Molecular studies on parental origin and cell stage of nondisjunction in sex chromosome aneuploidies / 中华预防医学杂志

To study the parental origin and cell stage of nondisjunction in sex chromosome aneuploidies. Retrospectiving and analyzing the results of 385 cases of SCA confirmed by QF-PCR and karyotype analysis in the prenatal diagnosis center of Guangzhou Women and Children Medical Center from January 2015 to December 2020. The types of samples and prenatal diagnosis indications were analyzed. The parental origin and cell stage of nondisjunction in sex chromosome aneuploidies analyzed by comparing the short tandem repeat (STR) peak patterns of samples from fetuses and maternal peripheral blood. The results show that (1) There were 324 cases of nonmosaic SCA, 113 cases (113/324, 34.9%) were 45, XO, 118 cases (118/324, 36.4%) were 47, XXY, 48 cases (48/324, 14.8%) were 47, XXX and 45 cases (45/324, 13.9%) were 47, XYY. 68 (45/324, 60.2%) cases of 45, X were detected in villus samples. The other SCA cases were mainly detected in amniotic fluid samples. There were 61 mosaic SCA samples, 58(58/61, 95.1%) of mosaic SCA samples were mosaic 45, X. (2) The top two indications of 45, X cases are increased nuchal translucency(53/113, 46.9%) and fetal cystic hygroma (41/113, 36.3%), while the most common indication of other types of SCA was high risk of NIPT(170/272, 62.5%). (3) Among 45, X cases, there were 88 cases (88/113, 77.9%) inherit their single X chromosome from their mother and 25 cases (25/119, 22.1%) from their father. In 47, XXY samples, 47 cases (47/118, 39.8%) of chromosome nondisjunction occurred in meiosis stage Ⅰ of oocytes, 51 cases (51/118, 43.2%) occurred in meiosis stage Ⅰ of spermatocytes, and 20 cases (20/118, 16.9%) occurred in meiosis stage Ⅱ of oocytes. Among 47, XXX samples, 29 cases (29/48, 60.4%) of X chromosome nondisjunction occurred in meiosis stage Ⅰof oocytes, 15 cases (15/48, 31.3%) occurred in meiosis stage Ⅱ of oocytes, and 4 cases (4/48, 8.3%) occurred in meiosis stage Ⅱ of spermatocytes. In summary , the cases of 45, X were mainly diagnosed by villous samples for abnormal ultrasound findings. The other cases of SCA were mainly diagnosed by amniocentesis samples for abnormal NIPT results. Different types of SCA, the origin and occurrence period of sex chromosome nondisjunction were different.

