Changes in anticoagulant prescription in Dutch patients with recent-onset atrial fibrillation: observations from the GARFIELD-AF registry.

BACKGROUND: For the improvement of AF care, it is important to gain insight into current anticoagulation prescription practices and guideline adherence. This report focuses on the largest Dutch subset of AF-patients, derived from the GARFIELD-AF registry. METHODS: Across 35 countries worldwide, patients with newly diagnosed 'non-valvular' atrial fibrillation (AF) with at least one additional risk factor for stroke were included. Dutch patients were enrolled in five, independent, consecutive cohorts from 2010 until 2016. RESULTS: In the Netherlands, 1189 AF-patients were enrolled. The prescription of non-vitamin K antagonist oral anticoagulants (NOAC) has increased sharply, and as per 2016, more patients were initiated on NOACs instead of vitamin K antagonists (VKA). In patients with a class I recommendation for anticoagulation, only 7.5% compared to 30.0% globally received no anticoagulation. Reasons for withholding anticoagulation in these patients were unfortunately often unclear. CONCLUSIONS: The data from the GARFIELD-AF registry shows the rapidly changing anticoagulation preference of Dutch physicians in newly diagnosed AF. Adherence to European AF guidelines in terms of anticoagulant regimen would appear to be appropriate. In absence of structured follow up of AF patients on NOAC, the impact of these rapid practice changes in anticoagulation prescription in the Netherlands remains to be established.

[Extent and quality of anti-coagulation treatment with coumarin derivatives by the Dutch Thrombosis Services]. / Omvang en kwaliteit van de antistollingsbehandeling met cumarinederivaten door de Nederlandse trombosediensten.

OBJECTIVE: To obtain an impression of the extent and quality of the anti-coagulation treatment with coumarin derivatives carried out by the Thrombosis Services in the Netherlands. DESIGN: Descriptive. METHOD: Data were drawn from the medical annual reports of 62 of the 63 Thrombosis Services in the Netherlands over the period 1998-2002. In 2002 the Thrombosis Services treated 325,072 patients and performed 4,469,730 INR laboratory tests. The half-yearly figures produced by the Thrombosis Services were calculated as an average percentage per year per thrombosis service and then recalculated as a percentage per year. RESULTS: Seventy-three per cent of the patients were treated for an arterial and 27% for a venous indication. Depending on the required intensity of anticoagulation a mean of 74-78% of the long-term treated patients fell within the therapeutic range and a mean of 6-10% below. The mean number of major bleedings per 100 treatment years was 1.0. A mean of 79% of the patients was treated with acenocoumarol and 21% with phenprocoumon. When acenocoumarol was used, a mean of 72-77% fell within the therapeutic range and in the case of phenprocoumon 79-82%. In the last few years the number of patients had increased due to a growing number of patients treated for atrial fibrillation. The percentages of INR within the therapeutic range were unchanged or showed a slight increase. CONCLUSION: The quality of the anticoagulation therapy with coumarin derivatives was good or acceptable.

SIHONSCORE: a scoring system for external quality control of leukaemia/lymphoma immunophenotyping measuring all analytical phases of laboratory performance.

For the diagnosis of leukaemia and leukaemic lymphoma, clinicians frequently have to rely on the results of immunophenotyping. To improve the quality of these results, the Dutch Foundation for Immunophenotyping of Haematological Malignancies (SIHON) initiated external quality rounds in 1986. Over a period of more than 10 years, this has led to improvements in the interpretation of immunophenotyping results. However, the evaluation of results focused mainly on the correctness of the interpretation of the immunophenotypical data, leaving the preceding analytical phases unevaluated. Therefore, in 1996 SIHON developed a more comprehensive scoring system, called SIHONSCORE, covering all three phases of immunophenotyping, namely the pre-analytical (i.e. choice of the staining panels), analytical (i.e. the technical part consisting of sample preparation, data acquisition and analysis) and the post-analytical phase (i.e. the interpretation) of the laboratory process. Here, we report how SIHONSCORE was successfully applied to three consecutive external quality rounds consisting of a total of nine different cases tested. For laboratory certification, participation in external quality control programmes is required. Evidently, criteria are needed to define the minimum acceptable performance of a certified laboratory. With SIHONSCORE, a useful instrument is obtained evaluating all phases of the performance of laboratories in leukaemia and lymphoma immunophenotyping.

