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In the phase 3 Evaluating Nilotinib Efficacy and Safety in Clinical Trials-Newly Diagnosed Patients (ENESTnd) study, nilotinib resulted in earlier and higher response rates and a lower risk of progression to accelerated phase/blast crisis (AP/BC) than imatinib in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP). Here, patients' long-term outcomes in ENESTnd are evaluated after a minimum follow-up of 5 years. By 5 years, more than half of all patients in each nilotinib arm (300 mg twice daily, 54%; 400 mg twice daily, 52%) achieved a molecular response 4.5 (MR(4.5); BCR-ABL⩽0.0032% on the International Scale) compared with 31% of patients in the imatinib arm. A benefit of nilotinib was observed across all Sokal risk groups. Overall, safety results remained consistent with those from previous reports. Numerically more cardiovascular events (CVEs) occurred in patients receiving nilotinib vs imatinib, and elevations in blood cholesterol and glucose levels were also more frequent with nilotinib. In contrast to the high mortality rate associated with CML progression, few deaths in any arm were associated with CVEs, infections or pulmonary diseases. These long-term results support the positive benefit-risk profile of frontline nilotinib 300 mg twice daily in patients with CML-CP.

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.

Background: We evaluated the clinical, laboratory, and prognostic factors in adolescent and adult patients with acute lymphoblastic leukemia (ALL)., Materials and Methods: In this observational, retrospective, cross-sectional study, we examined the medical records of all consecutive patients with ALL admitted to a public hospital in Brazil from 1990 to 2005., Results: Of the 102 patients included, 88 were treated with 2 protocols of chemotherapy (Berlin-Frankfurt-Münster [BFM] 86 modified [BFM-86M] and UCLA [University of California, Los Angeles] protocol). The complete remission (CR), disease-free survival, and overall survival (OS) rate was 70.6%, 27%, and 30.5%, respectively (median follow-up, 49 months). Age < 18 years and no leukemic infiltration in the central nervous system (CNS) at diagnosis were positively associated with CR (P = .03); no bleeding and hepatomegaly at diagnosis and age < 35 years were associated with better OS on multivariate analyses of the whole population (P = .01). OS at 4 years was superior with BFM-86M than with UCLA (49.5% vs. 16%; P = .004), especially in young adults without risk factors., Conclusion: We identified age as the most important prognostic factor in patients with ALL. CNS infiltration, hepatomegaly, and bleeding were associated with lower OS but must be validated in future research with South American populations and worldwide. The BFM-86M protocol can be considered a therapeutic option for young adults (age < 35 years) without adverse prognostic factors. For other patients with ALL, we emphasize the need for different therapeutic approaches., (Published by Elsevier Inc.)

Patients in complete cytogenetic response (CCyR) with detectable BCR-ABL1 after ≥2 years on imatinib were randomized to nilotinib (400 mg twice daily, n = 104) or continued imatinib (n = 103) in the Evaluating Nilotinib Efficacy and Safety in clinical Trials-Complete Molecular Response (ENESTcmr) trial. By 1 and 2 years, confirmed undetectable BCR-ABL1 was achieved by 12.5% vs 5.8% (P = .108) and 22.1% vs 8.7% of patients in the nilotinib and imatinib arms, respectively (P = .0087). Among patients without molecular response 4.5 (BCR-ABL1(IS) ≤0.0032%; MR(4.5)) and those without major molecular response at study start, MR(4.5) by 2 years was achieved by 42.9% vs 20.8% and 29.2% vs 3.6% of patients in the nilotinib and imatinib arms, respectively. No patient in the nilotinib arm lost CCyR, vs 3 in the imatinib arm. Adverse events were more common in the nilotinib arm, as expected with the introduction of a new drug vs remaining on a well-tolerated drug. The safety profile of nilotinib was consistent with other reported studies. In summary, switching to nilotinib enabled more patients with chronic myeloid leukemia in chronic phase (CML-CP) to sustain lower levels of disease burden vs remaining on imatinib. This trial was registered at www.clinicaltrials.gov as #NCT00760877., (© 2014 by The American Society of Hematology.)

