Determination of leukemia stem cells in childhood acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

The aim of this study was to detect the presence of human AML leukemia stem cells (LSC) in childhood patients with acute leukemia (AL) and analyze the correlation between LSC concentrations and minimal residual disease (MRD) levels in AML cases after remission. The multi-parameter flow cytometry (FCM) and a panel of monoclonal antibody combination were used to detect the AML LSC or AML LSC immunophenotype-identical cell (AML LSC-IPIC) concentrations in childhood AML or ALL leukemia both at new diagnosis and at remission and correlated AML LSC to the MRD levels at different time points after remission. The results indicated that the AML LSC or AML LSC-IPIC concentrations [in average 166 (range 14 - 1459)/100 000 mononuclear cells (MNCs)] in AML at initial diagnosis were significantly higher than those in ALL [7 (range 0 - 560)/100 000 MNCs, p < 0.017] and control [0 (range 0 - 6)/100 000 MNCs, p < 0.017], respectively. The AML LSC concentrations in AML at non-CR were in average 36 (range 5 - 224)/100 000 MNCs. No statistical difference (p > 0.05) was found between the AML LSC or AML LSC-IPIC concentrations in AML (in average 6 (range 0 - 41)/100, 000 MNCs) and ALL [10 (range 0 - 105)/100, 000 MNCs] after CR. The significantly negative correlation was noticed between AML LSC concentrations and MRD levels. It is concluded that the AML LSCs exist in newly diagnosed AML, which are significantly reduced when complete remission has achieved, but the low levels of these populations still remain. The phenotypically similar (CD34(+)CD38⁻CD123(+)) AML LSC populations have also been found in the bone marrow from ALL patients, but their concentrations are not significantly different when CR has achieved. The significantly negative correlation between AML LSC concentrations and MRD levels is observed in AML patients after remission.

Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.</p><p><b>METHODS</b>2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.</p><p><b>RESULTS</b>After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.</p><p><b>CONCLUSION</b>2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.</p>

Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14 / 中华儿科杂志

<p><b>OBJECTIVE</b>Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.</p><p><b>METHODS</b>Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).</p><p><b>RESULTS</b>The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.</p><p><b>CONCLUSIONS</b>The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.</p>

Preparation of the immunotoxin 2E8-norcantharidin and its targeting killing effect in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented immunogenicity to patients capable of inducing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule toxin. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent.</p><p><b>METHODS</b>A monoclonal antibody named 2E8 was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) against CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometry. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made.</p><p><b>RESULTS</b>The purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm-6 cells, while only 0.98% of K562 cells reacted with this antibody. The newly generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the growth of CD19+ Nalm-6 cells (P < 0.001), while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells. Since no significant inhibitory effects were identified among immunotoxin 2E8-NCTD, 2E8 antibody and control groups on CD19(-) K562 cells, a significant targeting effect of the 2E8-NCTD against Nalm-6 cells was confirmed.</p><p><b>CONCLUSIONS</b>The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19+ Nalm-6 leukemia cells in vitro, which provides some experimental data for the further development of this new targeting agent.</p>

Establishment of animal model with B lineage acute leukemia in nude mice for evaluation of new therapeutic agents / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish an acute leukemia animal model for testing new therapeutic agents in vivo.</p><p><b>METHODS</b>Nude mice were intraperitoneally injected with 2 mg cyclophosphamide, 24 h later 5 x 10(6) acute B-cell leukemia Nalm-6 cells was inoculated via the tail vein, then monitored daily. When animals were paralyzed or dying, the organs including the liver, spleen, lung, heart, kidney, brain, bone marrow, pancreas, testes were removed and fixed with formalin, examined by routine histopathology.</p><p><b>RESULTS</b>After Nalm-6 cells were inoculated the mean survival of mice were( 19.4+/-0.55)d (n=6). The paralysis of mice was followed by weight loss, bent spines, hogback, cachexia and death. Histopathological examination showed that the tumor cells infiltrated liver, spleen, kidney, lung, meninges, interior cerebrum, the liver and kidney were the most affected organs.</p><p><b>CONCLUSION</b>B lineage acute leukemia animal model has been successfully established in the nude mice, which is suitable for testing new therapeutic agents.</p>

Construction and expression of prokaryotic vector of single chain antibody derived from a new clone of anti-CD14 antibody ZCH-7-2F9 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.</p><p><b>METHODS</b>Primers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.</p><p><b>RESULT</b>The cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.</p><p><b>CONCLUSION</b>The ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.</p>

