Process of human platelets loaded with rehalose before lyophilization / 中国实验血液学杂志

The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37 degrees C in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists--thrombin, ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time (2 hours later) and incubation temperature (rang from 30 degrees C to 40 degrees C), respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37 degrees C for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration (< 50 mmol/L). Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively (P > 0.01). After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37 degrees C, 4 hours and the extracellular trehalose concentration < 50 mmol/L are the optimal conditions for loading with trehalose. The processing of loading with trehalose before platelet lyophilization has no significant effects on response of platelets to agonists and activation.

Collection of peripheral blood stem cells from ABO incompatible allogeneic donors by using blood cell separator / 中国实验血液学杂志

To evaluate the yield of the blood cell separator for collection of peripheral blood stem cells (PBSC) from ABO major and (or) minor incompatible allogeneic donors and the feasibility of PBSC component infusion to the recipients without removal of erythrocytes or plasma, the Cobe Spectra (Version 6.1) blood cell separator was utilized to collect PBSC component from 9 allogeneic donors. Of all the donors, 4 were ABO major incompatible, 2 were minor incompatible and the other 3 were both major and minor incompatible to corresponding recipients. In each cycle, different amount of PBSC component was harvested, and the variable volume plasma chased the cells into the bag was adjusted according to the ABO incompatibility. The nucleated cell count, percentage of mononuclear cell, number of CD34(+) cell and percentage of viable cell (trypan blue excluding rate) in the component were detected. At the time of infusion, a series of protective measures to the renal function of recipients were taken. The results showed that apheresis was twice performed on these eight donors to collected enough PBSC for transplantation or cryopreservation, except one apheresis was enough for cell amount needed by transplantation, as the donor's body weight was much heavier than that of the recipient. Altogether 17 apheresises were performed, the mean yield of nucleated cells was 3.77 x 10(10), in which 97% to 99% were mononuclear cells (MNC). The harvested number of CD34(+) cell was 8.62 x 10(6)/kg. All the trypan blue exclusion rate was 100%. In ABO major incompatible or both major and minor incompatible component, there were 8 - 10 ml packed erythrocytes; in ABO minor incompatible component, there were 80 - 120 ml of plasma. These components were infused into the recipients without removal of erythrocytes or plasma and no haemolytic reaction was observed in any recipient, and their hematopoietic functions soon recovered. Results suggest that enough PBSC can be acquired by using blood cell separator Cobe Spectra (Version 6.1), with the modified separation factors, and the collected PBSC component can be safely infused into the ABO incompatible recipients without removal of erythrocytes or plasma.

Optimization on trehalose loading technique as protective conditioning for lyophilization of human platelets / 中国实验血液学杂志

This study was aimed to further optimize trehalose loading technique including loading temperature, loading time, loading solution and loading concentration of trehalose, based on the established parameters. Loading efficiency in plasma was compared with that in buffer at 37 degrees C; the curves of intracellular trehalose concentration versus loading time at 37 degrees C and 16 degrees C were measured; curves of mean platelet volume (MPV) versus loading time and loading concentration were investigated and compared. According to results obtained, the loaing time, loading temperature, loading solution and trehalose concentration were ascertained for high loading efficiency of trehalose into human platelet. The results showed that the loading efficiency in plasma was markedly higher than that in buffer at 37 degrees C, the loading efficiency in plasma at 37 degrees C was significantly higher than that at 16 degrees C and reached 19.51% after loading for 4 hours, but 6.16% at 16 degrees C. MPV at 16 degrees C was increased by 43.2% than that at 37 degrees C, but had no distinct changes with loading time and loading concentration. In loading at 37 degrees C, MPV increased with loading time and loading concentration positively. Loading time and loading concentration displayed synergetic effect on MPV. MPV increased with loading time and concentration while trehalose loading concentration was above 50 mmol/L. It is concluded that the optimization parameters of trehalose loading technique are 37 degrees C (temperature), 4 hours (leading time), plasma (loading solution), 50 mmol/L (feasible trehalose concentration). The trehalose concentration can be adjusted to meet the requirement of lyophilization.