Background: In Brazil, malaria is endemic in the Amazon River basin and non-endemic in the extra-Amazon region, which includes areas of São Paulo state. In this state, a number of autochthonous cases of malaria occur annually, and the prevalence of subclinical infection is unknown. Asymptomatic infections may remain undetected, maintaining transmission of the pathogen, including by blood transfusion. In these report it has been described subclinical Plasmodium infection in blood donors from a blood transfusion centre in São Paulo, Brazil., Methods: In this cross-sectional study, representative samples of blood were obtained from 1,108 healthy blood donors at the Fundação Pró-Sangue Hemocentro de São Paulo, the main blood transfusion centre in São Paulo. Malaria exposure was defined by the home region (exposed: forest region; non-exposed: non-forest region). Real-time PCR was used to detect Plasmodium falciparum and Plasmodium vivax. Subclinical malaria cases were geo-referenced., Results: Eighty-four (7.41%) blood donors tested positive for Plasmodium; 57 of these were infected by P. falciparum, 25 by P. vivax, and 2 by both. The prevalence of P. falciparum and P. vivax was 5.14 and 2.26, respectively. The overall prevalence ratio (PR) was 3.23 (95% confidence interval (CI) 2.03, 5.13); P. falciparum PR was 16.11 (95% CI 5.87, 44.21) and P. vivax PR was 0.47 (95% CI 0.2, 1.12). Plasmodium falciparum subclinical malaria infection in the Atlantic Forest domain was present in the mountain regions while P. vivax infection was observed in cities from forest-surrounded areas., Conclusions: The presence of Plasmodium in healthy blood donors from a region known as non-endemic, which is important in the context of transfusion biosafety, was described. Infected recipients may become asymptomatic carriers and a reservoir for parasites, maintaining their transmission. Furthermore, P. falciparum PR was positively associated with the forest environment, and P. vivax was associated with forest fragmentation.

Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients., Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units., Findings: 14 patients had B. multivorans (one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred., Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.

Nilotinib has shown favorable safety in patients with Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic (CML-CP) or accelerated phase (CML-AP) who failed prior imatinib, and superior efficacy over imatinib in newly diagnosed Ph+ patients with CML-CP. Reported here are the efficacy and safety data for patients in CML-AP (n = 181) or blast crisis (CML-BC) (n = 190; myeloid BC, 133; lymphoid BC, 50; unknown, seven) enrolled in an expanded access phase IIIb study. Non-hematologic adverse events were mostly mild to moderate. Drug-related myelosuppression was generally manageable with dose reductions or interruptions and infrequently led to discontinuation of nilotinib. Drug-related grade 3/4 elevations in serum bilirubin and lipase were infrequent. While an analysis of efficacy was not the primary objective of this study, significant hematologic and cytogenetic responses were observed. These results support the safety and efficacy of nilotinib in patients with advanced CML in AP and BC.

Despite the beneficial effects of imatinib mesylate, some patients may either not respond or respond suboptimally. Here, we report two chronic myelogenous leukemia patients; one had a suboptimal response according to European LeukemiaNet criteria (a major molecular response was not achieved after 18 months of standard-dose imatinib therapy) and the other had failure with a standard dose of imatinib. At the time of the suboptimal response in patient 1 and the failure in patient 2, we were able to detect the F359I mutation in the BCR-ABL tyrosine kinase domain using DNA sequencing in both patients. Therefore, it was decided to change the therapeutic regimen to dasatinib at a dose of 100 mg once daily in both patients. This change resulted in the achievement of complete cytogenetic remission in patient 1 after 4 months and a major molecular response within 2 and 3 months in both patients. Detection of the F359I mutation in our two cases likely explains the suboptimal response to imatinib in case 1 and the failure in case 2. This implies that in such cases dasatinib should be considered to effectively suppress the mutated clones., (Copyright © 2011 S. Karger AG, Basel.)