Evaluation of the P-gp pump function on leukemic cell membrane and proper application of its reversal agents with Calcein-AM and flow cytometry / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common malignancy in children. Combined chemotherapy is currently the primary treatment modality. During the past decade, very high cure rates of childhood acute lymphoblastic leukemia (ALL) have been reported both at home and abroad. However, the cure rates of children with acute myeloid leukemia (AML) remain low due to the multiple-drug resistance (MDR). P-glycoprotein (P-gp) is one of the most important mechanisms of MDR for leukemia cells. However, the function of the protein, the clinical application of its reversal agents and the efficacy of the combination of the reversal agents remain to be elucidated. The present study aimed to evaluate the P-gp pump function on leukemia cell membrane and the effects of the combined administration of the reversal agents cyclosporin A (CSA) and verapamil (VER) through the observation of Calcein-AM (C-AM) metabolism in the cell line K562 and its multi-drug resistant subline K562/VCR.</p><p><b>METHODS</b>The mean fluorescence intensity (MFI) of C-AM inside the cytoplasm was analyzed with flow cytometry (FCM). The events of K562 and K562/VCR cells treated and untreated with CSA, VER and CSA + VER were acquired at time points 0, 30, 60, 90 and 120 minutes, respectively, and the data obtained were analyzed with CellQuest software.</p><p><b>RESULTS</b>The C-AM in the K562 and K562/VCR varied more apparently in the fist 24 hours. In addition, the MFI of the C-AM in K562 was significantly higher than that in K562/VCR cells indicating that the P-gp pump molecules were functioning. The MFIs of the CSA, VER and CSA + VER groups co-cultured with K562/VCR cells were 4014 +/- 219, 3879 +/- 116 and 4158 +/- 302, respectively after 120 min of incubation, significantly higher as compared to that of control group (3251 +/- 107, P < 0.05). On the other hand, significant inhibition of the efflux from the K562/VCR cell line was also noticed after the same time period of incubation with the MFIs of 2237 +/- 155, 1932 +/- 233 and 2231 +/- 147, respectively in the three groups, which was significantly higher than that of control group (1622 +/- 191, P < 0.05). CSA, VER and CSA + VER could increase the uptake and inhibit the efflux of C-AM by K562/VCR cells, while no evident influence on those functions inside the parental cell line K562 cells was noticed.</p><p><b>CONCLUSIONS</b>CSA, VER and CSA + VER could increase the uptake and reduce the efflux of C-AM by K562/VCR cells while no significant difference between the CSA + VER and CSA or VER was noticed. P-gp pump function and the effects of its reversal agents on leukemic cells can be rapidly and easily evaluated by using the C-AM and FCM.</p>

Long-term follow-up of treatment outcome and prognosis on 46 children with acute promyelocytic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Acute promyelocytic leukemia (APL) is a specific type of hematopoietic malignancy, accounting for 10% of the de novo acute myeloid leukemia (AML). The data on long-term outcome of APL in children are limited. The aim of this study was to investigate the clinical biological features, diagnosis, prognosis and long-term survival of childhood APL.</p><p><b>METHODS</b>A total of 46 children with newly diagnosed APL from April 1998 to October 2005 were enrolled into this study. Induction treatment containing all-trans retinoic acid (ATRA) plus daunorubicin (DNR) or pirarubicin (THP) was performed on these patients, followed by 6 courses of chemotherapy consolidation: DNR, homoharringtonine or etoposide plus Ara-C. A maintenance therapy was then administered once 3-6 months. The total period of treatment was 2.5 years.</p><p><b>RESULTS</b>Of the 39 patients who had completed the regular treatment, 36 (92.3%) achieved a complete remission. The 5-year cumulative incidence of relapse (CIR) was 28.6%. The estimated overall survival (OS) rates at 1, 3 and 5 years were (86.1 +/- 5.8)%, (76.1 +/- 7.5)% and (70.2 +/- 8.9)% respectively, while the event free survival (EFS) rates were (78.4 +/- 6.8)%, (63.6 +/- 8.7)% and (53.1 +/- 10.0)% respectively. The 5-year OS rate of patients with WBC less than or equal to 10.0 X 10(9)/L was (81.4 +/- 10.3)%, which was significantly higher than that with WBC greater than 10.0 X 10(9)/L[(51.6 +/- 14.7)%, P < 0.05]. Five patients with RT-PCR positive for PML/RARalpha S (short) subtype died eventually although all of them achieved CR, but none of the 13 patients with PML/RARalpha L (long) subtype died.</p><p><b>CONCLUSIONS</b>Remission induction therapy with ATRA + DNR or THP is effective and safe for newly diagnosed childhood APL. The remission induction therapy combined with chemotherapy containing high/intermediate dose Ara-C can improve the long-term survival rates of APL patients. High WBC count and S subtype of PML-RARa are two poor prognostic factors for children with APL.</p>