Determination and comparison of anti-SARS antibody in children and adults / 中国实验血液学杂志

To investigate the positive rate of anti-SARS antibody in children and adults without SARS, 197 paediatric patients under 14 years old from inpatient and outpatient department of our hospital, 156 healthy children pupils from primary school, 453 adult patients over 18 years old from inpatient and outpatient department of our hospital and other 502 healthy adult blood donors were selected. Anti-SARS antibodies were determined by anti-SARS specific antibody detection kit and ELISA method. The results showed that both the positive rates of IgG antibody in paediatric patients and healthy children were about 2% (4/197 and 3/156), while the positive rates in adult patients and healthy adults were about 0.2% (1/453 and 1/502). The difference between the positive rates of children and adults was significant (chi(2) = 11.61, P < 0.001). IgM antibody was negative in all the samples. It is concluded that the anti-SARS IgG antibody positive rate in children was obvious higher than that in adults. This may be the cause why the cases with SARS in children is much less than in adults.

Effects of preparation and quality control of cryopreserved platelets / 中国实验血液学杂志

The purpose of this study was to establish a set of techniques for cryopreservation of platelets with dimethylsulphoxide (DMSO) to insure high quality of cryopreserved platelet. The methods were as following: (1) DMSO was filtered in stead of being sterilized before infusion into the bag with platelets. (2) The whole blood was centrifuged immediately after blood collection and the attached tube was tied on the top of the bucket. (3) The related centrifugal force was 480 x g, the accelerating and braking grades of the centrifuge for acceleration and deceleration were 9 and 4 respectively. (4) The flow rate of platelet rich plasma (PRP) could not be too high, 80 - 100 ml PRP should be harvested at 1 minute or so. The infusion rate of DMSO into the PRP was 1 ml/min. After the infusion of DMSO, the PRP bag must be put into the -80 degrees C ultra low freezer at once to make the product to be freezed as soon as possible. The cryopreserved platelet should be thawed in the cycling warm water at the temperature of 38 - 40 degrees C. (5) After thawing of platelet, the platelet, red blood cell and white blood cell were counted, and the bacteria culturing, tests for HBsAg, anti-HCV, anti-HIV, TP and ALT were carried out. The results showed that altogether 14 800 units of cryopreserved platelets were prepared including 80 units collected with blood cell separator, of which quality control was accomplished in 300 units. The manually collected platelet mean count >/= 2.4 x 10(10)/unit, while the apheresis platelet count >/= 2.5 x 10(11)/unit. The yield was over 70%. The contaminated red and white blood cells were </= 1 x 10(9) and </= 1 x 10(7)/unit respectively. All the bacteria cultures were negative, while tests for HBsAg, anti-HCV, anti-HIV and TP were negative too. The ALT values were all in normal range. The transfusion of cryopreserved platelets showed obvious effect of haemostasis. In conclusion, the cryopreserved platelets prepared with this method were of high quality and efficaciousness in haemostasis clinically.

Comparison of platelet activators used in slide platelet aggregation test / 中国实验血液学杂志

The study was purposed to explore the suitable platelet activators to be used in slide platelet aggregation test. Experiments were as follows: (1) to detect the intensity and time in 15 healthy donors' platelet aggregation tests induced by cationic propyl gallate (c-PG) and the usual platelet activators: ADP, collagen, epinephrine, arachidonic acid and ristocentin, respectively; (2) to detect the time in platelet aggregation tests of 15 healthy donors induced by c-PG and the above usual platelet activators respectively after addition of PGI2, cAMP or EDTA; (3) to detect the time in 15 healthy donors' platelet aggregation tests induced by c-PG after addition of heparin; (4) to detect the intensity and time of platelet aggregation induced by c-PG at the platelet count of (240-15) x 10(9)/L, (5) to detect the time of platelet aggregation induced by c-PG in eight patients each of whom had taken 100 mg aspirin per day for five days. The results showed that (1) c-PG reduced the strongest intensity of platelet aggregation and the time taken was appropriate, (2) c-PG was the most effective activator to reveal the inhibitive effect on platelet by PGI2, cAMP or EDTA, (3) 0.5 - 3 U/ml heparin did not significantly change the platelet aggregation induced by c-PG, (4) 15 healthy donors' platelet aggregation induced by c-PG displayed clearly on the slide until the platelet count below 30 x 10(9)/L, (5) The platelet aggregation time induced by c-PG was significantly prolonged in eight patients who had taken aspirin. In conclusion, compared to the usual platelet activators, c-PG has remarkable potential advantages when used in slide platelet aggregation test.