Reduction of interlaboratory variability in flow cytometric immunophenotyping by standardization of instrument set-up and calibration, and standard list mode data analysis.

Two workshops addressed the question to which degree standardization of instrument set-up and calibration, and standard list mode data analysis would reduce interlaboratory variability of flow cytometric results on prestained peripheral blood mononuclear cells (PBMC). Standard instrument set-up included uniform positioning of the "windows of analysis" for the forward and sideward light scatter and fluorescence (FL) 1 (i.e., fluorescein isothiocyanate [FITC]) and 2 (i.e., phycoerythrin [PE]) parameters. Reference standards and PBMC, double-stained with FITC- and PE-conjugated monoclonal antibodies covering a wide range of FL intensities and coexpression patterns, were sent out to 25 laboratories in Workshop 1 and to 35 laboratories in Workshop 2 with the following requests: a) to set up instruments according to local and standard protocols, b) to acquire list mode data on the PBMC with both instrument settings, and c) to analyze both datasets according to local protocols. Standard analysis of the list mode data acquired with uniform instrument settings was performed centrally using so-called "latent class model" software (Van Putten et al., Cytometry 14:86-96, 1993). This software provides an automated, "no-gating" analytical method of lymphocyte immunophenotypes and employs fixed FL marker settings as defined prior to each analytical run. In Workshop 1, these markers were set in identical histogram channels for all instruments based on results obtained with a reference instrument. Standard analysis of list mode data acquired after uniform instrument set-up led only to a 13% reduction of interlaboratory variability of results as compared to data analysis using local protocols. The standard protocol for instrument set-up led to uniform positioning of relatively strong FL signals but variable positioning of unstained cells on the FL histogram scales. Hence, standard FL marker settings were inappropriate for some instruments. Therefore, instrument responses to FITC and PE signals in Workshop 2 were calibrated using microbeads labeled with FITC or PE in a range of predefined FL intensities expressed in MESF units (molecules of equivalent soluble fluorochrome). That approach allowed the positioning of the FL markers for the standard analysis on the basis of identical FL1 and FL2 intensities, expressed in MESF units, for all instruments. Standard analysis of list mode data acquired after uniform instrument set-up and calibrated FL marker settings led to a 43% reduction of interlaboratory variability as compared to data analysis to local protocols. We conclude that standard list mode data analysis using fixed FL marker settings reduces the interlaboratory variability of flow cytometric results on prestained PBMC, provided that the instruments have been set up in a uniform way and that FL markers have been standardized on the basis of calibration of each instrument's response to the corresponding FL signals.

Pitfalls in the immunophenotyping of leukaemia and leukaemic lymphomas: survey of 9 years of quality control in The Netherlands. Dutch Cooperative Study Group on Immunophenotyping of Haematological Malignancies (SIHON).

During the last decade, biannual quality controls were performed in the Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18-34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut-off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as 'correct', 'minor fault', 'major fault', 'not based upon the markers used', and 'no diagnosis', CD3, CD8, CD19, CD61 and Sm lambda had the lowest percentage discordancy (sum of total negative and positive discordant values 5-7.5% of assays): CD13, CD15, cyCD22, CD33 and TdT scored worst with 14-20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B-cell leukaemias, AMLs and common-ALL, and the poorest scores for T-cell malignancies which were correctly diagnosed in only 24-60% of specimens. Mature T-NHL and T-PLL were mistakenly diagnosed as T-ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation. In conclusion, both the technical performance of immunophenotyping of haematological malignancies in The Netherlands and the procedure by which a final diagnosis is obtained needs improvement, especially for T-cell malignancies.

Use of the direct RNA amplification technique NASBA to detect factor V Leiden, a point mutation associated with APC resistance.