Reactivity of a novel monoclonal antibody ZCH-2B8a on normal hematopoietic cells and malignant cell lines and its significance / 中国实验血液学杂志

ZCH-2B8a (IgG2a) is a novel monoclonal antibody (McAb) generated in laboratory of Children Hospital of Medical College, Zhejiang University recently using human myeloblastic leukemia cell line KG1a as immunogen. This antibody has been submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. The aim of this study was to investigate the reactivity of this antibody on normal blood cells and malignant cell lines and to explore its possible application in clinical practice. The multi-parameter flow cytometry was used to analyze the expression pattern of 2B8a antigen in triplicate on normal blood components including T cells, B cells, natural killers (NK), neutrophils, monocytes, dendritic cells (DC), red blood cells (RBC), platelets (Plt), hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34(+) cells and malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines. The amount of positive cells > or = 20% was considered as positivity. The results showed that 2B8a antibody reacted to 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive to neutrophils (23.72%) and negative for T cells, NK, DC, RBC and Plt. The antibody reacted to all 3 marrow CD34(+) cells with an average positive rate of 39.33% while it was negative for G-CSF-mobilized CD34(+) peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody notably reacted to three out of 4 cell lines (Raji, SMS-SB, Nalm-6 and Nall-1) with the positive rates of 98.78%, 98.61%, 94.93% respectively and weakly to one of them with 5.68% in B lineage cell lines and monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including Meg01 (33.40%), HL-60 (29.70%), K562 (28.19%), KG1a (16.23%) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia, 5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KCNR, BE, SK-N-SH, SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). It is concluded that 2B8a antibody primarily reacts to B lineage and monocytic lineage cells which may bear the diagnostic and therapeutic applications among different types of hematopoietic malignancies.

Changes in CD69, CD25 and HLA-DR expressions in peripheral blood T cells in Kawasaki disease / 中华儿科杂志

<p><b>OBJECTIVE</b>The study was designed to investigate the changes in CD(69), CD(25) and HLA-DR expressions in peripheral blood T cell in Kawasaki disease (KD).</p><p><b>METHODS</b>The authors detected CD(69), CD(25) and HLA-DR expressions in peripheral blood T cell by using flow cytometry. The patients who met the diagnostic criteria for KD comprised sixteen boys and fifteen girls (4 - 60 months of age; mean, 26 +/- 18 months). All received intravenous gammaglobulin at a dose of 1 g/(kg.d), for 2 days and oral aspirin at a dose of 30 - 50 mg/(kg.d). In case of persistent fever, a repeated dose of intravenous gammaglobulin or I.V. methylprednisolone at a dose of 20 mg/(kg.d) for three daily doses was attempted. The authors tested blood samples from 17 healthy controls consisting of nine boys and eight girls (3 - 84 months of age; mean, 25 +/- 18 months) and the samples from 31 patients.</p><p><b>RESULTS</b>The percentage of peripheral blood CD(3)(+) T lymphocyte was (54.4 +/- 9.0)% in acute stage of KD and (65.0 +/- 7.0)% in healthy controls. There was a significant difference between the two groups (P < 0.001). The values of CD(69)(+) [(11.2 +/- 12.6)%, vs. (0.6 +/- 0.4)%], CD(25)(+) [(9.2 +/- 3.5)% vs. (3.9 +/- 1.8)%] and HLA-DR(+) [(8.3 +/- 5.0)% vs. (4.3 +/- 2.3)%] in KD patients were markedly increased compared to those of the healthy controls. After intravenous gammaglobulin treatment, the percentage of CD(3)(+)CD(69)(+) and CD(3)(+)CD(25)(+) significantly decreased [CD(3)(+)CD(69)(+): (14.0 +/- 13.0)% vs. (1.6 +/- 1.2)%, P < 0.05; CD(3)(+)CD(25)(+): (7.8 +/- 4.1)% vs. (2.0 +/- 0.6)%, P < 0.01]. However, the CD(3)(+) T lymphocytes increased [(50.8 +/- 5.0)% vs. (64.9 +/- 5.5)%, P < 0.01]. There was no significant difference in expression of CD(3)(+) T lymphocyte cell activating markers between coronary artery disease group and normal coronary artery group in KD cases (P > 0.05).</p><p><b>CONCLUSION</b>CD(3)(+) T cell activation in the early and middle stages is involved in the mechanism responsible for cardiovascular injury.</p>