Pooled Umbilical Cord Blood Plasma for Culturing UCMSC and Ex Vivo Expanding Umbilical Cord Blood CD34⁺ Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of umbilical cord blood plasma (UCP) as a replacement for fetal bovine serum (FBS) for culturing mesenchymal stem cells (MSC) derived from umbilical cord, and to observe the supporting effects of these cells (served as a feeder layer) on ex vivo expanding of human umbilical cord blood CD34(+) cells.</p><p><b>METHODS</b>Umbilical cord blood (UCB) units were suitable if the Guangzhou cord blood bank donor selection criteria strictly were fulfilled. UCP were ready to use after the collection from the plasma depletion/reduction during the processing and pooling of suitable UCB units (at least 30 units were screened for pathogens and microorganisms, and qualified). Umbilical cord mesenchymal stem cells (UCMSC) were harvested from the umbilical cord tissue of health full-term newborns after delivery by enzyme digestion and divided into 3 groups: group 1 and 2 were cultured in the presence of DMEM/F12 containing either FBS or UCP; and group 3 was cultured in serum-free medium (StemPro® MSC SFM CTS™). Morphology, proliferation and surface marker expression were examined by flow cytometry, and the differentiation toward adipogenic and osteogenic lineages was used for investigating the effect of media on UCMSC after 3-5 passages. Next, the cells cultured in the three different media were cryopreserved and thawed, then prepared as feeder layers with the name of UCMSC(FBS), UCMSC(UCP), and UCMSC(SFM), respectively. The CD34⁺ cells were separated from UCB by magnetic activated cell sorting (MACS) and divided into 4 groups cultured in StemPro(-34) SFM medium added with hematopoietic cytokine combination (StemSpan® CC100). The control group included only CD34⁺ cells as group A (blank control) and experimental groups included UCMSC(FBS) + CD34⁺ cells as group B, UCMSC(UCP) + CD34⁺ cells as group C, UCMSC(SFM) + CD34⁺ cells as group D, and cells in all groups were cultured ex vivo for 7 days. The nucleated cell (NC) number was counted by cell counter, CD34⁺ cells were measured by flow cytometry, and clonogenic assay was conducted at day 0 and 7 of culture. The expansion efficiency was assessed.</p><p><b>RESULTS</b>The morphology (spindle-shaped and plastic-adherent), the immunophenotype (high positive percentage of CD73, CD90, CD105 and CD166) and the differentiation potential (osteogenic and adipogenic) were almost indistinguishable among the cells cultured in any of these three media except for the expression of CD105 in group 3 (serum-free medium) was lower than that in other 2 groups (P < 0.05). UCMSC grown in UCP medium demonstrated significantly higher proliferation rates than that in media containing FBS or commercial serum-free supplement (P < 0.05). After co-culture for 7 days, the CD34⁺ cell percentage decreased in all the groups, while NC were amplified effectively and the CD34⁺ cell number increased with the same order as group C or D group B or A (control group) (P < 0.05). As compared with the colony-forming unit (CFU) number at day 0, there was no significant difference in the expansion multiple between group C and D, while the expansion of CFU in group C were higher than that in group B and A.</p><p><b>CONCLUSION</b>The UCP can be used as a better animal-free serum supplement for growth, maintenance and differentiation of UCMSC, thus would be a safe choice for clinical-scale production of human MSC.</p>

Analysis of post-thaw infused cell dose for predicting engraftment after unrelated cord blood transplantation / 中国实验血液学杂志

This study was purposed to investigate the role of post-thaw infused donor cells for predicting engraftment and hematopoietic reconstitution after unrelated cord blood transplantation (UCBT). The retrospective analysis was performed on clinical data of 97 children with malignant or non-malignant diseases received single unit UCBT from August 1999 to April 2010. The impact of pre-freezing and post-thaw cell dose of total nucleated cells (TNC), CD34(+) cells and colony-forming units-granulocyte/macrophage (CFU-GM) on engraftment and hematological recovery after UCBT was analyzed. Unrelated donors were from Guangzhou cord blood bank (GZCBB) entirely. The results indicated that the pre-freezing TNC (/kg) (mean ± SD: 7.65 × 10⁷ ± 4.26 × 10⁷; median: 6.34 × 10⁷), CD34(+)cells (/kg) (mean ± SD: 4.64 × 10(5) ± 4.47 × 10⁵; median: 3.03 × 10⁵) and CFU-GM (/kg) (mean ± SD: 0.79 × 10⁵ ± 1.09 × 10⁵; median: 0.57 × 10⁵) showed a good correlation with their post-thaw counterparts including TNC(/kg) (mean ± SD: 6.98 × 10⁷ ± 4.12 × 10⁷; median: 6.00 × 10⁷), CD34(+)cells (/kg)(Mean ± SD: 6.86 × 10⁵ ± 8.56 × 10⁵; Median: 4.17 × 10⁵), and CFU-GM (/kg) (mean ± SD: 0.52 × 10⁵ ± 0.52 × 10⁵; median: 0.39 × 10⁵) (r = 0.952, p < 0.001; r = 0.794, p < 0.001; r = 0.478, p < 0.001). Either the pre-freezing or post-thaw number of infused CFU-GM was significant higher in patients who achieved engraftment (n = 70) than those who suffered graft failure (n = 22) (p = 0.023 and 0.011, respectively), but no significant difference of TNC and CD34(+) cells dose (pre-freezing or post-thaw) were found between these two groups. Pre-freezing CFU-GM, TNC, CD34(+) cell dose negatively correlated with the time of neutrophil engraftment (r = -0.285, p = 0.018; r = -0.396, p = 0.002; r = -0.373, p = 0.002), as well as the post-thaw number of TNC and CD34(+) cells (r = -0.260, p = 0.031; r = -0.483, p < 0.001), whereas only pre-freezing CD34(+) cells showed a significant correlation with platelet engraftment time (r = -0.352, p = 0.013). It is concluded that the CFU-GM amount is useful for predicting engraftment of UCBT, while pre-freezing hematopoietic cell doses show superior correlation with the speed of engraftment and hematopoietic reconstitution than their post-thaw counterparts in pediatric recipients, suggesting that it is essential to perform hematopoietic potency assay on each cord blood unit prior to listing or release for administration.

