【摘要】目的：建立检测细胞内柔红霉素（DNR）的荧光直方图诊断白血病细胞耐药的方法。材料与方法：选取15例临床耐药的急性髓性白血病（AML），5初治AML进行研究，用流式细胞仪（FCM）检测细胞内DNR的荧光强度，根据其强弱程度划分成耐药区和药敏区，其直方图主峰左移（LSMP）诊断为耐药；直方图右移（RSMP）诊断为药敏。耐药指数（DRI），即R/S（R为耐药细胞数，S为药敏细胞数）≥2为耐药，＜2为药敏。荧光指数（FI）="平"均荧光强度×DRI，FI≥200为耐药，FI＜200为药敏。结果：初治5例AML中，4例为RSMP，DRI="0.23～"1.13，FI="50～"160，诊断为药敏；另一例为LSMP，RI="3.60，"FI="367，"诊断为原发耐药。15例临床耐药的AML中，12例为LSMP，DRI="2.59～"17.72，FI="265～"1041，诊断为经典耐药；其余3例为RSMP，DRI="0.39～"0.67，FI="57～"83，诊断为再生耐药。结论：用FCM检测白血病细胞内DNR的荧光直方图，结合DRI、FI以及临床耐药表现和耐药细胞荧光强度的诊断界点的方法，我们可以快速、简便、直观地在临床上及时发现和评价AML耐药的类型和程度，可为制定个体化疗的方案提供依据，也为观察逆转耐药的疗效提供了一个方法。

中图号：R733.7文献标识码：A文章编号：1000－467X(1999)03－0246－04

Significance of fluorescent histogram of daunorubicin(DNR) in

diagnosis of drug resistance to leukemic cells

He Ming－sheng, CHEN Yan, WU Yu－dan, et al

Institute of Hematology, Xiehe Hospital Tongji Medical University, Wuhan 430022,China

【Abstract】Objective：To develop a fluorescent histogram of intracellular daunorubicin(DNR) as a diagnostic method for drug resistance in acute myeloid leukemia(AML).Methods：15 patients with clinically diagnosed drug resistant AML and 5 patients with previously untreated AML were studied. The intensity of fluorescence of intracellular DNR and the fluorescent histogram were determined by flow cytometer(FCM).According to a degree of the intensity, both drug－resistant and－sensitive zones were marked off. Left shift of main peak(LSMP) with the fluoresent histogram may be diagnosed as drug resistance, and right shift of main peak(RSMP) as drug sensitivity. R/S stood for drug resistant index(DRI);R for the number of drug resistant cells, and S for drug－sensitive cells. DRI≥ 2 was drug－resistant or DRI ＜2 was drug-sensitive. Fluorescent index(FI) was the product of mean fluorescent intensity (MFI) multiplied by DRI. FI≥ 200 meant drug-resistant, while FI＜ 200 meant drug-sensitive.Results：Among 5 patients with previously untreated AML, 4 patients were diagnosed as drug-sensitive for RSMP, DRI="0.23～" 1.13,FI="50～" 160,and one patient was diagnosed as primary drug-resistant for LSMP,DRI="3.60," FI="367." In 15 patients with clinically drug resistant AML,12 patients were diagnosed as classical resistance for LSMP,DRI="2.59～" 17.72,and FI="265～" 1041,and 3 patients as regrowth drug resistance for RSMP,DRI="0.39～" 0.67,and FI="57～" 83.Conclusion：Clinically, it was a rapid, convenient and visual way, which by FCM mapping the fluorescent histogram of intracellular DNR, in combination with DRI, FI, a clinically drug-resistant manifestation and a cut off point for diagnosis of the fluorescent intensity of drug-resistant cells, we may in good time find and value a type and a degree of drug resistance to AML. Moreover ,it may provide a basis for working out an individually therapeutic scheme and a method for an observation in drug-resistant reversal treatment as well.

Key words：Daunorubicin(DNR); Fluorescent histogram; Drug resistance; Flow cytometer (FCM); Leukemia

Drug resistance in leukemia was a leading cause underlying chemotherapeutic failure. Its degree and its type were close associated with the chemotherapeutic scheme for each patient. Still, an urgent problem to solve in clinic is how to find and evaluate the resistance quickly, conveniently and timely. Daunorubicin (DNR) produces red fluorescence with laser excitation at 488 nm wavelength. Utilizing it, drug resistance was detected through measuring DNR fluorescent intensity in the cells. It was only in a few minutes that flow cytometer(FCM) could analyse it for thousands of cells and draw into a fluorescent histogram that represented the marks of drug resistance to leukemic cells. Hence, we tried to investigate the diagonstic significance for the fluorescent histogram in classical and in regrowth drug resistance in leukemia.

1MATERIALS AND METHODS

1.1Patients

15 patients with acute myelogenous leukemia(AML),who manifested drug resistance clinically according to a clinically drug－resistant standard^〔1〕,were eligible for this study.9 males,6 females, age 17～ 52 years old. 5 patients with previously untreated AML were investigated as control.3 males,2 females, age 32～ 48 years old. All of them were diagnosed with a clinical, cell morphology and cell chemistry method in our hospital. Mononuclear cells were obtained with a standard way, i.e. Ficoll Hypaque density gradient separation, in which the number of leukemic cells were more than 90％.

1.2 Reagents

DNR(Farmitalia Carlo Erba,Italy) and RPMI 1640(Gibco－BRL,USA).

1.3Assay of DNR fluorescent intensity in leukemic cells

Leukemic cells were washed twice with RPMI 1640 which contained 10％ calf serum. The concentration of cells was adjusted to 1× 10⁶ /ml.1 μg/ml DNR was added and incubated for 1 hour at 37℃ ,5％ CO2.Then dissociated DNR was washed twice with RPMI 1640 at 4℃. 1 ml RPMI 1640 at 4℃ was added, the fluorescent intensity of cells was detected by FCM ( Becton Dickinson, USA ). 488nm wavelength was used. FCM was first adjusted with fluorescent balls , and CV value was lower than 3％.10 000 cells were assayed for each sample. Sample without DNR was regarded as control. The detected results were drawn into the histogram with software CELLQuest according to fluorescent intensity that represented intracellular DNR concentration. The ordinate was cell number and the abscissa was logarithmic fluorescent intensity.

1.4Data analysis

According to other article^〔2〕,the cut-off point of DNR fluorescent intensity in drug－resistant cells was 100. Passing through this point, fluorescent intensity was divided into two zones, namely, ＜100 was the zone of drug resistance, and ≥100 was the zone of drug sensitivity. The left shift of main peak(LSMP) in DNR fluorescent histogram meant that it located in the drug－resistant zone. The previously untreated patient was diagnosed as primary drug resistance. If the patient manifested drug resistance clinically, he was diagnosed as classical drug resistance. The right shift of main peak （RSMP）meant that it located in the drug－sensitive zone. If the patient manifested drug resistance clinically, he was diagnosed as regrowth drug resistance. R stood for cell number of drug resistance;S stood for cell number of drug sensitivity. The drug resistant index(DRI)="R/S." If it≥ 2,it meant drug resistant, and＜2 meant drug sensitive. Fluorescent index (FI)="mean" fluorescent intensity(MFI)× DRI. FI＜200 indicated sensitivity, and≥200 indicated resistance; the larger the value of them, the higher the degree of drug resistance.

2RESULTS

The DNR fluorescent histogram. 4 cases of previously untreated AML displayed RSMP in the fluorescent histogrom, in which the main peak were high－narrowed(Fig.1), DRI=0.23～1.13,and FI=50～160.These patients were diagnosed as drug sensitiv