Here we compare the management and survival outcomes of chronic myeloid leukemia (CML) patients who had early or late imatinib mesylate (IM) therapy. The cytogenetic and molecular responses of 189 CML patients were analyzed. Of this group, 121 patients were classified as the early chronic phase (ECP) group and started IM within 12 months of diagnosis. The other 68 patients were classified as the late chronic phase (LCP) group who had been treated with interferon (IFN)-alpha-2 and crossed over to IM more than 12 months after diagnosis. The overall rates of complete cytogenetic response (CCyR) and major molecular response (MMR) at last follow-up were 83.6 and 78.1% in the ECP and LCP groups, respectively. The CCyR rates were 89.3 (for ECP patients) versus 73.5% (for LCP patients; p < 0.0001). At last follow-up, 82.4% ECP and 64.2% LCP patients had achieved an MMR (p < 0.0001). No significant differences were noted between the two groups with regard to survival outcomes. Our experience reveals that IM is an effective rescue therapy in most CML LCP patients who are intolerant or in whom IFN-alpha therapy fails. Such therapeutic options should be considered in LCP patients, particularly in countries where IM may not be available., (Copyright © 2012 S. Karger AG, Basel.)

The purpose of this study was to evaluate outcomes such as success of the initial therapy, failure of outpatient treatment, and death in outpatient treatment during intravenous antimicrobial therapy in patients with febrile neutropenia (FN) and hematological malignancies. In addition, clinical and laboratory data and the Multinational Association for Supportive Care of Cancer index (MASCC) were compared with failure of outpatient treatment and death. In a retrospective study, we evaluated FN following chemotherapy events that were treated initially with cefepime, with or without teicoplanin and replaced by levofloxacin after 48 h of defervescence in patients with good general conditions and ANC>500/mm3. Of the 178 FN episodes occurred in 126 patients, we observed success of the initial therapy in 63.5% of the events, failure of outpatient treatment in 20.8%, and death in 6.2%. The success rate of oral levofloxacin after defervescence was 99% (95 out of 96). Using multivariate analysis, significant risks of failure of outpatient treatment were found to be smoking (odds ratio (OR) 3.14, confidence interval (CI) 1.14-8.66; p=0.027) and serum creatinine levels>1.2 mg/dL (OR 7.97, CI 2.19-28.95; p=0.002). With regard to death, the risk found was oxygen saturation by pulse oximetry<95% (OR 5.8, IC 1.50-22.56; p=0.011). Using the MASCC index, 165 events were classified as low risk and 13 as high risk. Failure of outpatient treatment was reported in seven (53.8%) high-risk and 30 (18.2%) low-risk episodes (p=0.006). In addition, death occurred in seven (4.2%) low-risk and four (30.8%) high-risk events (p=0.004). Ours results show that MASCC index was able to identify patients with high risk. In addition, non-smoking, serum creatinine levels≤1.2 mg/dL, and oxygen saturation by pulse oximetry≥95% were protection factors.

Background: The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment., Methods: The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols., Results: Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR., Conclusions: Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.

Background: Dasatinib 100 mg once daily achieves intermittent BCR-ABL kinase inhibition and is approved for chronic-phase chronic myeloid leukemia patients resistant or intolerant to imatinib. To better assess durability of response to and tolerability of dasatinib, data from a 2-year minimum follow-up for a dose-optimization study in chronic-phase chronic myeloid leukemia are reported here., Design and Methods: In a phase 3 study, 670 chronic-phase chronic myeloid leukemia patients with resistance, intolerance, or suboptimal response to imatinib were randomized to dasatinib 100 mg once-daily, 50 mg twice-daily, 140 mg once-daily, or 70 mg twice-daily., Results: Data from a 2-year minimum follow-up demonstrate that dasatinib 100 mg once daily achieves major cytogenetic response and complete cytogenetic response rates comparable to those in the other treatment arms, and reduces the frequency of key side effects. Comparable 2-year progression-free survival and overall survival rates were observed (80% and 91%, respectively, for 100 mg once daily, and 75%-76% and 88%-94%, respectively, in other arms). Complete cytogenetic responses were achieved rapidly, typically by 6 months. In patients treated with dasatinib 100 mg once daily for 6 months without complete cytogenetic response, the likelihood of achieving such a response by 2 years was 50% for patients who had achieved a partial cytogenetic response, and only 8% or less for patients with minor, minimal, or no cytogenetic response. Less than 3% of patients suffered disease transformation to accelerated or blast phase., Conclusions: Intermittent kinase inhibition can achieve rapid and durable responses, indistinguishable from those achieved with more continuous inhibition.

While chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) are common diseases in the elderly, they rarely occur simultaneously in the same patient. Here we present the case of a 77-year-old patient diagnosed with CML in the chronic phase who showed an optimal response to 400 mg/day of imatinib. This patient progressed to Binet B-CLL with an 11q22.3 deletion and CD38 positivity in the 4th month of treatment. During the follow-up, his lymphocyte number doubled in <6 months. Based on previous reports, dasatinib was chosen instead of imatinib. After 6 months of treatment with 100 mg/day of dasatinib, the patient demonstrated a partial response, characterized by the regression of lymph node enlargement, a hemoglobin level of 10.7 g/dl, neutrophils of 1.7 × 10(9)/l, a 82% reduction in the lymphocyte number and an increase in cytotoxic CD8+ and large granular lymphocytes. This partial response has persisted to the present time. While little data have been published regarding the in vitro effect of dasatinib monotherapy for CLL, this case report provides some evidence of the clinical activity of dasatinib in CLL., (Copyright © 2010 S. Karger AG, Basel.)

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.

The occurrence of chronic myeloid leukemia in pregnancy is rare and its management poses a clinical challenge for physicians treating these patients. We report a 30-year-old woman with chronic myeloid leukemia who became pregnant twice successfully. Philadelphia-positive CML in its chronic phase was diagnosed at 16 weeks of her first gestation. At that time, she received no treatment throughout her pregnancy. At 38 weeks of gestation, a normal infant was delivered by cesarean section. At six weeks postpartum, the patient underwent imatinib mesylate therapy but she could not tolerate the treatment. The treatment was then changed to nilotinib at 400 mg orally b.i.d. Two years later, she became pregnant again while she was on nilotinib 200 mg b.i.d. The unplanned pregnancy was identified during her 7.4 weeks of gestation. Because the patient elected to continue her pregnancy, nilotinib was stopped immediately, and no further treatment was given until delivery. Neither obstetrical complications nor structural malformations in neonates in both pregnancies were observed. Both babies' growth and development have been normal. Although this experience is limited to a single patient, the success of this patient demonstrates that the management of chronic myeloid leukemia in pregnant women may be individualized based on the relative risks and benefits of the patient and fetus.

One limiting factor for automated two-red blood cells collections (2-RBC) is its potential iron depletion. We analyzed hematological parameters and iron balance before, two and four months after 2-RBC of 96 non-supplemented male donors. Four months after 2-RBC, ferritin level was significantly lower (P<0.01) than baseline levels and the number of donors who presented ferritin <30 ng/ml increased from 18 to 47. We concluded that four months was not sufficient for iron recuperation in the population studied. In an attempt to avoid iron depletion after 2-RBC, we recommend augmentation in the interval between blood donations and pre-donation ferritin measurement.

Background: Anemia screening before blood donation requires an accurate, quick, practical, and easy method with minimal discomfort for the donors. The aim of this study was to compare the accuracy of two quantitative methods of anemia screening: the HemoCue 201(+) (Aktiebolaget Leo Diagnostics) hemoglobin (Hb) and microhematocrit (micro-Hct) tests., Study Design and Methods: Two blood samples of a single fingerstick were obtained from 969 unselected potential female donors to determine the Hb by HemoCue 201(+) and micro-Hct using HemataSTAT II (Separation Technology, Inc.), in alternating order. From each participant, a venous blood sample was drawn and run in an automatic hematology analyzer (ABX Pentra 60, ABX Diagnostics). Considering results of ABX Pentra 60 as true values, the sensitivity and specificity of HemoCue 201(+) and micro-Hct as screening methods were compared, using a venous Hb level of 12.0 g per dL as cutoff for anemia., Results: The sensitivities of the HemoCue 201(+) and HemataSTAT II in detecting anemia were 56 percent (95% confidence interval [CI], 46.1%-65.5%) and 39.5 percent (95% CI, 30.2%-49.3%), respectively (p < 0.001). Analyzing only candidates with a venous Hb level lower than 11.0 g per dL, the deferral rate was 100 percent by HemoCue 201(+) and 77 percent by HemataSTAT II. The specificities of the methods were 93.5 and 93.2 percent, respectively., Conclusion: The HemoCue 201(+) showed greater discriminating power for detecting anemia in prospective blood donors than the micro-Hct method. Both presented equivalent deferral error rates of nonanemic potential donors. Compared to the micro-Hct, HemoCue 201(+) reduces the risk of anemic female donors giving blood, specially for those with lower Hb levels, without increasing the deferral of nonanemic potential donors.