Study of the HLA-A, B, DRB1 allele and haplotype frequencies of 4194 cord blood samples in Guangzhou / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the distribution of the human leukocyte antigen (HLA)-A, B and DRB1 allele and haplotype in cord blood samples preserved in Guangzhou Cord Blood Bank collected in the last 10 years.</p><p><b>METHODS</b>The HLA-A, B and DRB1 genotyping of 4194 cord blood samples were detected by Special Monoclonal Tray, PCR-sequence specific promer (PCR-SSP), PCR-sequence specific oligonucleotide probe (PCR-SSO) and sequence based typing (SBT). Frequencies of HLA-A, B and DRB1 allele and haplotype were calculated by Arlequin software.</p><p><b>RESULTS</b>The total numbers of HLA-A, B and DRB1 alleles are 18, 43, 13 respectively. The obviously high frequency alleles are A*11, A*02, A*24, A*33, B*40, B*15, B*46, B*13, DRB1*12, DRB1*15, DRB1*09 and DRB1*04, with accumulative frequency of each locus being more than 50%. The most common haplotypes are A2-B46, B46-DR9, A11-DR12 and A2-B46-DR9.</p><p><b>CONCLUSION</b>The distribution of HLA-A, B and DRB1 allele and haplotype of cord blood in Guangzhou Cord Blood Bank has typical characteristics of southern Chinese Han population. Authors' data may help in searching for appropriate donors.</p>

Clinical application of HLA sequencing in unrelated umbilical cord blood transplantation / 中国实验血液学杂志

From June 1998 to July 2004, Guangzhou umbilical cord blood bank provided unrelated umbilical cord blood for 54 patients to more than 21 transplantation centers. HLA sequencing-based typing (SBT) was used to re-analyze the results of HLA antigens and alleles so as to investigate the relationship between HLA alleles and GVHD. The information about 48 out of 54 patients was obtained after 6 months of follow up. SBT was used to identify HLA-A, B, DRB1 alleles in 48 patients received the unrelated umbilical cord blood units, and the obtained results were compared with the results of HLA-SSP Low Resolution Typing. The results showed that the difference of GVHD incidence between less than 2 mismatched HLA sites and less than 3 sites was statistically significant (P < 0.05). In the results from single factor analysis and high-resolution typing of HLA-A, B and DRB1 alleles, the mismatch between HLA-B and HLA-DRB1 alleles was found to be a significant factor for the occurence of GVHD. It is concluded that SBT plays an important role in umbilical cord blood transplantation, and the incidence of GVHD is higher in the transplantation with HLA-DRB1 alleles mismatching.