APC resistance is a common and strong hereditary risk factor for venous thrombosis. This plasma abnormality appears to be almost always caused by the same defect in the coagulation factor V gene (a G --> A transition at nucleotide 1691 leading to replacement of 506 Arg by Gln; factor V Leiden). Therefore, it is possible to consider a simple and specific genetic test as an alternative to a plasma APC resistance test that is compromised by treatment and other factors. We have investigated whether a new amplification procedure, NASBA, together with the detection procedure ELGA would provide a simple protocol for the nucleotide specific detection of the factor V mutation.

Multicentre evaluation of the EBIO plus glucose analyser.

The EBIO plus, a newly developed dedicated glucose analyser, was tested at three different sites for its analytical characteristics and practicability. Results were linear over a range of 2-50 mmol/l, precision was satisfactory (overall CV < 5.3% at a concentration level of 1.8 mmol/l and < 3.7% at a concentration level of 4.5-21 mmol/l). Calibration was stable up to 90 minutes. No significant influences were observed for several potential interfering substances at physiological or therapeutical levels. Practicability was found to be good. Sample through-put depends on calibration frequency and is maximally 166 per hour. Some problems with outliers had to be overcome in the beginning of the evaluation period, but in the end all evaluators concluded that the EBIO plus is user friendly with good analytical characteristics.

'Oversaturation' of transferrin after intravenous ferric gluconate (Ferrlecit(R)) in haemodialysis patients.

BACKGROUND: Chronic haemodialysis causes blood loss and iron-deficiency. This can be corrected with intravenous preparations, e.g. sodium ferric-gluconate (FeGl). In two patents complaints of hypotension and malaise during FeGl infusion coincided with high levels of serum iron and a calculated transferrin iron saturation above 100%. Iron toxicity could be the cause of these complaints. Free iron is known to aggravate the toxicity of free radicals and other reactive oxygen products that are constantly formed in the body. We compared four rates of FeGl infusion with regard to iron parameters. METHODS: 20 dialysis patients received a total of 26 infusions of FeGl. A rapid infusion of 135 mg (Protocol A (n=10)) or 62.5 mg (Protocol B (n=7)) of FeGl was given during the last 30 min of dialysis. A slow infusion of 125 mg (Protocol C (n=9)) or 62.5 mg (Protocol D (n=10)) was given during 4 or 4.5 h of dialysis. Blood was taken at regular intervals, before, during, and after dialysis for determination of serum iron, transferrin, ferritin, haematocrit, total protein, albumin, and lactate dehydrogenase (LDH). Transferrin saturation was calculated from transferrin and serum iron. RESULTS: With rapid infusion A (125 mg) the highest levels of serum iron (median 120 (range 40-159) micromol/l) and transferrin saturation (207 (84-331)%) were seen at the end of the infusion. These were significantly higher than the peak levels with B, C, and D (P

Ultrasound examination of pathological cervical lymph nodes in patients with non-Hodgkin's lymphoma and Hodgkin's disease.

The choice of treatment of malignant lymphoma may vary considerably and is mainly determined by the pathology and clinical stage. Ultrasonography is currently one of the most sensitive techniques for evaluation of the cervical area. We set out to assess the value of ultrasound imaging when added to conventional staging (physical examination, laryngoscopy, computer tomography of thorax and abdomen, bone marrow cytology and histology) of malignant lymphoma in 47 patients with untreated lymphoma. Hodgkin's disease was present in 14 patients and non-Hodgkin's lymphoma in 33 individuals. The ultrasound results were independently compared with physical examination of the neck. Ultrasonography revealed additional pathological lymph nodes in 6/47 cases (13%). Furthermore, the diagnosis non-Hodgkin's lymphoma could be established in one patient merely as a result of ultrasonography. Ultrasonography of the neck may reveal more pathological lymph nodes in a significant number of patients and may be of value in the initial staging of patients with malignant lymphoma.

tal-1 deletions in T-cell acute lymphoblastic leukemia as PCR target for detection of minimal residual disease.

