ISSN 1866-8836
Клеточная терапия и трансплантация

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C. Hematopoietic stem cell sources

Low and high-resolution HLA typing of cord blood samples of register of donors in St. Petersburg

Elena A. Kotelevskaya, Svetlana A. Smirnova, Alexander B. Smolyaninov

Importance of CFU-GM number for prediction of hematological reconstitution after low CD34+ cell dose autotransplantation in children

Yanina I. Isaikina, Valery I. Martinevsky, Nina V. Minakovskaya, Yury S. Strongin, Olga V. Aleinikova

Selection of optimal cryopreservation strategy for hematopoietic stem cells

Leonid I. Savelyev1,2,3, Yulia A. Yakovleva1, Grigory A. Tsaur1,2, Tatyana Yu. Verzbitskaya1,2, Alexander M. Popov1,2,3, Andrey A. Igumenshev1,2, Larisa V. Vakhonina1,2, Igor N. Vyatkin1,2, Egor V. Shorikov1,2, Larisa G. Fechina1,2

Effectiveness of hematopoietic progenitor cell separation with the help of the Sepax S100 cell separator

Alexander B. Smolyaninov, Dmitry A. Ivolgin, Shirzod F. Adylov, Elena A. Kotelevskaya

Virus markers antibodies detection in umbilical cord blood

Olga V. Supilnikova, Irina I. Maslenikova, Alexander B. Smolyaninov

Features of a stem cell’s cryopreservation

Alexander B. Smolyaninov, Ksenia V. Korovina, Shirzod F. Adylov, Elena A. Kotelevskaya

First experience of CD34+ cell selection to high-risk neuroblastoma patients

Yulia A. Yakovleva1,2, Grigory A. Tsaur1,2, Alexander M. Popov1,2,3, Tatyana Yu. Verzhbitskaya1,2, Igor N. Vyatkin1,2, Andrey A. Igumenshev1,2, Natalya G. Maisheva1,2, Anton Yu. Zadoya1,2, Egor V. Shorikov1,2, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

C. Hematopoietic stem cell sources

Is young matched unrelated donor comparable or superior to older sibling donor? An analysis of 76 allografted patients aged above 50 years from the Czech Acute Leukaemia Clinical Register (ALERT)

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Vladimir Koza1, Pavel Jindra1, Karel Indrak2, Fedor Alsabty3, Sarka Hrabetova4

1Dpt. Haematology & Oncology, Univ. Hosp., Pilsen, Czech Republic; 2Dpt. Haematology & Oncology, Univ. Hosp., Olomouc, Czech Republic; 3Dpt. Haematology, Univ. Hosp., Bratislava, Slovak Republic; 4Dpt. Haematology, Faculty Hosp., Prague, Czech Republic

Allo-SCT can be the only curative option even in the elderly. However, the siblings of elderly pts are naturally older and often ineligible for stem cell collection. The question is whether younger unrelated donors could compensate for the higher immune incompatibility.

Method

We retrospectively analyzed 76 AML pts aged above 50 (median 55y) from 5 Czech and Slovak centres allografted either with related (SIB group, n=47) or unrelated, 9–10/10 HLA allele-level matched donors (MUD group, n=29). Groups were comparable in terms of age, remission status pre-SCT, cytogenetic risk, donor sex, CMV D-/R- status, and conditioning. As expected, the MUDs were significantly younger and received more CD34+.

Results

After a median follow-up of 18 months, 39 pts (51%) are alive. The 3-y
Kap.-Meier OS and EFS probabilities were 42% and 40%, respectively, while overall NRM and relapse rates were 25% (19/76) and 26% (20/76), respectively. After splitting the pts into SIB vs. MUD, the figures were as follows: number of surviving pts 24/47 vs. 15/29, resulting in median OS probabilities for SIB and MUD of 16 and 21 months (p=0.817), respectively. The relapse rates were not significantly higher in the SIB group (30% vs. 21%, p=0.433); whereas the NRM rate was similar (23% vs. 28%, p=0.77). The incidences of aGVHD were identical both for SIB and MUD, comparable results were also reported for extensive cGVHD incidences.