Application of flow cytometry to detect PP65 antigenemia for diagnosis and monitoring of human cytomegalovirus infection / 中华儿科杂志

<p><b>OBJECTIVES</b>To evaluate the clinical significance of flow cytometry (FCM) to detect the cytomegalovirus (CMV) PP65 antigen in patients with CMV infection.</p><p><b>METHODS</b>Samples from 35 patients without CMV infection were used as negative control. The definite diagnosis of CMV infection was based on the national criteria for CMV infection. All 136 patients with CMV infection were examined with the FCM to detect CMV PP65 antigen, real-time fluorescence quantitative-polymerase chain reaction assay (RFQ-PCR) to detect CMV-DNA and ELISA to measure the serum level of IgM antibody against CMV. The results of these 3 assays in 2 groups (isolated organ involvement and disseminated diseases) were compared and the significance of PP65 antigenemia was evaluated. A short-term follow-up was undertaken in 18 patients.</p><p><b>RESULTS</b>The percentages of PP65 positivity in blood mononuclear cells (MNC) and polymorphic nuclear leukocyte (PMNL) from 35 negative control patients were 0.21% +/- 0.09% with a range of 0 - 0.41% and 0.24% +/- 0.10% with a range of 0.12% - 0.48%, respectively, which were not significantly different (t = 0.425, P > 0.05). The 95(th) percentiles (P(95)) of PP65 in MNC and PMNL were 0.39% and 0.45%, respectively, so a cutoff value of >/= 0.50% was set. Of the 136 patients with CMV infection, 118 samples from 118 patients were positive for PP65 antigenemia with a positive rate of 86.8%, which was not statistically different from that (90.4%, chi(2) = 0.91, P > 0.05) of CMV-DNA detected by RFQ-PCR assay but it was significantly higher than that (45.6%, chi(2) = 51.50, P < 0.005) of the detection by IgM measurement. PP65 detection was correlated with urine CMV DNA amplification (chi(2) = 63.78, P < 0.01) while the different detection rates between the two assays were not statistically significant (chi(m)(2) = 1.78,P > 0.05). PP65 detection was not correlated with serum IgM measurement while the detection rates between the two were significantly different (chi(m)(2) = 52.92,P < 0.01). No significant difference was found between the detection rates of CMV infection in MNC (45/53, 84.9%) and PMNL (43/53, 81.1%) (chi(m)(2) = 0.25, P > 0.05). Higher PP65 antigenemia level was correlated with systemic CMV infection, while lower level of PP65 was either in the patients with isolated organ involvement by CMV (chi(2) = 38.51, P < 0.005) or less severe in patient's situation. PP65 antigenemia of CMV infection returned to lower level or negative in recovery stage and increased when condition of patients deteriorated.</p><p><b>CONCLUSIONS</b>PP65 antigenemia detection by FCM is effective in the diagnosis of the active CMV infection. Quantitative monitoring of PP65 antigenemia is useful in the evaluation of patients with CMV infection.</p>

Study on elimination delay in high dose methotrexate therapy in childhood acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the incidence of elimination delay in high dose methotrexate (HDMTX) therapy, its side effects and influence to next course of chemotherapy and analyze the relationship between the dosage, the duration of MTX infusion and the morbidity of the elimination delay.</p><p><b>METHODS</b>A total of 121 childhood acute lymphoblastic leukemia (ALL) (497 infusions of HDMTX) were analysed in this study. The elimination delay rate and the adverse effects in different dose groups (3 g/m2 vs 5 g/m2) and different infusion duration groups (7 h vs 24 h) were compared. The adverse effect evaluation was based on the World Health Organization (WHO) Toxicity Grading Criteria. The rescue dosages of calcium folinate (CF) among these groups were compared through CF/MTX index.</p><p><b>RESULTS</b>The overall morbidity of elimination delay was 12.1% with a relative risk of 30.6% for the first time. The relative risk for the second time of occurrence was increased to 45.9% (P < 0.01) and it was not significantly increased for the third time (35.3%). Children with elimination delay had lower platelet count (P < 0.01) and higher CF rescue dosage (P < 0.01), while the damage of oral mucous membrane was more severe (P < 0.05) and the next course of chemotherapy would be postponed for a median of 4 days in 3 g group. There was no significant difference in elimination delay rates between 3 g and 5 g groups (12.1% vs 12.0%, P > 0.05), and between 7 h and 24 h MTX infusion groups (13.6% vs 11.9%, P > 0.05). The only side effect occurred in 5 g group was gastrointestinal morbidity. The CF/MTX index of 5 g group without elimination delay was less than that of 3 g group (P < 0.01).</p><p><b>CONCLUSION</b>Elimination delay in HDMTX therapy accompanies the suppression of bone marrow and damage of oral mucous membrane, which need more CF rescues and will postpone the following course of chemotherapy. Elimination delay is not associated with the duration of the infusion and the dosage of MTX within the range of 3 approximately 5 g/m2 but there are individual differences.</p>