Polymerase chain reaction (PCR) techniques based on amplification and identification of leukemia-specific DNA sequences provide a sensitive diagnostic method for detection of minimal residual disease (MRD) with a detection limit of 10(-5) to 10(-6) (1-10 malignant cells in 10(6) normal cells). To date, the main leukemia-specific DNA sequences used as PCR targets in detection of MRD are breakpoint fusion regions of chromosome translocations and junctional regions of rearranged immunoglobulin (Ig) or T-cell receptor (TcR) genes. The recently identified tal-1 deletions involving the sil and tal-1 genes, provide a potential MRD-PCR target. tal-1 deletions are site-specific because they are mediated via recombination signal sequences homologous to Ig/TcR genes. In line with this homology, tal-1 deletions also show random insertion and deletion of nucleotides at their breakpoints, resulting in highly variable breakpoint fusion regions. The fusion region diversity can be applied to design patient-specific oligonucleotide probes. Our Southern blot analyses of a large series of 313 acute leukemias with a specific tal-1 deletion probe (SILDB) demonstrated that tal-1 deletions exclusively occur in T-cell acute lymphoblastic leukemia (T-ALL) and not in precursor B-ALL or acute non-lymphocytic leukemias. In addition, we did not detect tal-1 deletions in normal blood cells and normal thymocytes by PCR analysis. The diversity observed in tal-1 deletion fusion regions with an average insertion and deletion of approximately 7 and approximately 6 nucleotides, respectively, allowed us to design fusion-region-specific probes. The specificity of the fusion-region probes was proven and the detection limit of the MRD-PCR technique was tested in a series of dilution experiments. The observed detection limit of 10(-5) indicates that tal-1 deletions in T-ALL represent ideal leukemia-specific PCR targets for detection of MRD.

High expression of the multidrug resistance P-glycoprotein in high-risk myelodysplasia is associated with immature phenotype.

The expression of the multidrug resistance (MDR-1) gene product, P-170 glycoprotein (P-170) was investigated in 26 patients with low-risk (n = 9) or high-risk (n = 17) myelodysplastic syndrome (MDS), using a panel of monoclonal antibodies to P-170 (C219, JSB1, C494, MRK16) and quantitative analysis of MDR-1 mRNA. P-170 membrane staining was demonstrated in bone marrow blast cells of 14/17 HR-MDS and in 2/9 LR-MDS patients (p < 0.01). P-170 expression was associated with the presence of blast cells characterized by an immature or early myeloid phenotype as defined by CD34 expression (p = 0.034), CD13 or CD33 expression (p = 0.0006), or CD13/33 plus terminal deoxynucleotidyl transferase (TdT) double expression (p = 0.04). With double fluorescence analysis, P-170 expression was observed in a subset of CD34+ cells, but not in CD34- cells. P-170 expression was present in 13/15 (86%) patient samples with an abnormal karyotype as compared with 3/10 samples (30%) with a normal karyotype (p < 0.05). Nine of these 15 patients had a loss or a deletion of chromosome 7. Thirteen out of 16 (81%) MDR-1 positive patients developed acute leukemia versus two of ten (20%) MDR-1 negative patients (p = 0.025). It is concluded that MDR-1 expression in MDS is present in cells with an immature phenotype and is frequently observed in patients who have an abnormal karyotype and a high risk of leukemic transformation.

Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a specific immunophenotype with CD2 expression.

Extensive immunologic marker analysis was performed to characterize the various leukemic cell populations in eight patients with inv(16)(p13q22) in association with acute myeloid leukemia with abnormal bone marrow eosinophilia (AML-M4Eo). The eight AML cases consisted of heterogeneous cell populations; mainly due to the presence of multiple subpopulations, which varied in size between the patients. However, the immunophenotype of these subpopulations was comparable, independent of their relative sizes. Virtually all AML-M4Eo cells were positive for the pan-myeloid marker CD13. In addition, the AML were partly positive for CD2, CD11b, CD11c, CD14, CD33, CD34, CD36, CDw65, terminal deoxynucleotidyl transferase (TdT), and HLA-DR. Double immunofluorescence stainings demonstrated coexpression of the CD2 antigen and myeloid markers and allowed the recognition of multiple AML subpopulations. The CD2 antigen was expressed by immature AML cells (CD34+, CD14-) and more mature monocytic AML cells (CD34-, CD14+), whereas TdT expression was exclusively found in the CD34+, CD14- cell population. The eight AML-M4Eo cases not only expressed the CD2 antigen, but also its ligand CD58 (leukocyte function antigen-3). Culturing of AML-M4Eo cell samples showed a high spontaneous proliferation in all three patients tested. Addition of a mixture of CD2 antibodies against the T11.1, T11.2, and T11.3 epitopes diminished cell proliferation in two patients with high CD2 expression, but no inhibitory effects were found in the third patient with low frequency and low density of CD2 expression. These results suggest that high expression of the CD2 molecule in AML-M4Eo stimulates proliferation of the leukemic cells, which might explain the high white blood cell count often found in this type of AML.