We describe the response of imatinib as lifesaving treatment of chronic myeloid leukemia (CML) relapse in seven patients who underwent allogeneic bone marrow transplantation (alloBMT) at our institution over a period of 4 years. Retrospective analysis of their medical records revealed that a mean age at transplant was 45.2 years. The median time to diagnosis was 7.4 years after transplant. At relapse, four, two, and one patients were classified as having hematologic, major molecular, and cytogenetic relapse, respectively. At imatinib initiation, five had CML in a chronic phase, while one patient was diagnosed as having accelerated phase and blast crisis. All these patients could be evaluated for the therapeutic efficacy. At a mean of follow-up of 1.9 years of therapy, all evaluable patients achieved major molecular response without compromising safety. Consistent with available data, our results indicate that imatinib is safe and effective treatment option for patients with relapse after BMT.

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.

Background: The purpose of this retrospective study was to investigate the efficacy, toxicity and mobilization rate after modified Magrath IVAC (mIVAC) chemotherapy regimen prescribed in relapsed disease (RD) or primary refractory disease (PRD) in aggressive non-Hodgkin lymphoma (NHL)., Patients and Methods: Twenty-four patients (16 males, 8 females) aged 18-59 years (median age 37 year) were analyzed. The most frequent histopathological subgroup was diffuse large B-cell lymphoma (DLCL-B) (n=21/24), 13 (54%) were considered RD and 11 (46%) PRD. The mIVAC consisted of ifosfamide (IFM), high dose cytarabine and etoposide repeated every 28 days., Results: The overall response (OR) after three cycles of mIVAC was 66. 6%. Among the patients with PRD, OR was 45.5% (5 out of 11) and with RD was 86.4%, p>0.05, however, it was observed in RD better complete response (CR) than PRD 53.8x9.1% (p<0.05). Eighty-eight percent (14 out of 16) of patients with chemosensitive disease to mIVAC underwent autologous stem cell transplantation (ASCT). The median number of collected CD34+ cells was 2.86x10(6) (range 2.17x10(6) to 4.9x10(6)). The median overall survival rate (OS) for chemosensitive to mIVAC was 16.3 months, with a median follow-up of 16 months. Grades III-IV neutropenia was observed in 85.6% per cycles and grades III-IV thrombocytopenia in 87.5%. Grades III-IV febrile neutropenia was the most common nonhematological toxicity, it occurred in 28% of the cycles and no deaths by toxicity were observed., Discussion: Although a statistic comparative study was not carried out for these 24 patients, the rate of OR to mIVAC was alike the other second-line infusion regimens. The mobilization failure rate was 57.1% and it was similar to other regimens with high dose cytarabine, but it did not limit performed ASCT.

Purpose: Infection is the leading complication of long-term central venous catheters, and its incidence may vary according to catheter type. The objective of this study was to compare the frequency and probability of infection between two types of long-term intravenous devices., Methods: Retrospective study in 96 onco-hematology patients with partially implanted catheters (n = 55) or completely implanted ones (n = 42). Demographic data and catheter care were similar in both groups. Infection incidence and infection-free survival were used for the comparison of the two devices., Results: In a median follow-up time of 210 days, the catheter-related infection incidence was 0.2102/100 catheter-days for the partially implanted devices and 0.0045/100 catheter-days for the completely implanted devices; the infection incidence rate was 46.7 (CI 95% = 6.2 to 348.8). The 1-year first infection-free survival ratio was 45% versus 97%, and the 1-year removal due to infection-free survival ratio was 42% versus 97% for partially and totally implanted catheters, respectively (P <.001 for both comparisons)., Conclusion: In the present study, the infection risk was lower in completely implanted devices than in partially implanted ones.