Correlative analysis between HLA high resolution typing and GVHD in 48 cases received unrelated umbilical cord blood transplantation / 中国实验血液学杂志

Unrelated umbilical cord blood units for 54 cases in 21 transplant centers were provided by Guangzhou Cord Blood Bank in China from 1998 to 2003. This study was aimed to identify HLA-DRB1 alleles by means of PCR sequencing based typing methods (SBT) and to analyze the correlation between HLA-DRB1 alleles and GVHD in unrelated umbilical cord blood transplantation (UCBT). 48 out of 54 patients received UCBT were followed up. DNA were extracted from cryopreservation blood of recipients/donors with UCBT, HLA-DRB1 alleles typing were done by SBT. Compared with low resolution results of HLA-DRB1 alleles, high resolution results were analyzed to see any correlation between HLA-DRB1 alleles and GVHD. by double-blind statistically analysis of HLA-DRB1 high resolution in 48 donor/recipient typings in UCBT. The results showed that the incidence of GVHD (25%) in patients who has HLA-DRB1 alleles matched with donors significantly lower (65.6%) than that in the patients with HLA-DRB1 alleles mismatched (P = 0.008). It is concluded that HLA-DRB1 by high resolution typing method is important in clinical application in UCBT.

Modified guanidine hydrochloride method for DNA extraction from cord blood used in HLA genotyping / 中国实验血液学杂志

To compare two different methods for extracting genomic DNA from cord blood and to evaluate their applications for HLA genotyping, the genomic DNA from 72 samples was extracted by guanidine hydrochloride (Gu * HCl) and modified guanidine hydrochloride, the DNA yield and purity were evaluated by spectrophotometry and detected by PCR with sequence-specific primers. The result showed that the genomic DNA was successfully isolated from whole blood by both methods. The modified Gu * HCl method used was better than Gu * HCl method as the modified method produces better quality of DNA and less ambiguous bands in PCR. It is concluded that modified Gu * HCl method has the advantages of low-cost, simple operation, high quality output and clear positive bands in HLA-genotyping, the modified method is optimal for extracting DNA from multiple samples of cord blood bank.

Results of unrelated umbilical cord blood stem cell transplantation for 65 patients in China / 中华儿科杂志

<p><b>OBJECTIVE</b>From December 1998 to April 2004, 3960 umbilical cord blood units were stored in Guangzhou cord blood bank, which provided 100 umbilical cord blood units to 25 transplant center for 83 patients with malignant or non-malignant diseases. To study the related factors affecting unrelated umbilical cord blood stem cell transplantation, the authors analyzed retrospectively the results of transplantation of unrelated umbilical cord blood stem cells for 65 patients.</p><p><b>METHODS</b>ALL (acute lymphocytic leukemia) cord blood units were obtained from full term normal vaginal and cesarean deliveries in Guangzhou Women and Infants Hospital. The fractionation, cryopreservation and thawing of the cord blood were performed according to the regulations of New York umbilical cord blood bank and pertinent literature. The selection of cord blood was based on HLA typing and the number of nucleated cells. The sex and HLA antigens of donors were defined as the evidence of engraftment. Time to engraftment was recorded when the absolute number of neutrophil ANC (absolute neutrophil count) was higher than 5.0 x 10(8) for three days. Event-free survival and graft versus host disease (GVHD) were provided by transplant centers.</p><p><b>RESULTS</b>Out of 65 patients who received unrelated cord blood stem cell transplant, 49 patients were diagnosed as having malignant diseases [including 23 with ALL, 16 with AML (acute myeloid leukemia), 7 with CML (chronic myelogenous leukemia), 3 with lymphoma and one with MDS (myelodysplastic syndrome)], 16 patients had non-malignant disease. The 65 transplanted patients (42 male, 23 female) had a median age of 10 years (range 1 - 33 years) and a median body weight of 27 kg (range 10 - 67 kg). The patients received cord blood stem cells from unrelated 0-locus (n = 9) or 1-locus (n = 43) or 2-locus (n = 13) HLA mismatched donor. The median dose of infused cells was: total neutrophil count (TNC) 5.7 x 10(7), CD(34)(+) 5.1 x 10(5), CFU-GM 3.8 x 10(4). Fifty of 65 (77%) patients had engraftment. GVHD occurred in 41 patients (63%), including acute grade I - II GVHD in 31 patients (76%), acute grade III - IV GVHD in 8 patients (20%) and chronic GVHD in 2 patients (5%). Fifty patients had engraftment (ANC > 5.0 x 10(8)) after a median time of 17 (range 7 to 44) days after transplant, while an autologous hematopoietic reconstitution was observed in 6 patients; 24 patients died of severe pneumonia (n = 8), acute GVHD (n = 4), or sepsis (n = 12) and the disease-free survival probability was 61%.</p><p><b>CONCLUSIONS</b>Unrelated allogeneic umbilical cord blood transplantation may be a good substitution for unrelated allogeneic bone marrow transplantation with a good prospect.</p>