Establishment and evaluation on the method for platelet intracellular trehalose determination / 中国实验血液学杂志

This study was aimed to establish an applicable, convenient and rapid method for platelet intracellular trehalose determination, so that the technique of trehalose loading could be optimized and used in research on freeze-drying platelets. Protein from loaded-trehalose platelets was deposited by using trichloroacetic acid, trechalose concentration in platelets was determinated by sulfuric-anthrone reaction and was analyzed by high performance liquid chromatography (HPLC). The results showed that when platelets were loaded with 1.7% of trehalose in loading solution, intracellular concentration determinated by sulfuric-anthrone reaction was 0.22% and derterminated by HPLC was 0.2%. The recovery rate of trehalose determinated by sulfuric-anthrone reaction was 100.7% and stability and repeatability of results were good. In conclusion, the method is convenient, rapid, accurate and highly sensitive for determination of platelet intracellar trehalose.

Qualitative analysis of batch preparing cryopreserved fresh platelet rich plasma / 中国实验血液学杂志

To evaluate the efficiency and effectiveness of batch preparing cryopreserved fresh platelet-rich plasma (cryo-FPRP) derived from the volunteer donors, platelet count (Plt), mean platelet volume (MPV), platelet distribution width (PDW), plasma pH, plasma lactic acid concentration, and lactic dehydrogenase (LDH) concentration, germiculture, CD62p positive rate, PAC-1 positive rate, and the fluorescence intensity of platelet GPIb-IX-V were detected in ACD whole blood, fresh platelet-rich plasma (FPRP), FPRP with 5% dimethyl sulphoxide DMSO (DMSO-FPRP), and thawed cryopreserved FPRP (cryo-FPRP); the procoagulant activity of FPRP and cryo-FPR was determinated. The results showed that (1) 70 percentage of platelet were separated from the whole blood into FPRP, and the counts of residual erythrocyte and leucocyte were below 1 x 10(9), and below 1 x 10(7) per unit respectively. (2) The plasma pH, lactic acid concentration and PAC-1 positive rate retained a stable level during the preparing, storing and thawing process. (3) Plasma LDH concentration, platelet CD62p positive rate and GPIb-IX-V concentration in platelet surface were enhanced significantly after being frozen and thawing. (4) The plasma clotting time induced by cryo-FPRP were significantly shorter than that induced by FPRP. It is concluded that: (1) The batch platelet preparing process can efficiently obtain platelet from whole blood donated by volunteer, and the process didn't activate the platelet. (2) Cryopreservation can prevent lactic acid accumulation, pH reduce and activation of GPIIb/IIIa. (3) The membrane of partial platelets are affected by freezing and thawing. (4) The density of GPIb-IX-V complexes in platelet surface and its procoagulant activity are enhanced significantly after the FPRP freezing and thawing process.

Measuring the shelf life of CPDA and ACD whole blood stored at above 4 degrees C in vitro / 中国实验血液学杂志

In order to measure the shelf life of whole blood stored at above 4 degrees C and provide experimental data for blood preservation and transportation in battle fields, 200 ml whole blood was collected from each of the 10 donors and anticoagulated by CPDA or ACD, then 50 ml whole blood was separated from each one and marked as control group, the rest was marked as test group. The control group was stored at 4 degrees C and RBC ATP concentrations was measured at the end of its shelf life which signed as critical ATP. The test group was stored at above 4 degrees C condition, some items as ATP, FHb (free hemoglobin), serum K(+) and germiculture were tested daily and ensured all of them eligible. When RBC ATP decreased to the level of critical ATP, the time of preservation was considered as shelf life. The results showed that at temperatures from 10 to 33 degrees C, the shelf life of CPDA whole blood ranges from 2.5 days to 18 days, while shelf life of ACD whole blood ranges from 1 day to 13 days. It is concluded that CPDA whole blood stored at above 4 degrees C condition can be sent to the front hospital in effective shelf life so that the wounded can be cured in time.