Comparison between CD19 and CD20 expression patterns on acute leukemic cells / 中国实验血液学杂志

In order to provide the evidences for CD19 as a better antibody targeting molecule for B lineage acute leukemias than CD20 through the multi-parameter flow-cytometry analysis of leukemia cells, the samples from 321 patients with acute leukemia (AL) were immunophenotyped by multi-color flow cytometry and CD45/SSC gating strategy followed by the analysis of CD19 and CD20 expression. The results showed that the positive rate of CD19 (115/116, 99.1%) in 116 cases with B lineage acute lymphoblastic leukemia (B lineage ALL) was significantly higher than that of CD20 (33/116, 28.4%) (P < 0.01); in 17 patients with B lineage/Myeloid (B/My) acute mixed lineage leukemia (AMLL), the former positive rate (17/17, 100%) was also higher than the latter (5/17, 29.4%) (P < 0.01). Both of the two antigens were negative in 29 patients with acute T lymphoblastic leukemia and 7 patients with T/My AMLL. The positive rates of CD19 and CD20 in 152 patients with acute myeloid leukemia (AML) were 7.2% and 2.0%, respectively. The difference of the fluorescence intensity between the two antigens on the cells from each patient with B lineage ALL or B/My AMLL was statistically significant (t = 20.68, P < 0.001). The specificity of CD19 and CD20 in B lymphocytic lineage was 92.3% (132/143) and 92.7% (38/41), respectively, while the sensitivity was 99.2% (132/133) and 28.6% (38/133), respectively, the former sensitivity was significantly higher than the latter (chi(2) = 144.018, P = 0.001). It is concluded that CD19 continuously and steadily express on almost all subtypes of B lineage leukemic cells with homogeneous pattern while only a small number of leukemias express CD20. Both the specificity and sensitivity of CD19 were very high with a much broader reaction pattern than that of CD20 on this group of diseases. These indicate that CD19 may be a better antibody targeting molecule than CD20 for patients with B-lineage acute leukemia.

Preparation and characterization of a directly labeled mouse anti-human CD14 monoclonal antibody ZCH-2F9-FITC / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare fluorescein isothiocyanate (FITC) directly conjugated to monoclonal antibody (McAb) anti-human CD14, ZCH-7-2F9 (2F9-FITC).</p><p><b>METHODS</b>After generation and purification, the purity and the murine immunoglobulin subtype of the antibody were evaluated with SDS-PAGE and multicolor flow cytometry (FCM). 2F9 McAb was directly labeled with FITC through modified Marsshall's method and the positive rate of the 2F9-FITC on different types of leukemic cells were compared with the standard CD14-FITC by FCM.</p><p><b>RESULT</b>A large quantity of purified 2F9 McAb was prepared. The subtype of 2F9 was murine IgG1kappa. 2F9-FITC was successfully manufactured with A295/A280 ratio of 0.44. The positive cell percentages of 2F9-FITC and CD14-FITC on the monocytes were 84.50% and 90.08%, respectively, while those on lymphocytes were only 0.52% and 1.01%. There was no significant difference between the CD14 expressions with 2F9-FITC and CD14-FITC on each type of leukemia (n=23, t=0.922, P=0.367).</p><p><b>CONCLUSION</b>2F9-FITC has been successfully prepared and it can be applied in diagnosis and differentiation of monoblastic leukemias.</p>