Early diagnosis of smoldering acute lymphoblastic leukemia using immunological marker analysis.

During a period of 9 years, we performed immunological marker analysis in 164 children with acute lymphoblastic leukemia. In four children the diagnosis acute leukemia could not be established by cytomorphological analysis of bone marrow and peripheral blood samples at initial presentation. In two of these four children a hypoplastic bone marrow was found, whereas the bone marrow of the other two children was normocellular. Using double immunological marker analysis, we detected high frequencies of CD10+, TdT+ cells in bone marrow (range: 18-53%) as well as peripheral blood (range: 0.04-19.5%). In control bone marrow and peripheral blood samples from healthy children, the frequency of CD10+, TdT+ cells does not exceed 10% and 0.03%, respectively. Based on the immunological data, a common acute lymphoblastic leukemia (ALL) was suspected. In addition, chromosome analysis revealed a high hyperdiploid (> 50 chromosomes) karyotype in three patients and t(9;22) in one patient. At 18 to 68 days after initial presentation, an ALL was diagnosed according to cytomorphological criteria in all four patients. At that time the percentage of CD10+, TdT+ cells in bone marrow and peripheral blood had increased significantly. One patient could be monitored frequently from initial presentation onwards. First a decline in the percentage of CD10+, TdT+ cells was found, although treatment consisted only of red blood cell transfusion and antibiotics. Subsequently the percentage of CD10+, TdT+ cells gradually increased until the morphological ALL diagnosis. These results illustrate that CD10, TdT double immunological marker analysis is a useful tool for early diagnosis of smoldering ALL in patients with a suspicious bone marrow, even when the bone marrow is hypoplastic.

Detection of residual disease in AML patients by use of double immunological marker analysis for terminal deoxynucleotidyl transferase and myeloid markers.

In the majority of patients with acute myeloid leukemia (AML) immature leukemic subpopulations expressing myeloid markers and terminal deoxynucleotidyl transferase (TdT) are present. The normal counterparts of these double-positive cells are rare in bone marrow (BM) (< 0.03%; if they occur at all) and are not detectable in peripheral blood (PB). In 14 patients with TdT+ AML at diagnosis, we have performed a prospective follow-up study to monitor the myeloid-marker+, TdT+ cells during and after chemotherapy. One patient did not obtain complete remission (CR), a second patient relapsed under therapy, whereas the other 12 patients were in cytomorphological CR at the end of chemotherapy. During subsequent follow-up, seven of these 12 patients developed one or two relapses (total of ten relapses). Nine of these ten relapses were preceded by a gradual increase of myeloid-marker+, TdT+ cells in BM and PB samples over a period of 14-38 weeks. Based on comparable results in BM and PB samples and doubling times of 15-20 days, we propose that monitoring of AML patients should include PB sampling each 4-6 weeks. In one patient the relapse was not preceded by a gradual increase of double-positive cells. This false negative result was caused by a phenotypic shift, since at relapse the AML cells did not express TdT. In the five AML patients who still are in continuous cytomorphological CR for 32-46 months we repeatedly detected relatively high percentages of myeloid-marker+, TdT+ cells in BM (up to 0.1%) and PB (up to 0.02%). Although we could not prove the leukemic origin of these double-positive cells, they might represent residual dysplastic AML cells which survived chemotherapy but which are not capable of causing leukemia regrowth as yet. This would be in line with recent polymerase chain reaction studies, which could demonstrate the persistence of leukemic clones in the majority of AML patients in continuous CR. It is concluded that double immunofluorescence labeling for myeloid markers and TdT is useful for detection of residual disease in TdT+ AML patients. A gradual increase of double-positive cells is suggestive for leukemic cell growth and can be used to predict relapse.