Conclusions

Unrelated donors did not adversely affect the SCT outcome in AML patients aged >=50, and the younger allele-matched unrelated donor is – at least – the equivalent of an older HLA-identical sibling. We should not be reluctant with MUD in older pts, and molecularly-matched young unrelated donors should be tested in appropriately designed trials with the larger patient’s cohort.

Keywords

allo-SCT, donor, age, HLA compatibility

C. Hematopoietic stem cell sources

Low and high-resolution HLA typing of cord blood samples of register of donors in St. Petersburg

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Elena A. Kotelevskaya, Svetlana A. Smirnova, Alexander B. Smolyaninov

Stem Cell Bank Pokrovski, St. Petersburg, Russia

Umbilical cord blood (CB) contains hematopoietic stem cells (HSC) and can be used as an alternative to bone marrow transplantation in certain cases. Currently, the first register of cord blood donors is be created by the Stem Cell Bank Pokrovski. It aims to resolve the problem of deficiency the of HSC units in the country. The most important factor of effective transplantation is the degree of HLA matching. The target of this work is to HLA type each cord blood unit by low-resolution and then transition to high-resolution typing by sequence based typing (SBT).

During the work, 20 samples of cord blood were analyzing by polymerase chain reaction (PCR) with sequence specific primers (PCR-SSP). DNA was isolated from 0.7 ml of the entire quantity of blood by the PROTRANS DNA Box 500 (PROTRANS, Germany) DNA extraction column. HLA-A, HLA-B, and HLA-DRB1 loci were analyzed using the PROTRANS HLA-A*, -B*,
-DRB1* (PROTRANS, Germany) Cyclerplate System according to the manufacturer’s instructions. HLA-DRB1 high-resolution genotyping of 5 units was performed by the PROTRANS S4 system (PROTRANS, Germany), specially designed for the Beckman Coulter SEQ 8000 Genetic Analyzer (Beckman Coulter, USA). The program software Sequence Pilot for sequence result analyzing was also provided by PROTRANS.

The most frequently identified alleles were: HLA-A*02 (25%), *01 (20%), *24 (17.5%); HLA-B*08 (17.5%), *18 (12.5%), *35 (12.5%); HLA-DRB1*15 (17.5%), *07 (15%), *03 (12.5%).

The results of low-resolution HLA typing could be applied to the primary selection of donors when an HSC transplant HSC is required. Also they significantly simplify the high-resolution typing operation. The advantages of a national HLA-identified cord blood register in Russia are accessibility and lower costs of samples. But the major positive is the increased likelihood of HLA-matching in the same population.

Keywords

hematopoietic stem cells, umbilical cord blood, register of cord blood donors, transplantation, HLA typing

C. Hematopoietic stem cell sources

Importance of CFU-GM number for prediction of hematological reconstitution after low CD34+ cell dose autotransplantation in children

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Yanina I. Isaikina, Valery I. Martinevsky, Nina V. Minakovskaya, Yury S. Strongin, Olga V. Aleinikova

Belarusian Research Centre for Pediatric Oncology and Hematology, Minsk, Belarus

Correspondence
Isaikina Yanina I., Belarusian Research Center for Pediatric Oncology and Hematology, Pos. Lesnoe-2, 223040, Minsk reg., Belarus, Phone: +375-17-265-40-89, Fax: +375-17-265-42-22
E–mail: yaninai@mail.ru

Objective

The insufficiency of CD34+ cell numbers at the time of peripheral blood stem cell (PBSC) collection is frequently observed in children after multiple blocks of chemotherapy.

Aim

To detect the additional parameter of transplant quality for the prediction of successful hematological reconstitution after auto-PBSC transplantation with low doses of CD34+ cells in graft.

Methods

Forty-one children with malignances – who had received CD34+cells/kg <2х106 – were included in our study evaluating the correlations between CD34+cell/kg and CFU-GM/kg at different dose levels ≥2х105/kg and <2х105/kg and the time of hematopoietic recovery. A Mann-Whitney U test and multivariate correlation method were used to evaluate the importance of each graft parameter for the prediction of engraftment.