Gel microcolumn assay (GMA) is a modified serological technique that has been used for ABO and Rh typing, direct antiglobulin test (DAT), detecting alloantibodies, red cell phenotyping, and other applications. However, for DAT, the role of GMA is controversial. The purpose of this large study was to compare the performance of the conventional tube test (CTT) to GMA for detecting potentially significant antibodies coating red blood cells in vivo. From January 1996 to May 2002, we performed DATs by GMA and CTT on 9,862 blood samples submitted to our reference laboratory, using LISS/Coombs cards (DiaMed-Latino America, Lagoa Santa-MG, Brazil) for GMA and polyspecific and monospecific anti-IgG reagents for CTT. Acid eluates were prepared from all positive DAT samples. The specificity of eluates was determined by GMA. We detected nonconcordant results in 2,079 out of 3,163 positive DATs (65.7%). All of these tests were only positive in GMA. Sensitivity and specificity for DATs was 100% and 83.0% for gel, and 50.7% and 97.8% for tube, respectively. Based on this study GMA showed to be more sensitive than CTT for detecting potentially significant antibodies coating red blood cells in vivo., (Copyright 2004 Wiley-Liss, Inc.)

Imatinib mesylate is a specific inhibitor of protein tyrosine kinase activity secondary to bcr-abl, mostly indicated for the treatment of patients with Philadelphia chromosome positive chronic myeloid leukaemia (CML). Generally, the undesirable effects of imatinib administration observed in clinical trials were of mild-to-moderate degree, and no haemolysis has been associated with this drug. We report here a case of immune-mediated haemolytic anaemia associated to imatinib mesylate successfully treated with prednisone in a patient with CML. Laboratory investigation showed anaemia [haemoglobin (Hb) of 59 g/L], reticulocyte of 61 x 10(9)/L and a positive direct antiglobulin test. Anti-drug in vitro studies revealed a positive result with gel microcolumn assay by an adsorption mechanism. Seventy-four days after prednisone therapy, the patient's Hb level was of 110 g/L with negative direct antiglobulin test and drug in vitro studies. This case demonstrated that patients treated with imatinib mesylate can present immune-mediated haemolysis and adequate management of this event can be done maintaining the drug and associating corticosteroids.

Anti-U is a rare red blood cell alloantibody that has been found exclusively in blacks. It can cause hemolytic disease of the newborn and hemolytic transfusion reactions. We describe the case of a female newborn presenting a strongly positive direct antiglobulin test due to an IgG antibody in cord blood. Anti-U was recovered from cord blood using acid eluate technique. Her mother presented positive screening of antibodies with anti-U identified at delivery. It was of IgG1 and IgG3 subclasses and showed a titer of 32. Monocyte monolayer assay showed moderate interaction of Fc receptors with maternal serum with a positive result (3.1%). The newborn was treated only with 48 hours of phototherapy for mild hemolytic disease. She recovered well and was discharged on the 4th day of life. We conclude that whenever an antibody against a high frequency erythrocyte antigen is identified in brown and black pregnant women, anti-U must be investigated.

Anti-D titration is the first step in the evaluation of the RhD-sensitized patient. Traditionally, anti-D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti-D titer needs to be established. Seventy-nine known blood samples with anti-D (titers 1-32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R0r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2-2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti-D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4-fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti-D titration by the gel microcolumn assay.

Background: Primary immune response against red blood cell (RBC) antigens often takes weeks or months to be detected. In previous reports, for children receiving multiple units of blood components, ranging from five to 81 units, the elapsed time between the first RBC transfusion and antibody detection ranged from 18 to 78 days. Cytomegalovirus (CMV) is sometimes associated with immunohaematologic findings and may modulate immune response., Case Report: A 24-week-old male infant with interstitial pneumonia and hepatitis because of CMV developed an RBC auto antibody and two RBC alloantibodies: anti-Jka, detected in tube 11 days after a single RBC transfusion, and anti-K, detected only in papain gel test 18 days later., Conclusion: As anti-Jka is not a naturally occurring antibody, this is the most rapid primary immune response against an RBC antigen after a single RBC transfusion ever described, in the youngest child ever described.

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.