The expression of CD19 in 210 cases of childhood acute leukemia and its significance / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of CD19 on childhood acute leukemia (AL) and its significance, and to provide evidence for the diagnosis and differential diagnosis as well as monoclonal antibody-targeting treatment of leukemia.</p><p><b>METHODS</b>There were 210 cases of childhood AL, of which 130 cases were male and 80 were female with a mean age of 9 years old. Using a panel of 27 fluorochrome directly labeled monoclonal antibodies, 210 samples from the patients were analyzed with CD45/SSC double parameters and multi-color flow cytometry to determine the expression of CD19.</p><p><b>RESULTS</b>In the 93 cases of B lineage acute lymphoblastic leukemia (ALL), the positive rate (98.9%, 92/93) of CD19 was significantly higher than that of the other B cell related antigens, such as CD10 (88.2%, 82/93, P = 0.003), CD20 (24.7%, 23/93, P = 0.001) and CD22 (60.2%, 56/93, P = 0.001). CD19 was expressed on all 8 cases of B/myeloid (My) hybrid acute leukemia (HAL) and 1 case of B/T HAL, but was not expressed on all 24 cases of T lineage leukemia and 5 cases of T/My HAL. In the 79 cases of acute myeloid leukemia (AML), only 5 (6.3%) cases expressed CD19. The positive rate (6.3%) of CD19 on AML was significantly lower than that on B lineage ALL (98.9%, P = 0.001). The percentage of CD19 positive cells in B/My HAL (41.6% - 88.7% with a mean of 73.8%) was significant higher than that in CD19(+)-AML (21.4% - 50.4% with a mean of 24.2%; Run Sum test, P = 0.0084). Of the 210 cases, 102 were B lineage related AL including B lineage ALL, B/My HAL and B/T HAL. In B lineage related AL, the sensitivity and the specificity of CD19 was 99.0% (101/102) and 95.4% (13/108) while the positive predictive and the negative predictive values to B lineage were 95.3% (101/106) and 99.0% (103/104), respectively. Using CD19(+) as a single reagent to diagnose B lineage, the false positive rate was 4.6% (5/108) and the false negative rate was 1.0% (1/102) with a general diagnosis index (GDI) of 94.4% [GDI = 1-(false positive rate + false negative rate)].</p><p><b>CONCLUSION</b>CD19 is continuously and stably expressed on all stages of B lineage differentiation. It is a reliable cell membrane marker for diagnosing B lineage ALL and an ideal target for antibody-targeting treatment of leukemia as well; the expression degree of CD19 can be used to distinguish B/My HAL from CD19(+)-AML; CD19 didn't express on normal myeloid cells but did on some AML cells. Therefore it can be used to detect the minimal residual disease.</p>

Expression of CD56 in acute leukemia and its clinical significance / 中国实验血液学杂志

In order to investigate the expression of CD56 on acute leukemia cells and its clinical significance, samples from 70 patients with acute leukemia were analyzed with multicolor flow cytometry to determine the CD56 and other leukocyte differentiation antigens. The results showed that 16 of 70 cases (22.86%) were identified to express CD56, of which 1/35 (2.86%) patient with acute lymphocytic leukemia (ALL) and 15/31 (48.39%) with acute myelocytic leukemia (AML) were CD56 positive. The positive rate of CD56 in AML was significantly higher than that in ALL (P < 0.01). The expression of CD56 varied in AML subtypes. The positive rate (11/15) of CD56 in AML-M(0), -M(1) and -M(2) was significantly higher than that in AML-M(3), -M(4) and -M(5) (4/16) (P = 0.013). 13 of 15 AML with CD56 expression were also positive for HLA-DR (41.94%), and a significant positive correlation was found between the expression of CD56 and HLA-DR (r = 0.439, P = 0.014). It was concluded that CD56 mainly expressed in AML cells. The analysis of CD56 expression on acute leukemia is of great value in the diagnosis, prognosis prediction and monitoring of minimal residual disease in AML patients.

Establishment of models for purging leukemic cells from the grafts in vitro / 中国实验血液学杂志

Using a fluorochrome Calcein-AM, leukemia cells were labeled and seeded into cell lines or bone marrow cells to establish three cell-models of grafts with leukemia. These cell-models were engaged with CD34 immunomagnetic beads and the purging efficacy was evaluated using both fluorescence microscopy and flow cytometry. The results showed that the cell-models established in this study could be evaluated successfully not only with fluorescence microscopy but also flow cytometry. After CD34 positive selection, KG1a cells were removed by (0.98 +/- 0.09) log in model II and NALM-6 cells were removed by (1.82 +/- 0.51) log in model III, respectively. It is concluded that the models established in this study are stable and direct with an excellent reproducibility and an accuracy, which can be used to evaluate purging efficacy of leukemia cells in model graft using immunomagnetic selection and the experimental studies on tumorcidal effect in vitro.