Collection and processing of umbilical cord blood for cryopreservation.

Umbilical cord blood (UCB) is being increasingly used as an alternative source of hematopoietic stem cells for allogeneic bone marrow transplantation. UCB transplantation has been successfully used to treat a variety of genetic, hematological, and oncological disorders in children and adults. The objectives of this study was to establish a closed-system technique for UCB collection and buffy coat separation by Optipress I device. Thirty-four UCB were collected by triple-bag system from pregnant mothers whose fetuses were not affected by thalassemic diseases after prenatal diagnosis. The mean volumn of UCB collection were 120 +/- 5 ml (range 65-180 ml). Total WBC, CD34+ cells, the progenitor cell erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) in the UCB units were (9.36 +/- 0.84) x 10(8), (3.61 +/- 0.52) x 10(6), (9.12 +/- 1.60) x 10(5), and (5.32 +/- 1.23) x 10(5), respectively. Good correlation between the nucleated cell and net cord blood volume could be demonstrated (p < 0.0001). The correlation between CD34+ cells and the following parameters: nucleated cell, BFU-E or CFU-GM were also demonstrated (p = 0.001, 0.0105 or 0.0001, respectively). Buffy coat was subsequently separated from 18 UCB units by Optipress I device. 70 +/- 3 ml of buffy coat were collected and cryoprocessing was done by automatic controlled-rate freezer. Good recovery of total WBC, CD34+ cells, progenitor cells BFU-E and CFU-GM after buffy coat separation were observed 89 per cent, 95 per cent, 109 per cent, and 102 per cent respectively. There was no aerobic bacterial or fungal contamination in the separated blood products. By using this technique, the UCB units were easily collected, rapidly separated within one hour, and high recovery of the hematopoietic progenitor cells could be obtained.

Distribution of blood group systems in Thai blood donors determined by the gel test.

The rapid availability of fully typed donor blood is of great advantage, especially for patients requiring repeated blood transfusions. Only limited blood group antigen typing has been carried out using the conventional tube technique. This study aimed to examine the distribution of blood group systems in Thai blood donors by the gel test. The ABO, Rh, MNSs, Duffy, Lewis, P, Kell, Lutheran and Kidd blood groups were examined in 500 Thai blood donors by the gel test. The distribution of blood group systems using the gel test was compared with other studies in the Thai population. Results : For the ABO System, group O was the most common (42.6 %) followed by group B (30.8 %), group A (20.2 %) and group AB (6.4 %). The most common Rh gene complex was CCDee (53.8 %) which was similar to other studies. The MMss and MNss gene complexes were the most common in the MNSs System. Fya was very common as in other Asians. In the Lewis System, the incidence of Le (a-b-) was 21.0 %, which is consistent with other findings in the Thai population. One hundred and forty-five (29 %) were positive for anti-P1. For the Kell System, 1 out of 500 (0.2 %) had the Kk type, 99.8 % had the kk types and only Kpb positive types were observed in this study, as well as Lu (a-b+) in the Lutheran System. The Jk (a-b-) was not found since it is a rare phenotype among Thai people. Discussion : This study shows the blood group distribution in 500 Thai volunteers using the gel test. Because of its simplicity and efficacy, this test is practical in population studies. Additionally, it is useful for mass screening and can be applied in emergency situations.

A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR.

A novel molecular method for HIV-1 proviral DNA detection comprising two main techniques: nested PCR, amplifying a target sequence of the ENV-gene of HIV-1, and nonradioactively-reversed probe hybridization for the detection of the amplified target sequence. The dual amplification of inserted HIV-1 proviral DNA in each DNA sample to be tested was performed by nested PCR in two steps: firstly with two outer primers covering the target sequence of the ENV-gene of HIV-1; secondly with two 5'-biotinylated primers specific to the target sequence. The biotinylated PCR product could be visualized as a single band of 141bps in length on agarose gel stained with ethidium bromide. For the confirmation of the primary result, a method of reversed probe hybridization, using a nylon membrane immobilized with the oligonucleotide probe specific to the target sequence, was established. The oligonucleotide probe was given a homopolymer tail with terminal deoxyribonucleotidyl-transferase; the tail was spotted onto a nylon membrane and bound covalently by UV irradiation. Owing to its length, the tail bound to the nylon, leaving the oligonucleotide probe free to hybridize. Hybridization of the amplified target sequence to the immobilized probe was accomplished by a simple colorimetric reaction involving the enzymatic oxidation of a colorless chromogen that yielded a purple color wherever hybridization occurred.

Reference values of serum anti-streptolysin O in adults by nephelometry.

The anti-streptolysin O (ASO) test, which depends upon age and geographic location, is used to provide evidence for antecedent streptococcal infection in patients suspected of having rheumatic fever. Reference values of ASO have not previonsly been determined by nephelometric assay for laboratory use at Siriraj Hospital. A total of 402 serum specimens were collected from healthy adults aged between 16 and 63 years in Siriraj Hospital. They consisted of 235 blood donors, 150 subjects who were going to study aboard and 17 dentists. ASO concentrations from 5 groups according to five age ranges were compared and it was found that two age ranges which were 25 years old and younger and 26 years and older were appropriate to determine reference values. The reference values of ASO for these two age ranges, with 90% confidence intervals, were 486 (401-673) IU/ml and 385 (362-500) IU/ml, respectively.

Preoperative autologous blood collection and haemodilution with dextran.

Thirty patients, ASA class I-II with haemoglobin level > 12.5 gm percent, who underwent elective gynaecological surgery were included in the study. Control blood samples for haematocrit, haemoglobin, electrolytes, serum osmolarity, coagulogram and platelet function were investigated before the autologous blood was collected and haemodiluted with 3 percent dextran-40 solution. The cardiovascular responses, arterial blood gases, urine output and central venous pressure were also recorded. After the end of haemodilution, another blood samples (as study samples) were once collected for analysis. Haematocrit, haemoglobin, sodium and potassium showed to decrease significantly. However, the serum osmolarrity, coagulogram and platelet function had no any significant differences. All parameters were within normal limits. In conclusion, the autologous blood collection and haemodilution technique was suitable and possibly practical in ever elective surgical patients without any undesirable side effects.