Characterization of the translocation breakpoint sequences of two DEK-CAN fusion genes present in t(6;9) acute myeloid leukemia and a SET-CAN fusion gene found in a case of acute undifferentiated leukemia.

The t(6;9) associated with a subtype of acute myeloid leukemia (AML) was shown to generate a fusion between the 3' part of the CAN gene on chromosome 9 and the 5' part of the DEK gene on chromosome 6. The same part of the CAN gene appeared to be involved in a case of acute undifferentiated leukemia (AUL) as well, where it was fused to the SET gene. Genomic sequences around the translocation breakpoint were determined in two t(6;9) samples and in the case of the SET-CAN fusion. Although coexpression of myeloid markers and terminal deoxynucleotidyl transferase was shown to be one of the characteristics of t(6;9) AML, no addition of random nucleotides at the translocation breakpoint could be found. In addition, the breakpoint regions did not reveal heptamer-nonamer sequences, purine-pyrimidine tracts, a chi-octamer motif, or Alu repeats. The sequence in which the translocation breakpoints occurred was enriched in A/T. Notably, the specific introns in which clustering of breakpoints occurs in DEK and CAN both contain a LINE-I element. As LINE-I elements occur with a moderate frequency in the human genome, the presence of such an element in both breakpoint regions may be more than coincidental and may play a role in the translocation process.

Quality assessment of immunological marker analysis and the immunological diagnosis in leukaemia and lymphoma: a multi-centre study. Dutch Cooperative Study Group on Immunophenotyping of Leukaemias and Lymphomas (SIHON).

In order to standardize and assess the quality of immunophenotyping of leukaemias and lymphomas for diagnostic purposes, a cooperative study group in the Netherlands, SIHON, has formulated guidelines for the composition of antibody panels to be applied and guidelines for the interpretation of the marker analysis. To assess the value of these guidelines frozen cell samples of three patients with different haematological malignancies were sent to the 26 participating laboratories twice a year. Here we present the results with respect to the marker analysis and to the immunological diagnosis on 387 samples from 18 patients. A large inter-laboratory variation was seen in the percentage of positive cells for each marker, which influenced the valuation of a marker to be discordant positive in up to 23% and discordant negative in up to 40%. No single major factor could be traced to explain the large variation in the results. However, probably due to the balanced composition of the antibody panel and to the application of the guidelines for interpretation, this variation did not much influence the agreement in immunological diagnosis. In only 13/387 samples (3.3%) differences in the percentage of positive cells caused disagreement in the final diagnosis. In 23 samples (5.9%) the disagreement was due to an incorrect application of the guidelines. Quantitative data of single observations obtained from different laboratories, in which the materials and methods are not standardized, cannot be compared; but standardization of guidelines for marker sets and for interpretation contributes to a high grade of agreement in immunological diagnosis.

High expression of the mdr3 multidrug-resistance gene in advanced-stage chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by an often indolent course with a poor therapeutic response at advanced stage. We investigated the expression of the human multidrug resistance genes mdr1 and mdr3 in 31 patients with CLL. Using specific probes for mdr1 and mdr3 mRNA, respectively, expression of both genes could be found in 29 of 31 patients. Of those, nine had high expression of mdr1 and 13 of mdr3. Although 29 of 31 patients showed coexpression of mdr1 and mdr3, the mRNA levels were not interrelated. Prior treatment did not significantly influence the level of mdr1 or mdr3 expression. In patients with advanced CLL (Rai stage 3 + 4) the mdr3 expression was significantly higher than in early-stage CLL (Rai stage 0 to 2) (mean +/- SEM, 25.4 +/- 4.2 U v 4.2 +/- 1.1 U; P less than .0001). Such a difference was not present for mdr1 expression (21.5 +/- 4.3 U v 10.7 +/- 3.1 U; P = .09). These data indicate that advanced-stage CLL is associated with an increased mdr3 expression, which may concur with a decreased sensitivity to chemotherapy.