Detection of the anti-SARS-coronavirus specific antibody levels in 156 SARS patients / 中国实验血液学杂志

The objective of this study was to explore the development of IgG and IgM against SARS CoV and characteristics of changes of antibody titers in patients with severe acute respiratory syndrome (SARS) and to search the opportunity for collecting specific anti-serum from convalescent patients with SARS. The anti-SARS-coronavirus specific antibody levels in 156 SARS patients were measured with ELISA. The results showed that the total positive rates of IgG and IgM were 75.6% and 41.7% respectively, and the negative rate of both IgG and IgM was 23.7%. The average titers of IgG and IgM antibody in positive samples were 18.23 +/- 24.72 and 2.18 +/- 1.13, respectively. There was no significant correlation between the titers of IgG/IgM and sex, age, course of diseases and duration of body temperature recovery. It was concluded that not all SARS patients could produce the anti-SARS-coronavirus specific antibody. The titers of the anti-body are diversified even if the antibodies have been emerged in them. In order to obtain effective anti-serum, the titers of antibody must be tested just before collection of convalescent serum, and it ensures the therapeutic effect and provides a measurable index for clinical transfusion.

Studies on the blood group serologic characters of enzyme-only red cell antibody / 中国实验血液学杂志

The objective of this study was to investigate the serologic characters of enzyme-only red cell antibody and its clinical significance, and to provide basis for the safety of blood transfusion. The patient serum containing enzyme-only antibody was used to react with the red cells of donors, panel cells and auto-cells in various medium. Absorption and elution test were also per formed. The results showed that this blood sample was found to contain an antibody that reacted with donor red cells and panel cells only in papain medium, but was not demonstrable by indirect antiglobulin test and other method s. Decline of antibody titers was observed after absorption test, but antibody activity was not detected in the elute. The patient underwent transfusion with 600 ml of Rh type identical RBCs, without any hemolytic transfusion reaction. In conclusion, enzyme-only antibody usually doe s not lead to hemolytic transfusion reaction.

Effects of leukocyte elimination before storage on quality of red blood cell concentration / 中国实验血液学杂志

The objective of this study was to explore the possible effects of leukocyte elimination by filteration before storage on the quality of red blood cell concentrations (RCC) that prepared through two procedures. Eight units of red blood cell concentrations derived from whole blood after plasma separated (RCC1) and eight units of red blood cell concentrations derived from whole blood after platelet-rich plasma separated (RCC2) were divided randomly into filtered group and control group respectively. The RCC of filtered group were filtered by leukocyte deplete filter before storage. The control group didn't have any other treatments. These two groups were stored for five weeks at 4 degrees C according to AABB standard. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and plasma concentration of K(+) and lactate dehydrogenase (LDH), free hemoglobin (FHb), adenosine triphosphate (ATP) of red blood cell of all RCC were evaluated weekly, and bacteria contamination of all RCC was also detected after five weeks of storage. The results showed that there was no difference of MCV, MCH and MCHC and ATP level of red blood cell in all RCC of two groups, the ATP of red blood cell was lower than the control group on week 4 and 5. The average concentration of K(+) of the filtered group was less than the control group. The differences are significant except that of RCC1 stored till the third week. The plasma LDH concentration of filtered group was less than the control group, and the differences were exacerbate during the storing time prolonged. FHb release in the filtered group of RCC2 was significant less than that of control, but no significant difference was found between the two groups of RCC1. It was concluded that leukocyte elimination by filter before storage could be benefit to RCC preservation.

Micro-column gel indirect anti-globulin technique for screening and identification of irregular antibody / 中国实验血液学杂志

The purpose of this study was to design an antibody screening method based on the micro-column gel indirect anti-globulin technique (MGIAT), using pooled cells and plasma, by comparison with the conventional indirect anti-globulin technique (CIAT) combined with a two-stage papain technique, and to explore the feasibility of the use of plasma instead of serum as test material. The samples of blood recipients in our hospital were screened for irregular antibody using pooled test cells. Screening of the antibodies was identified both by MGIAT and CIAT combined papain technique respectively. The results showed that the irregular erythrocyte antibodies were detected in 20 cases from 5,000 recipients screened by MGIAT, using pooled cells, the positive rate was 0.4%. The specificity of 20 cases of irregular antibodies was as follows: 2 cases of anti-D, 8 cases of anti-E, 1 cases of anti-C, 2 cases of anti-c, 2 cases of anti-Mi(a), 2 cases of anti-Jk(a), 1 case of anti-Le(a) and 2 cases of anti-Fy(a). Antibody was detected from 19 cases using CIAT. Anti-Le(a) was detected with adding complement from Le(a-b-) person. Only 13 cases antibody were found by papain technique. It was concluded that irregular antibody screening by MGIAT using pooled cells can take place of the CIAT combining with papain technique in clinical application. Plasma is superior to serum in antibody screening test.