Results

There was no correlation between CD34+ cell dose and the time of engraftment in these pts. A high level of correlation was detected between the number of CFU-GM/kg infused and the time of  neutrophil (r=-0.67, p<0.05), platelet (r=-0.3, p<0.05), and/or first reticulocyte ›2‰ (r=-0.66, p<0.05) reconstitution. Infusion of CFU-GM/kg ≥2х105/kg (n=26) resulted in rapid short–term neutrophil >0.5x109/l and platelet >20x109/l recovery (10 days (5–13) and 22 days (8–57), respectively) compared with CFU-GM/kg <2х105/kg (n=15) (13 days (11–21) and 25 days (10–95), respectively) (p<0.05).

Long-term hematopoietic recovery platelet counts >50x109/l were 31 days (range: 9–81) in CFU–GM/kg ≥2х105/kg infusion.

We conclude that the number of CFU-GM/kg was the most important parameter for predicting the auto-PBSC with CD34+ cell dose <2x106/kg engraftment. The minimal recommended number of CFU-GM is 2x105/kg for the efficacy of hematopoietic recovery after auto-PBSCT.

Keywords

autotransplantation, CFU-GM, insufficiency of CD34+ cells, engraftment

C. Hematopoietic stem cell sources

Selection of optimal cryopreservation strategy for hematopoietic stem cells

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Leonid I. Savelyev1,2,3, Yulia A. Yakovleva1, Grigory A. Tsaur1,2, Tatyana Yu. Verzbitskaya1,2, Alexander M. Popov1,2,3, Andrey A. Igumenshev1,2, Larisa V. Vakhonina1,2, Igor N. Vyatkin1,2, Egor V. Shorikov1,2, Larisa G. Fechina1,2

1Regional Children’s Hospital №1, Yekaterinburg, Russia; 2Research Institute of Medical Cells Technologies, Yekaterinburg, Russia;
3Ural State Medical Academy, Yekaterinburg, Russia

Background

The results of hematopoietic stem cell (HSC) transplantation depend primarily on transplant quality. The temperature compensation provided during controlled rate preservation for the release of latent heat results in improved post-cryopreservation cell viability. The major variables involved are the rate of chamber temperature decrease, hold temperature and duration, the rate of temperature increase, and the temperature at which chamber cooling is reinitiated.

Aim

To create – on the basis of international recommendations – an optimal program of freezing for HSC using cryoprotective compounds – available in Russia – and the IceCube 15M programmed freezer.

Methods

Peripheral blood mononuclear cells (PBMC) were used as a test model. The cryoprotective solution contained high–molecular weight dextran, autologous serum, and DMSO with a final concentration of 1.6%, 5%, and 10%, respectively. Results were estimated by the shape of freezing curves and cells’ viability with 7-AAD staining on FacsCanto II.

Results

We tested 2 different volumes (185 ml and 100 ml) of PBMC. After adjusting freezing programs to the selected volume, the 2 different freezing curves gave optimal results. Afterwards created programs were used for the programmed freezing of patients’ HSC (24 samples). Thirteen samples were thawed for autologous transplantation. The median viability of HSC after thawing was 94% (range 77.5–98.9%) and the median time to hematopoietic recovery was 10 days (range 9–13).

Conclusion

It was shown that various volumes of PBMC/HSC need different freezing conditions. Thus, 2 different freezing curves were constructed for volumes less than 100 ml and more than 100 ml, respectively, that gave optimal results when assessed by viability and time to hematopoietic recovery (“take”).

Keywords

hematopoietic stem cells, cryopreservation, program of freezing, transplantation

C. Hematopoietic stem cell sources

Effectiveness of hematopoietic progenitor cell separation with the help of the Sepax S100 cell separator

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Alexander B. Smolyaninov, Dmitry A. Ivolgin, Shirzod F. Adylov, Elena A. Kotelevskaya

Stem Cells Bank Pokrovski, Bolshoi Prospekt V.O., Saint Petersburg, Russia

Correspondence
Alexander B. Smolyaninov, Stem Cells Bank Pokrovski, Bolshoi Prospekt V.O., 85, 199106, Saint Petersburg, Russia,
Phone: +7 (812) 908-77-27
E-mail: stemcellbank@inbox.ru

Cord blood processing for hematopoietic progenitor cell (HPC) separation had a quantum jump through the last few years. Only 5 years ago the cord blood bank in Singapore, which separated buffy coat practically by hand, had been accredited by the AABB. But now there is equipment that simplifies this step of cord blood processing. Confirmation of the effectiveness of the method of HPC separation using the Sepax S100 cell separator is our study purpose.

