23%, P less than 0.025). Since the levels observed in these patients are above those reported for congenital deficiencies, the reduction in plasma levels of anticoagulant proteins may be the result of chronic intravascular coagulation. Furthermore, normal levels of plasminogen and fibrin degradation products suggest a localized disorder or an acquired decrease in fibrinolytic activity.

PAF-acether, superoxide anion and beta-glucuronidase as parameters of polymorphonuclear cell activation associated with cardiac surgery and cardiopulmonary bypass.

1. Seven patients submitted to myocardial revascularization surgery with cardiopulmonary bypass were studied. Blood samples were obtained immediately before and 24 h after surgery. The parameters studied were the production of platelet activating factor (PAF-acether) and superoxide anion, cellular beta-glucuronidase activity as well as polymorphonuclear cell (PMN) and platelet counts. 2. Twenty-four h after surgery, there was a 54% decrease in platelet number (P less than 0.005), a 121% increase in PMN number (P less than 0.005), a 353% increase in PAF-acether (P less than 0.01), a 211% increase in superoxide anion (O2-) and a 104% increase in beta-glucuronidase (P less than 0.05) levels when compared with the pre-surgery levels. 3. The present results indicate that PMN are more reactive after surgery with cardiopulmonary bypass.

Increased plasma removal of microemulsions resembling the lipid phase of low-density lipoproteins (LDL) in patients with acute myeloid leukemia: a possible new strategy for the treatment of the disease.

A microemulsion of lipid composition resembling low-density lipoprotein (LDL), but devoid of apolipoproteins and labeled with [14C]-cholesteryl oleate was injected into 16 healthy subjects and into 15 patients with acute myeloid leukemia (AML). Removal from plasma of the lipid label was higher in the leukemic group compared to healthy subjects in terms of fractional clearance rate (0.185 +/- 0.205 and 0.080 +/- 0.030 h-1, respectively, P < 0.03). When the emulsion was again injected into 10 of the AML patients after complete hematological remission, the fractional clearance rate of cholesteryl ester was reduced to one third of the value observed prior to treatment (0.061 +/- 0.038 h-1) and was not different from that obtained for the healthy subjects. Also, in untreated AML patients, serum LDL-cholesterol levels inversely correlated with the values of fractional clearance rate of the microemulsion. This correlation was no longer observed after treatment. These data suggest that the LDL-like microemulsion was selectively taken up by the neoplastic cells presumably by interaction with LDL receptors. Therefore, microemulsions may function as potential carriers for anticancer drugs that are targeted to tumor cells for patients with acute myeloid leukemia. Unlike native LDL, microemulsions are suitable for utilization in routine clinical practice.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells.

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

Diclofenac plasma protein binding: PK-PD modelling in cardiac patients submitted to cardiopulmonary bypass.

Twenty-four surgical patients of both sexes without cardiac, hepatic, renal or endocrine dysfunctions were divided into two groups: 10 cardiac surgical patients submitted to myocardial revascularization and cardiopulmonary bypass (CPB), 3 females and 7 males aged 65 +/- 11 years, 74 +/- 16 kg body weight, 166 +/- 9 cm height and 1.80 +/- 0.21 m2 body surface area (BSA), and control, 14 surgical patients not submitted to CPB, 11 female and 3 males aged 41 +/- 14 years, 66 +/- 14 kg body weight, 159 +/- 9 cm height and 1.65 +/- 0.16 m2 BSA (mean +/- SD). Sodium diclofenac (1 mg/kg, im Voltaren 75 twice a day) was administered to patients in the Recovery Unit 48 h after surgery. Venous blood samples were collected during a period of 0-12 h and analgesia was measured by the visual analogue scale (VAS) during the same period. Plasma diclofenac levels were measured by high performance liquid chromatography. A two-compartment open model was applied to obtain the plasma decay curve and to estimate kinetic parameters. Plasma diclofenac protein binding decreased whereas free plasma diclofenac levels were increased five-fold in CPB patients. Data obtained for analgesia reported as the maximum effect (EMAX) were: 25% VAS (CPB) vs 10% VAS (control), P < 0.05, median measured by the visual analogue scale where 100% is equivalent to the highest level of pain. To correlate the effect versus plasma diclofenac levels, the EMAX sigmoid model was applied. A prolongation of the mean residence time for maximum effect (MRTEMAX) was observed without any change in lag-time in CPB in spite of the reduced analgesia reported for these patients, during the time-dose interval. In conclusion, the extent of plasma diclofenac protein binding was influenced by CPB with clinically relevant kinetic-dynamic consequences.

Absence of hemolysis after a kidney transplant in an E+ recipient from a donor with anti-E.

A 12-year-old Caucasian male with cystinosis received a kidney from his mother, whose red blood cells typed as group O, D+, E-. Her serum contained an anti-E with an IgG1 titer of 16 (score 31). The recipient's type was group O, D+, E+, with a negative antibody screen in the pretransplant period. The recipient and donor Rh phenotypes were most likely DCcEe and Dccee, respectively. Because the recipient's mother had no transfusion history, she was probably immunized by the fetal red blood cells of her one pregnancy (the recipient). The kidney had been immediately perfused with saline after removal from the donor. No acute or delayed hemolysis was observed clinically or in laboratory tests performed immediately after the transplant and at 7, 15, and 30 days after the transplant. Antibody screens were still negative at 6 months. In this case, anti-E was not present in the transplanted kidney in sufficient concentration to cause hemolysis of the recipient's red blood cells and transplanted lymphocytes did not synthesize sufficient anti-E to be detectable or to cause hemolysis.