CD62p expression in platelet during the preparation course of Cryopreservated platelet-rich plasma / 中国实验血液学杂志

In order to explore the factors that affect CD62p expression in platelet during the whole course of cryopreservated platelets preparation, CD62p expression of platelet was evaluated by flow cytometry assay. The whole course of cryopreservated platelets preparation in order included whole blood collection, centrifugation for fresh platelet-rich plasma preparation, addition of dimethyl sulfoxide, and freeze in -80 degrees C refrigerator and thaw in 38 degrees C water bath. Result showed that the CD62p expressed slowly from whole blood collection to addition of dimethyl sulfoxide, but expressed abruptly after freeze and thaw and it occupied 82 per cent of the whole expression. It was concluded that the whole blood collection, centrifugation for fresh platelet enriched plasma preparation and addition of dimethyl sulfoxide were optimized in the whole course, but the damage to platelets in the whole course of cryopreservation could not be avoided. It suggests that improved techniques are needed to reduce the damage to cryopreservated platelets.

Comparison of leucocyte-reduced platelet concentrates produced with spectra version 5.1 and version 7.0 blood cell separators / 中国实验血液学杂志

In the present study, the performance of a new blood cell separator (COBE Spectra LRS Turbo Version 7.0) and that of the previous version LRS version 5.1 in the collection efficiency (CE), collection rate and residual white blood cells during platelet collection from donors were compared. 232 units of platelet concentrates (n = 232) were evaluated and 163 units were collected with the Spectra LRS version 5.1 (Group A) and 69 units with the LRS turbo version 7.0 (Group B). Donor's blood cell counts and parameters, platelet yield, collection efficiency and residual leukocytes in platelet concentrates were analysed. Results showed that the platelet yield was higher in group B than that in group A: (2.90 +/- 1.1) x 10(11) versus (2.58 +/- 1.2) x 10(11), P < 0.001; residual WBCs were less than 5 x 10(6) in 99.4% of group A platelet concentrates and in 97.1% of group B platelet concentrates. Collection efficiency was higher in group B than in group A: 51.4 +/- 8.7 versus 43.6 +/- 6.3. A correlation between platelet count before collecting blood and platelet yield was observed in both groups. In conclusion, the Spectra LRS Turbo version 7.0 showed a higher platelet yield than that with LRS version 5.1. Higher platelet counts before collection allow a higher platelet yield.

Flow cytometry combined assay for phosphatidylserine and CD62p expressed by preserved platelets / 中国实验血液学杂志

Human platelets have distinct characters when preserved by different methods. A efficient flow cytometric assay for different preserved platelets expression of CD62p and phosphatidylserine(PS) is in dire need. Efficient flow cytometric assay for CD62p and PS expressed by preserved platelets was established and the major conditions were optimized. The platelets need not to be washed to wipe off plasma and can be labelled diredtly during the sample preparation. It is efficient for flow cytometric analysis when fresh platelet riched plasma (FPRP) was set as negative control, thrombin actived FPRP, and liquid nitrogen treated FPRP were set as positive control respectively. Gly-Pro-Arg-Pro acetate salt (GPRP) was applied to prevent platelets aggregation and fibrin formation, stabilize platelets and minimize the artificial platelets activation. This is also the key to conquer difficulty of flow cytometric quantitive analysis when platelet, Ca(2+) and plasma coexist. This flow cytometric method is specially suitable for the multi-parameter assay including PS expression for cryopreserved platelets. Minimal sample manipulation, no fixation, and GPRP application resulted in minor artifacts and good sample stability. Results suggested, this flow cytometric assay for preserved platelets is simple and efficient. In addition, the author prepared four different methods treated platelets that can be easily distinguished through this flow cytometric assay. It not only makes sure the practicability of this flow cytometric assay, but also suggests the value of the treated platelets applied in preserved platelets flow cytometric ass