Background: Few immunohematological studies have been done in myelodysplastic syndrome (MDS)., Methods: Twenty-nine MDS patients were retrospectively evaluated with a direct antiglobulin test (DAT), antibody screening, serum electrophoresis and immunoelectrophoresis. Clinical and laboratory studies (hemoglobin level, reticulocyte count, DHL, total and indirect bilirubin) were done simultaneously, as well as the French-American-British subtype and bone marrow biopsy findings., Results: Alloantibodies were demonstrated in 17 patients (58.6%), autoantibodies in 10 (34.4%) patients and cold agglutinin in 18 (62%) patients. DAT was mediated by only IgG in 8 patients (80%), by IgG and C3 in 1 patient (10%) and by IgG, IgA and C3 in 1 (10%) patient. No hemolytic disease occurred in patients with autoantibodies. Increased serum gammaglobulin was observed in 16 (54.4%) patients. There was no correlation between the incidence of allo-/autoantibodies and the gammaglobulin level (p = 0.937) and the presence of lymphocyte infiltrates in bone marrow biopsies (p = 0.156). No significant difference was observed when the incidence of autoantibodies and number of red blood cell transfusions were compared (p = 0.334). Patients with refractory anemia and refractory anemia with ringed sideroblasts subtypes had a higher incidence of allo-/autoantibodies than other MDS subtypes (p = 0.03)., Conclusion: Patients with MDS, in particular refractory anemia and refractory anemia with ringed sideroblasts have a high incidence of allo- and autoantibodies, probably related to intrinsic immune disorder, without clinical or laboratory hemolysis., (Copyright 2001 S. Karger AG, Basel)

Serologic ABO blood typing is routinely performed using anti-A and anti-B sera to distinguish four phenotypes (A, B, AB, and O). Restriction fragment length polymorphisms (RFLPs) and DNA sequence studies offer the possibility of direct ABO genotyping. We used polymerase chain reaction-RFLP analysis to determine the frequency of O(1) and O(2) alleles in 82 unrelated blood donors in São Paulo, Brazil, known to be group O. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. Different genotypes (O(1)O(1), O(1)O(2), O(2)O(2)) were identified after digestion with restriction enzymes KpnI, HpaII, and AluI, followed by agarose gel electrophoresis. Of 82 samples analyzed, 74 were O(1)O(1), 7 were O(1)O(2), and 1 was O(2)O(2). These results showed the frequency of O(1)O(1), O(1)O(2), and O(2)O(2) genotypes to be 90.24 percent, 8.53 percent, and 1.22 percent, respectively, in blood donors in São Paulo, Brazil.

There are several methods for antibody detection and each technique has advantages and limitations. We compared the performance of the tube (polyethylene glycol-indirect antiglobulin test [PEG-IAT]) and gel test technique for antibody identification. From January to May 1999, we performed antibody screening tests by gel and tube techniques on 10,123 random blood samples submitted to our reference laboratory. Six hundred and twenty-eight (6.2%) reactive samples were tested for antibody specificity by both methods. One hundred and ninety-six were reactive only by gel: 25 anti-D, 33 anti-C, 76 anti-E, 13 anti-c, 5 anti-e, 18 anti-K, 7 anti-Jka, 2 anti-Dia, 3 anti-S, 8 combination Rh antibodies (1 with anti- K), and 6 other antibody specificities. Two samples were reactive only by PEG-IAT: 1 anti-K and 1 anti-Dia. Four hundred and thirty were positive by the two methods: 156 anti-D, 9 anti-C, 68 anti-E, 15 anti-c, 6 anti-e, 61 anti-K, 12 anti-Jka, 17 anti-Dia, 5 anti-S, 73 combination Rh antibodies (2 with anti-K), and 8 other antibody specificities. Based on this study, the gel test is more sensitive (p <.01) than the tube test for identifying potentially clinically significant antibodies.