Using the established Stem Cells Bank Pokrovski procedure, we added the HES solution to the cord blood harvest and then placed it into the Sepax S100. We tested the leukoconcentrate after processing and before cryopreservation.

We processed 135 samples of cord blood using this technique. The average volume of processed cord blood was 89.45±23.52 ml. The average volume of leukocytal fraction was 21.35±5.35 ml (we used different protocols according to the cord blood harvest volume). The total amount of leucocytes in the concentrate was equal to 34.6±4.87*106cell/ml and the CD34+ cell fraction was 0.072±0.014 cell/ml – 0.2% of mononuclear cells on average. The vitality of HPC amounted to 91.2±8.4% on average. The tests had been carried out using a Beckman Coulter flow cytometer. We also found bacterial contamination in 4 samples (2.9%).

Our conclusion is that the effectiveness of cord blood processing using the Sepax S100 is equal to and – in some cases – better than other popular methods of cell separation. The comfort, processability, and accordance with international standards of that technique can significantly simplify cord blood processing. The Sepax S100 is capable of making this processing more available for our developing branch of stem cell banking and cell therapy.

Keywords

cord blood, hematopoietic progenitor cells, cell separation

C. Hematopoietic stem cell sources

Virus markers antibodies detection in umbilical cord blood

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Olga V. Supilnikova, Irina I. Maslenikova, Alexander B. Smolyaninov

Stem Cells Bank Pokrovski, Saint Petersburg, Russia

In accordance with the Ministry of Health’s Order № 325 – “About the developing of cells technologies,” 25 June 2003 – 120 samples of cord blood were tested at the Stem Cell Bank Pokrovski for the presence of infectious agents (anti-HIV 1 and 2, p24 HIV1, anti-HTLV I and II, anti-НВcorAg, HBsAg, anti-HCV, anti-CMV, anti-Toxoplasma gondii, and RW) using the ELISA method.

We detected none of the listed markers in 29.2% of samples. НВcorAg antibodies were detected in 6.6% of samples, HCV antibodies in 1.6%, CMV antibodies in 62.5%, and T. gondii antibodies in 20% of samples. The absence of the positive detection of HIV antibodies, р24 HIV1 antigen, and HbsAg depends on the isolation of the epidemic cause of mothers infected with HIV and HBV.

We also detected  T. pallidum antibodies in 2 cases with negative RW using the ID-PaGIA method. Those patients had a history of treating the disease caused by T. pallidum. HTLV I/II antibodies were not detected in the explored samples. Simultaneous detection of 2, rarer to 3 causative agents have been observed. Simultaneous presence of antibodies have been detected to CMV – 5 cases of 120, anti-НВcorAg and anti-HCV – 1 case of 120. However, T. gondii antibodies were not detected together with НВcorAg, HCV, and T. pallidum antibodies.

Thus, considering that cord blood derived from stem cells can be placed in long-term storage on condition of negative results of testing for the presence of anti-HIV 1 and 2, anti-HTLV I and II, anti-НВcorAg, HbsAg, and anti-HCV, 110 samples from the explored samples were deemed fit for storage and usage.

Keywords

hematopoietic stem cells, umbilical cord blood

C. Hematopoietic stem cell sources

Features of a stem cell’s cryopreservation

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Alexander B. Smolyaninov, Ksenia V. Korovina, Shirzod F. Adylov, Elena A. Kotelevskaya

Stem Cell Bank Pokrovski, St. Petersburg, Russia

Correspondence
Alexander B. Smolyaninov, Stem Cell Bank Pokrovski, St. Petersburg, 199106, Russia, Bolshoi Prospekt V.O., 85
Phone: +7 (812) 908-77-27
E-mail: stemcellbank@inbox.ru

Stem cells are the unique source for the renewal of all kinds of tissue. The umbilical cord blood (UCB) is well known to be a rich source of stem cells with practical and ethical advantages, but it can be received only at childbirth. That is what raised the question about the preservation of stem cells from cord blood.