HLA-B gene polymorphism detected by high-resolution sequence-based typing in Guangdong Han populations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the genetic polymorphism of HLA-B locus in Guangdong Han population and compare the characteristic of the allele frequency distribution with that in other populations.</p><p><b>METHODS</b>A total of 562 cord blood samples from Guangzhou Cord Blood Bank were analyzed by sequence-based typing. Then the sequences encompassing exons 2, 3, and 4 for HLA-B gene were analyzed by direct sequencing of PCR products. The allele frequency distribution of HLA-B in this population was compared with that in other populations.</p><p><b>RESULTS</b>A total of 59 different HLA-B alleles were detected, and among them were 6 HLA-B alleles with frequencies higher than 5%: HLA-B*4601 (14.5%), HLA-B*400101 (14.4%), HLA-B*1502 (11.5%), HLA-B*1301 (8.6%), HLA-B*5801 (8.1%) and HLA-B*380201 (6.4%); the total frequency of these six alleles was 63.5%. At the same time, there were 30 kinds of HLA-B allele with frequencies lower than 0.5%; the total frequency of these alleles was 4.9%. Maximum variation at HLA-B was seen in the HLA-B*15 allele family (nine alleles). Comparison of the HLA-B frequencies in different populations showed a close relationship of Guandong Han population with the Chinese populations from Hong Kong and Singapore, respectively.</p><p><b>CONCLUSION</b>The results have shown the characteristic of HLA-B distribution and provided more accurate genotypic data that may serve as normal reference value for the Han population in Guangdong, China.</p>

Retrospective analysis of 54 cases of unrelated umbilical cord blood transplantation / 中国实验血液学杂志

In order to research the related factors of umbilical cord blood transplantation, 54 cases of unrelated umbilical cord blood transplantation were analyzed retrospectively, which were performed from June 1998 to July 2003. All cord blood units were obtained from full term normal vaginal deliveries in Guangzhou Maternal-Neonatal Hospital. The fractionation, cryopreservation and thawing of cord blood have been done according to the regulation of New York umbilical cord blood bank and pertinent literature. The selection of cord blood is based on HLA typing and the number of nucleated cells. The results showed that from June 1998 to July 2003, 3 475 units of cord blood were collected in Guangzhou Umbilical Cord Blood Bank and 99 units were provided for therapy of 85 patients in 21 transplantation centers, including 11 sibling and 74 unrelated cord blood transplantations. 54 cases of unrelated cord blood transplantation were reported, including 43 malignant diseases and 11 non-malignant diseases. The median age of recipients was 9.5 (range 1.2 - 33) years, the median weight was 27 (range 10 - 60) kg, the median number of TNC was 6.82 x 10(7)/kg, 43 cord blood were implanted (ANC > 500/microl) at day 60 after transplantation (79.6%, median 17). The time of nuclear cell reconstitution after cord blood transplantation was statistically related with nucleated cells and the type of disease, not related with HLA matching. Acute GVHD was present in 8 patients (21.6%) and chronic GVHD occurred in 2 patients (5.4%), 6 patients suffered from graft failure (11.1%). The total survival rate was 42.6%. It is suggested that unrelated umbilical cord blood transplantation seems to be a good substitute for bone marrow transplantation and has good prospects especially in children.