RAS mutations can be detected in a variable number of patients with myeloproliferative disorders such as myelodysplastic syndromes and acute myeloid leukemia, but are rare events in chronic myelogenous leukemia in chronic phase. However, there is good evidence supporting the involvement of RAS signalling pathway in CML and this could be due to alterations in RAS activity regulatory proteins. The neurofibromatosis (NF1) gene down-regulates the RAS signal transduction pathway through the inhibitory function of its GAP-related domain (GRD) on RAS protein. The loss or alteration of neurofibromin (the NF1 protein) may produce a disfunction similar to point mutations in the RAS gene resulting in the permanent stimulation of the RAS signal transduction pathway. Mutations involving the GRD region of the NF1 gene (GRD-NF1) have been described in a variety of tumors such as colon carcinoma and astrocytoma. Germline mutations and deletions in the NF1 gene, as seen in neurofibromatosis type 1, are also associated with certain myeloid disorders. In the present work, we sought to identify mutations in the codons 12/13 and 61 of RAS gene and in the Lys-1423 codon of GRD-NF1, which are well known hot spots in these genes, in a group of 36 adults and ten children with chronic myelogenous leukemia in chronic phase and blast crisis. Using the PCR-SSCP and the allele-specific restriction assay (ASRA) techniques, we were not able to observe any RAS or NF1 detectable mutation. These findings suggest that RAS and GRD-NF1 mutations are not involved either in chronic phase or in the progression to blast crisis in chronic myelogenous leukemia in adults and children.

K (Kell) is one of the most immunogenic of the red blood cell (RBC) antigens. In order to select K- RBC units, we developed K phenotyping on the Olympus PK-7200 equipment to save labor, time, and costs. The Olympus PK-7200 is fully automated equipment used primarily for blood typing and syphilis screening. We tested 3,587 blood donor samples in EDTA using a commercial anti-K serum diluted in HP Hemagen Power Solution(1:40). The equipment was set to prepare a 1.7% RBC suspension in bromelain and to dispense 25 microL of the mixture (diluted serum and HP Hemagen Power Solution) in terraced microplates. After mixing, the microplates were incubated for 1 hour at 30 degrees C. Reading was performed by a C.C.D. camera and the results were automatically transferred to the mainframe computer. We found 185 K+ blood samples and 3,402 K- samples. Four samples, K+ by the PK-7200, were confirmed as K- by tube test. The use of bromelain with the PK-7200 may have caused the falsely positive tests. The Olympus PK-7200, used for K phenotyping, saves labor time and costs. It also reduces handling and thus promotes less contamination risk for laboratory personnel.

The use of polymerase chain reaction (PCR) for routine detection of clonal immunoglobulin heavy-chain (IgH) gene rearrangements represents an attractive alternative to Southern hybridization analysis not only because PCR protocols are quicker and simpler, but also because of the ability to analyze very small population of cells in search of minimal residual disease. This can be especially important for the detection of clonal malignant cells in locations other than bone marrow or peripheral blood. We describe a case in which central nervous system involvement, a very rare complication of chronic lymphocytic leukemia, was confirmed by PCR analysis for IgH genes rearrangement of the lymphocytes found in cerebrospinal fluid. The cerebrospinal fluid and the peripheral blood lymphocytes (obtained from archival cytospins stored at the time of diagnosis, 5 years before) presented an identical IgH gene rearrangement, as shown by sequence analysis. Thus, the use of PCR for IgH genes rearrangement can be very useful in the detection of monoclonality in samples with a small number of cells and in the confirmation of the common origin of B cells in different specimens of the same patient.

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.

A 22-year-old man in his first relapse of T-acute lymphoblastic leukemia developed fever and a pulmonary infiltrate after 23 days of granulocytopenia. Although having been under amphotericin B for 10 days, productive purulent cough ensued, with right lobe atelectasis and acute ventilatory failure that resolved after the elimination of a thick gelatinous bronchial plug. Sputum cultures yielded Candida Albicans and Staphylococcus epidermidis, and microscopic examination of the sputum plug disclosed Aspergillus hyphae. The patient died 9 days after, of a disseminated Aspergillus infection, confirmed by necropsy.

A patient with myasthenia gravis and a thymoma did not respond to thymectomy. He was submitted to radiotherapy concurrent with steroid therapy followed by an alkylating based chemotherapy. Four years later, he developed an otherwise typical Philadelphia chromosome/BCR-ABL positive chronic myelogenous leukemia (CML) that quickly evolved to a blast crisis. We discuss the possible cause-effect mechanism between the previous treatment and CML, and suggest that a distinct mechanism, albeit unknown, could be involved in the development and progression of secondary CML.