To solve this problem, cryopreservation in liquid nitrogen is used. The storage of biopreparations – after freezing – is performed in cryobanks in liquid nitrogen with cryoprotectant. DMSO is used as a cryoprotectant to prevent cell destruction at low temperatures. The received cells are poured  into cryobags (20 ml) or cryoampules (4.5 ml), frozen with DMSO, and placed in liquid nitrogen (-196°С) for long-term storage. The process of cryoconservation consists of three stages: cooling from +20ºС to +1ºС using Coolmix (Biosafe, Switzerland), then freezing from +1ºС to -100ºС using Planer (UK), and finally transferring into storage with a temperature of -196ºС.

For optimal freezing conditions, Planer controller freezer (Planer, UK) was used. The freezing program has been optimized for stem cells and the speed of freezing is from 1 to 3 K per minute, which  avoids cell damage.

To avoid cross contamination, all samples of cord blood are placed in a specialized Dewar quarantine vessel until the results of an analysis on infections are received. Then samples are removed to “clear” Dewar vessels. There are currently more than 150 samples in the cryobank at the Stem Cell Bank Pokrovski.

According our results, after unfreezing the cell decreases only by 21.35±2.64% (p<0,001). The duration of UCB samples stored in a frozen state was 1 day and 1, 6, and 12 months; there was no difference in viability that depended on the storage duration. Therefore, the selected method of cryoconservation is rather effective and suitable for stem cells from umbilical cord blood. As a result, at the Stem Cell Bank Pokrovski the method of cord blood cryopreservation was optimized and adopted successfully.

Keywords

cryopreservation, stem cell, controller freezer, viability, crystallization point

C. Hematopoietic stem cell sources

First experience of CD34+ cell selection to high-risk neuroblastoma patients

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Yulia A. Yakovleva1,2, Grigory A. Tsaur1,2, Alexander M. Popov1,2,3, Tatyana Yu. Verzhbitskaya1,2, Igor N. Vyatkin1,2, Andrey A. Igumenshev1,2, Natalya G. Maisheva1,2, Anton Yu. Zadoya1,2, Egor V. Shorikov1,2, Leonid I. Savelyev1,2,3, Larisa G. Fechina1,2

1Regional Children Hospital 1, Ekaterinburg, Russia; 2Research Institute of Medical Cell Technologies, Ekaterinburg, Russia;
3Ural State Medical Academy, Ekaterinburg, Russia

Objective

To estimate the applicability of CD34+ cell selections using CliniMACS plus equipment (Milteni Biotec).

Methods

From June 2007 to April 2008 eight selections were performed on 7 children with high-risk neuroblastoma (stage 4). In 4 patients peripheral blood stem cells (PBSC) were mobilized with G-CSF (10 µg/kg/day) for 6 days and leukapheresis was performed on days 5 and 6 of G-CSF administration by the “Cobe Spectra” cell separator (Gambro). In 2 patients bone marrow (BM) was used for the CD34+ cell selection. In 1 patient PBSC and BM were used consecutively. The total number of nucleated cells was measured by Sysmex KX 21 (Roche) before and after CD34+ cell selections. The number and viability of cells were assessed by the “FACSCanto II” flow cytometer (BD, USA).

Results

Before CD34+ selection, the median percentage of CD 34+ cells in harvested cells was 1.64% (0.23–5.5). The median dose of CD34+ cells per 1 kg of patients body weight was 10.64*106 (1.61–42.37*106). After CD34+ selection, the median of selected CD34+ cells per 1 kg of weight was 4.93*106 (1.42–14.5*106) with a median purity of 96.74% (88.2–97.7%).
The viability of isolated CD34+ cells was 91.8% (71.6–99.6%). The median time to achievement of 500/µl ANC was 34 days (28–40).

Conclusion

Immunomagnetic selection of CD34+ cells by CliniMACS allows the obtainment of viable and highly purified autologous CD34+ cells. CD34+ selection leads to delayed hematopoietic recovery in our patients.

Keywords

high-risk neuroblastoma, children, CD34+ cell selection