Characterization of a novel CYP2C9 mutation (1009C>A) detected in a warfarin-sensitive patient.

Warfarin is the most frequently prescribed anticoagulant for the long-term treatment in the clinic. Recent studies have shown that polymorphic alleles within the CYP2C9, VKORC1, and CYP4F2 genes are related to the warfarin dosage requirement. In this study, a novel non-synonymous mutation (1009C>A) in CYP2C9 was detected in a warfarin-hypersensitive patient, while the other two candidate genes were both found to be homozygous for the wild-type alleles. The newly identified point mutation results in an amino acid substitution at position 337 of the CYP2C9 protein (P337T) and has been designated as the novel allele CYP2C9*58. When expressed in insect cell microsomes, the relative intrinsic clearance values of the CYP2C9.58 variant for tolbutamide and losartan were quite similar to those of the typical defective variant CYP2C9.3, whereas the clearance value of CYP2C9.58 for diclofenac was slightly higher than that of another typical defective variant CYP2C9.2. These data suggested that when compared with wild-type CYP2C9.1, the enzymatic activity of the novel allelic variant has been greatly reduced by the 1009C>A mutation. If patients carrying this allele take drugs metabolized by CYP2C9, their metabolic rate might be slower than that of wild-type allele carriers and thus much more attention should be paid to their clinical care.

[Clinical evaluation of leukocyte differential count in peripheral blood by five-color flow cytometry].

OBJECTIVE: To explore the clinical application values of five-color flow cytometry for leukocyte differential count in peripheral blood. METHODS: Leukocyte differentiation in 265 peripheral blood samples collected at Peking University First Hospital from September 2010 to December 2010 was detected by standard microscopic cytology as a reference method. Meanwhile, Beckman-Coulter LH750 hematology analyzer and FC500 flow cytometer were performed. Then the correlations were analyzed between microscopic cytology, hematology analyzer and flow cytometry. Forty blood samples collected at Peking University First Hospital, Beijing Daopei Hospital and General Hospital of Beijing Military Command from August 2010 to November 2010 were analyzed by standard microscopic cytology, Beckman-Coulter LH750 hematology analyzer and NAVIOS flow cytometer. Then the correlations between microscopy, hematology analyzer and flow cytometry were explored to analyze the clinical diagnostic efficiency of flow cytometry. RESULTS: Correlation of leukocyte differential count between FC500 flow cytometer and standard microscopic cytology was significant (all P < 0.01) . And it was superior in the detection of lymphocytes, neutrophils and eosinophils (r = 0.955, 0.928, 0.876). Moreover, the correlation of leukocyte differential count between NAVIOS flow cytometer via manual gate and standard microscopic cytology was significant (r > 0.700, all P < 0.01) except for basophils. And it was superior in the detection of neutrophils, lymphocytes and blasts (r = 0.950, 0.915, 0.852). When 1% was set as the cut-off value of immature granulocytes on standard microscopic cytology, the sensitivity and specificity of flow cytometry was 87% and 76% respectively. When 0.5% was set as the cut-off value of blasts on standard microscopic cytology, the sensitivity and specificity of flow cytometry stood at 100% and 92% respectively. CONCLUSION: Five-color flow cytometry is well-correlated with standard microscopic cytology for leukocyte differential count in peripheral blood with different flow cytometers, and the sensitivity of detecting blasts and immature granulocytes is very excellent.

Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials.

BACKGROUND: White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review. However, this last step is time consuming and requires experienced personnel. We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials. METHODS: A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials. WBC differentials of 622 samples were also determined using both a cell counter and FCM. These results were then confirmed using manual microscopic methods. RESULTS: The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%. The resulting WBC differentials were well correlated between FCM and the cell counter (r > 0.88, P < 0.001), except in the case of basophils. Neutrophils, lymphocytes, and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r > 0.80, P < 0.001). The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%, respectively) were higher than those of the cell counter method (44.92% and 11.11%, respectively). The specificities of FCM were all above 85%, substantially better than those of the cell counter method. CONCLUSION: These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

[Establishment and evaluation of review criteria for ADVIA 120/2120 and different series of hematology analyzers].

OBJECTIVE: To establish the suitable review criteria for ADVIA 120/2120 and those for different series of hematology analyzers. METHODS: A total of 2400 samples, including 6 blood neoplasms, were detected with ADVIA 120/2120 hematology analyzer, in which 1200 samples were detected by Sysmex XE-2100 and Beckman-Coulter LH750 hematology analyzers. In the meantime, blood smears were reviewed, and the results were analyzed statistically. The new review criteria were established by consulting and modifying the one as recommended by an international consensus group. Finally 300 samples were selected to validate the new review criteria. RESULTS: The results of 2400 samples detected by ADVIA 120/2120 hematology analyzer were analyzed statistically according to the international consensus review rules and blood smear positive criteria formulated by Chinese experts. The true positive rate was 22.1% (n = 530), false positive rate 28.1% (n = 675), true negative rate 44.3% (n = 1063), false negative rate 5.5% (n = 132), and the smear review rate 50.2% (n = 1205). The false negative rate was over the acceptable limit of 5%. The new review criteria were established by amending the blood smear positive criteria, i. e. increasing the percentage of band neutrophils, eosinophils, basophils and monocytes and adjusting the international consensus review rules. Then the results were re-analyzed. The true positive rate, false positive rate, true negative rate and false negative rate were 15.5% (n = 371), 18.7% (n = 449), 61.6% (n = 1479) and 4.2% (n = 101) respectively. The smear review rate was 34.2% (n = 821) and no specimen of blood neoplasms was missed. On that basis, the current review criteria for ADVIA 120/2120, XE-2100 and LH750 hematology analyzer were proposed by adding some specific parameters. The results of 1200 samples with three instruments were analyzed according to the current criteria. And the false negative rates were 4.3%, 4.6% and 4.6%, and false positive rate 14.7%, 17.5% and 12.7% respectively. And no specimen of blood neoplasm was missed. The false negative rates of three instruments were 3.8%, 4.3% and 4.0% in validation teses. CONCLUSION: The review criteria for three different series of hematology analyzers have been formulated for large general hospitals.