STEM CELL LEUCOCYTE BIOLOGY. Setting up of an Umbilical cord blood stem cell bank at Institute of Immunohaematology

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1 STEM CELL LEUCOCYTE BIOLOGY Setting up of an Umbilical cord blood stem cell bank at Institute of Immunohaematology Objectives: 1. To establish umbilical cord blood stem cell bank, which can be used as a source of haemopoietic stem cells (HSC) in bone marrow transplantation. 2. To compare different combinations of cryoprotective agents to improve viability after cryopreservation. 3. To find out whether cryopreservation at 80 o C in long term basis is feasible for the purpose of transplant. One hundred blood samples were collected from full term normal deliveries in Wadia maternity hospital. All pregnant females were screened antenatally for HIV, HbSAg and VDRL. After the delivery of the fetus, the umbilical cord was clamped and cut as per the hospital protocol. Umbilical vein was punctured while placenta still in utero, using 16 G needle of a triple blood bag set containing 25 ml CPDA and blood was allowed to drain in the bag by gravity. Once the placenta was separated and delivered, it was placed on a sterile sheet on a stand. Additional blood was collected by puncturing the prominent veins on the placenta by 18G needle & syringe. Blood aspirated with syringe was used for various tests like complete blood count, HLA typing and HIV, HbSAg and HCV testing. UCB was processed within 1 hour of collection. Testing: Before Cryopreservation UCB was tested for: 1. UCB was tested for complete blood count, blood grouping and HLA typing. 2. Serological assay (HIV, HbSAg and HCV), Progenitor assay (CD34 count and CFC), Bacteriological cultures. 3. Cell viability was checked by Trepan blue dye exclusion. RBC Depletion Technique:

2 The standard protocol for RBC depletion used worldwide includes use of 6 % Hydroxy ethyl starch (HES). However when we tried 6% Hydroxy Ethyl Starch, results were very poor with less than 70% nucleated cell recovery. Hence we tried different methods to improve the mononuclear cell yield. High molecular weight Dextran (Pharmacia), Gelatin (Sigma) and HES (Stem cell tecnlologies Ltd) with dilution of UCB with equal volume of phosphate buffer saline were tried with good results. Table 10: Results of RBC sedimentation using different techniques. 3%Dextran 3%Gelatin 6% HES 6% HES +PBS % WBC Recovery % RBC Depletion % Viability Since 6% HES with dilution of UCB with equal volume of PBS gave best results, this method was adopted for the cryopreservation of UCB units. The collected UCB was diluted with equal volume of PBS and 6% HES was added to make the final concentration to 1.5%. This mixture was allowed to stand for 1 hr for RBC sedimentation and the leukocyte rich supernatant was transferred to the second bag. This was centrifuged to reduce the final volume of cryopreservation to 20 ml. The cryopreservative solution (1/4 volume of cell suspension of 50 % DMSO and 5%dextran 40 in 0.85 % NaCl) was added slowly to cell suspension, in the cold with calibrated syringe. This was transferred to cryobags and stored at 80 0 C Results of Cryopreservation: Parameters 3 months 6 months One year % Recovery

3 % Viability by dye exclusion test Colony forming capacity % CD34 + recovery Study of effect of membrane stabilizers and bioantioxidants on cryopreservation of UCB HSCs: Optimization of cryopreservation method is important aspect in the banking of UCB. Out of multiple factors responsible for cryodamage of the cells. Cell membrane damage as a result of intracellular ice formation and subsequent leakage are one of the important factors. Generation of oxygen free radicals is another important factor that damage the cells at lower temperatures. We studied the effect of certain additives, like membrane stabilizer (Trehalose and Butanediol and bioantioxidants (asorbic acid and catalase) to conventional freezing medium on viability, nucleated cell recovery, CD34 + cell recovery and clonogenic potential of frozen cells. Freezing: The MNCs were frozen at a density of 10 7 / ml/ vial. The vials were frozen in 80 0 C mechanical freezers without any controlled rate freezing. The freezing medium contained 70% IMDM, 20% autologous plasma and 10% DMSO with or without additives. The concentration of additives used per milliliter was as follows: ascorbic acid (80µg), trehalose (25µg), 2-3 butanediol (8%). Thawing: The vials were removed and quickly thawed in the water bath at 37 0 C. The cells were diluted with thawing medium (90% IMDM +10% FCS). After centrifugation at 40C, the pellet was resuspended in medium containing 80% IMDM +20% FCS. Studies performed on fresh and frozen cells: Viability and nucleated cell recovery: Viability was determined by Trepan Blue dye exclusion test and nucleated cell count was taken using a Sysmex automated counter. CFU assays:

4 CFU assays were performed on fresh and frozen cells using methyl cellulose semisolid assays. CD34+ cell recovery: The CD34+ cell count on the fresh and frozen cells was performed using CD34- PE conjugated and CD45-FITC monoclonal antibodies conjugated using the International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines. Results: Viability and nucleated cell recovery: The samples showing % viability by Trypan blue dye exclusion study were used for cryopreservation experiments. The samples were tested for viability and nucleated cell recovery after 15 days, one month and 3 months after freezing at 80 ο C. The viability and nucleated cell recovery ranged from 80-80% and 50-90% respectively. The viability with Butanediol as an additive was better than other two combinations after 3 months 100 Viability % DMSO 10% DMSO 10%+T+AA DMSO 10%+B+AA No. of days Fig 22: shows Addition of Butanediol to the freezing medium results in better viability after 3 months of cryopreservation after 3 months. Effect of cryoprotective agents on Colony Forming Capacity (CFC): The semisolid methyl cellulose assay method was adopted for studying CFC as described earlier. The samples were tested for CFC at 15 days, 1 month and 3 months duration. The colony counts with or without additives were compared. The addition of

5 additives to conventional freezing medium resulted in better protection of Clonogenic cells. The combination of Butanediol and Ascorbic acid with 10% DMSO was better than other two combinations. CFU/2x105 MNC Platlet DMSO 10% DMSO 10%+T+AA DMSO 10%+B+AA No. of days Fig: 23 Addition of 2,3 Buatnediol to the conventional freezing medium of 10% DMSO results in increased recovery of colony forming cells after 3 months of cryopreservation. Conclusion: During the first year of the project standardization of the procedure for final cryopreservation was carried out. At present 50 units of cord blood have been cryopreserved at 80 o C. However due to the very low budget of the project a large scale cryopreservation in liquid nitrogen was not undertaken. The preliminary results of addition of different cryopreservation agents to the standard 10% DMSO solution have been encouraging. However, effect on long-term storage at 80 o C is in progress. Study of cytogenetic, immunophenotypic and biologic characteristics of AML with study of FLT3 gene mutations Objectives:

6 1. Study FLT3 mutations and expression of multi-drug resistance protein, P- glycoprotein (P-gp) in patients of Acute Myeloid Leukemia (AML) to identify the high-risk population. 2. Study correlation between FLT3 gene mutations, expression of P-gp and immunophenotypic and cytogenetic abnormalities in patients of AML Total 68 patients with diagnosed de novo acute myeloid leukemia attending the haematology department, K.E.M. Hospital during year were included in this study. Diagnosis was based on the peripheral blood and bone marrow examination for morphology, cytochemistry and immunophenotypic studies. All clinical details including presenting features, treatment given and response to treatment were a recorded. Immunophenotyping: A muitiparameter analysis based on Tripple colour immunophenotypic study of the surface antigens will be performed using combinations of Phycoerythrine (PE), Flurescein Isothyocynate (FITC) and PerCP conjugated monoclonal antibodies antibodies. The folloing antibodies were used Myeloid markers: CD13, CD14, CD 15, CD 33, cmpo Lymphoid markers: CD2, CD3, CD4, CD5, CD 7, CD8,CD10, CD19, CD20, CD22, CD23, Cd79a Non lineage markers: CD34, CD45, and HLA-DR Cytogenetic studies: Cytogenetic analysis was performed using standard Giemsa banding techniques to stain metaphases obtained from unstimulated and after short-term cultures. The definition of a cytogenetic clone and descriptions of karyotypes will follow the International System for Human Cytogenetic nomenclature. FISH analysis will be carried out using different specific probes. FLT3 mutation studies:

7 Genomic DNA was extracted using a salting out procedure from fresh bone marrow or peripheral blood cells after Ficoll separation of mononuclear cells of all these 68 cases. Exon 11 and 12 of the FLt3 gene will be amplified by genomic polymerase chain reaction. The direct nucleotide sequencing of the amplified fragment will be carried out further. The standardization of this procedure is being done. P-gp expression studies: P-gp expression was studied by Flowcytomerty using fluorescent label monoclonal antibodies to P-gp protein. Results: Table 11: Age and sex distribution of patients of AML Age Males Females Total Children (1yr-12 yrs) 8 (66.6 %) 4(33.3%) 12 Adults (12-65 yrs) 29 (51.7%) 27(48.2%) 56 Total 37 (54.4%) 31 (45.5 % ) 68 Table12: Hematological profile of patients with AML Hb (gm%) TC (x10 9 / L) Platelet count (x10 9 / L) Children 6.2 ( ) 28.4 ( ) 54.5 (10-120) Adults 7.5 ( ) ( ) (2-3600) Table 13: FAB classification of AML patients: AML M0/M1 AML-M2 AML-M3 AML-M4 AML-M5 AML-M6 AML-M7

8 Children 2 (16.6%) 2(16.6%) 4(33.3%) 3(25%) Adults 9(14.28%) 15(26.7%) 7(12.5%) 8(14.28%) 8(14.28%) 4 (7.14%) 1(1.78%) Total 10(16.17) 17(25) 11(14.70) 11((16.17) 8(11.76) 4(5.88) 1(1.47) Immunophenotypic characteristics: Table 14: Aberrant expression of lymphoid markers in AML patients Markers Number of patients (%) CD 19 6 (11.76) CD 7 7 (13.72) CD 10 2 (3.92) CD 3 1 (1.96) CD56 7/34 (20.58) P-gp expression studies: Ten out of 28 (35.7%) patients studied for expression of P-gp was found to be positive for P-gp. All these patients were adults. Cytogenetic studies: Table 15: Chromosomal abnormalities in 68 patients with AML S No Type of chromosomal abnormality Number of patients 1 Hypodypliody 6 (8.82%) 2 45, XY, -7 2 (2.94%) 3 47, XX,+4 1 (1.47%) 4 46,X, (1.47%) 5 46, XY, del(7), (q21.1-qter) 1 (1.47%) 6 46, XY, inv (16) (p12q24 2 (2.94%) 7 46,XY,del(9) (q12-qter) 2 (2.94%)

9 8 Hypodiplody / del (9) (q12-qter) / (1.47%) 9 PML/RARA fusion 7 (10.2%) 10 Normal karyotype 40 (58.8%) 11 Unsuccessful kayotype 6 ((8.82%) Total 68 CELL BIOLOGY Generating murine monoclonal antibody pertaining to institutes interest remained an important activity. During the year the following antibodies were produced. a. Monoclonal antibody to H antigen The H antigen is present in varying amounts in A,B, AB, and O group erythrocytes and is maximum in O group red cells. The H antigen is absent in Bombay phenotype individuals. The presence of H antigen is usually detected by either monoclonal or polyclonal anti-h reagent. Last year while attempting to raise monoclonal antibody to A antigen, using A group erythrocytes as immunogen, two clones were isolated having anti-h specificity. However the agglutination titre of this antibody was 1:16 suggesting dosage effect. To verify this, it was suggested by SAC to raise a similar antibody using O group erythrocytes as immunogen. Following a conventional protocol, we isolated two stable clones (3E8H11, 3E8A10) which showed anti-h specificity and has an agglutinating titre of 1:128 with O group erythrocytes. The reactivity is also confirmed with A1, A2, A1B, A2B and B group erythrocytes Table 16: Titre of anti-h antibody with various cells. Cells Clones A1 A2 A1B A2B B O Oh 3E8A10 1:2 1:32 1:2 1:8 1:16 1: Neg b. Monoclonal antibody to Factor VIII antigen Monoclonal antibody to Factor VIII was generated by fusing spleen cells of Balb c/mice immunized with purified recombinant factor VIII with an Sp 2/0 mouse

10 myeloma cell line in presence of HAT selective medium and polyethylene glycol using a conventional protocol. We have isolated two clones 4E2F3 and 4E2C9 that showed anti factor VIII activity. This antibody reacted by ELISA; will be useful in screening patients diagnosed as hemophilia developing inhibitors. b. Maintenance of cell lines Different cell lines used for various studies are obtained from National Centre for Cell Sciences, Pune and maintained in liquid nitrogen. They are as follows: 1. Sp2/0 Ag.14 - Mouse myeloma cell line 2. Namalwa - Human lymphoblastoid 3. K Human leukemia 4. W1TR2 - Human glyoma cell line 5. NCCS 1 - Human glyoma line 6. W1Trf1 - Human glyoma line