A retrospective analysis on multiple cord blood transplantation in 13 cases / 中国实验血液学杂志

In order to study the feasibility of multi-umbilical cord blood transplantation (multi-UCBT) in adult, 13 cases of unrelated allogeneic multi-UCBT performed from June 1998 to July 2003 were analyzed retrospectively. All cord blood units were obtained from full term normal vaginal and cesarean deliveries in Guangzhou Maternity and Neonatal Hospital. The fractionation, cryopreservation and thawing of cord blood were done according to the regulation of New York Umbilical Cord Blood Bank and pertinent literatures. Donors of HLA 1/6-2/6 mismatch were accepted at registry search. The results showed that from June 1998 to July 2003, 28 umbilical cord blood units were selected by 7 transplantation centers for 13 cases. The median age of recipients was 22 (8 - 41) years, and the median weight was 50 (21 - 75) kg, the median infused dose of total nuclear cells was 2.91 x 10(7)/kg. Six out of thirteen cases were engrafted after cord blood infusion with absolute neutrophil count of > 5.0 x 10(8)/L at 19 days post-infusion. Only one case suffered from graft versus host disease, the total survival of multi-UCBT was 46.2% (6/13). It is concluded that good prospects in the field of multi-umbilical cord blood transplantation is likely to be realized.

Analysis of HLA matching probability in Guangzhou Cord Blood Bank / 中国实验血液学杂志

The HLA matching results for 1 060 patients searching donors within 3 000 units of umbilical cord blood in Guangzhou Cord Blood Bank from 1998 to 2002 were analyzed. There were 119 (11.23%) and 992 (93.58%) patients found 6/6 and more than 4/6 of HLA matched loci of unrelated cord blood donors respectively. 61.29% of probability in all patients could find one or more cord blood with 4/6 or more matched loci and total nucleated cell (TNC) dose of >or= 3.7 x 10(7)/kg. The highest mean body weight in these supplied patients was 79 kg. The probability was 89.79% for those patients with TNC dose of >or= 2.0 x 10(7)/kg and >or= 4/6 of HLA loci matched. In these patients, the highest weight was 175 kg. In conclusion, a cord blood bank with 3 000 units or more of cord blood in stock shows a high probability of HLA matching and can meet the requirement of TNC >or= 3.7 x 10(7)/kg dose in child and part of adult patients. The umbilical cord blood is a good alternative stem cell source for all patients including adults.

Establishment of a method for HLA-DRB genotyping in cord blood by reverse dot-blot hybridization technique / 中国实验血液学杂志

The HLA system was discovered by virtue of the fact that it was polymorphic. The impetus for its discovery was the search for polymorphic antigens to match for transplantation, by analogy with the human red cell blood groups. The most usually DNA method of HLA typing is sequence specific oligonucleotides (SSO) and PCR sequence specific primers (SSP). SSO technique is perfectly suited for analyzing large number of samples, it is not suitable for individual or small numbers. The SSP method is ideal for typing individual samples, but it is costly and requires high capacity thermal cycles for larger numbers of samples. To set up a simple, quick, cheap and high resolution DNA method, were collected sixty-three cord blood samples from Guangzhou Cord Blood Bank, got DNA from blood by the traditional guanidine hydrochloride distillation method. Each sample was simultaneously typed by SSOP, PCR-SSP and reverse dot-blot hybridization (RDB) methods. All of typed is success. The results of three DNA methods are consistent each other. 60 HLA-DRB1 alleles could be accurately distinguished with the RDB method. Our results show that RDB method is a simple, quick, cheap and high resolution method for HLA-DRB types. It can be used in any HLA typing.