Reference interval establishment of full blood count extended research parameters in the multi-ethnic population of Malaysia.

Haematological cellular structures may be elucidated using automated full blood count (FBC) analysers such as Unicel DxH 800 via cell population data (CPD) analysis. The CPD values are generated by calculating volume, conductivity, and five types of scatter angles of individual cells which would form clusters or populations. This study considered 126 CPD parameter values of 1077 healthy Malaysian adults to develop reference intervals for each CPD parameter. The utility of the CPD reference interval established may range from understanding the normal haematological cellular structures to analysis of distinct cellular features related to the development of haematological disorders and malignancies.

Gene expression in obstetric antiphospholipid syndrome: a systematic review.

BACKGROUND: Antiphospholipid syndrome (APS) is a multisystem disease that may present as venous or arterial thrombosis and/or pregnancy complications with the presence of antiphospholipid antibodies. Until today, heterogeneity of pathogenic mechanism fits well with various clinical manifestations. Moreover, previous studies have indicated that genes are differentially expressed between normal and in the disease state. Hence, this study systematically searched the literature on human gene expression that was differentially expressed in Obstetric APS. METHODOLOGY: Electronic search was performed until 31st March 2015 through PubMed and Embase databases; where the following Medical Subject Heading (MeSH) terms were used and they had been specified as the primary focus of the articles; gene, antiphospholipid, obstetric, and pregnancy in the title or abstract. From 502 studies retrieved from the search, only original publications that had performed gene expression analyses of human placental tissue that reported on differentially expressed gene in pregnancies with Obstetric APS were included. Two reviewers independently scrutinized the titles and the abstracts before examining the eligibility of studies that met the inclusion criteria. For each study; diagnostic criteria for APS, method for analysis, and the gene signature were extracted independently by two reviewers. The genes listed were further analysed with the DAVID and the KEGG pathways. RESULTS: Three eligible gene expression studies involving obstetric APS, comprising the datasets on gene expression, were identified. All three studies showed a reduction in transcript expression on PRL, STAT5, TF, DAF, ABCA1, and HBEGF in Obstetric APS. The high enrichment score for functionality in DAVID had been positive regulation of cell proliferation. Meanwhile, pertaining to the KEGG pathway, two pathways were associated with some of the listed genes, which were ErBb signalling pathway and JAK-STAT signalling pathway. CONCLUSION: Ultimately, studies on a genetic level have the potential to provide new insights into the regulation and to widen the basis for identification of changes in the mechanism of Obstetric APS.

MicroRNA expression in antiphospholipid syndrome: a systematic review and microRNA target genes analysis.

INTRODUCTION: Antiphospholipid antibodies (aPL) are autoantibodies that attack phospholipid through anti-beta 2-glycoprotein 1. The actions of aPL are associated with events leading to thrombosis and morbidity in pregnancy. Antiphospholipid syndrome (APS) is diagnosed when a patient is persistently positive for aPL and also has recognised clinical manifestations such as recurrent pregnancy losses, arterial or venous thrombosis and in a catastrophic case, can result in death. Unfortunately, the pathogenesis of APS is still not well established. Recently, microRNA expressed in many types of diseased tissues were claimed to be involved in the pathological progression of diseases and has become a useful biomarker to indicate diseases, including APS. OBJECTIVE: This systematic review aims to search for research papers that are focussing on microRNA expression profiles in APS. METHOD: Three search engines (Ebcohost, ProQuest and Ovid) were used to identify papers related to expression of specific microRNA in antiphospholipid syndrome. RESULTS AND DISCUSSION: A total of 357 papers were found and screened, out of which only one study fulfilled the requirement. In this particular study blood samples from APS patients were tested. The microRNAs found to be related to APS were miR-19b and miR-20a. No data was found on specific microRNA being expressed in obstetric antiphospholipid syndrome. Analysis on the microRNA target genes revealed that most genes targeted by miR-19b and miR-20a involve in TGF-Beta Signalling and VEGF, hypoxia and angiogenesis pathways. CONCLUSION: In view of the limited data on the expressions of microRNA in APS we recommend further research into this field. Characterization of microRNA profile in blood as well as in placenta tissue of patients with APS could be useful in identifying microRNAs involved in obstetric APS.

Gene expression in obstetric antiphospholipid syndrome: a systematic review

Background: Antiphospholipid syndrome (APS) is a multisystem disease that may present as venous or arterial thrombosis and/or pregnancy complications with the presence of antiphospholipid antibodies. Until today, heterogeneity of pathogenic mechanism fits well with various clinical manifestations. Moreover, previous studies have indicated that genes are differentially expressed between normal and in the disease state. Hence, this study systematically searched the literature on human gene expression that was differentially expressed in Obstetric APS. Methodology: Electronic search was performed until 31st March 2015 through PubMed and Embase databases; where the following Medical Subject Heading (MeSH) terms were used and they had been specified as the primary focus of the articles; gene, antiphospholipid, obstetric, and pregnancy in the title or abstract. From 502 studies retrieved from the search, only original publications that had performed gene expression analyses of human placental tissue that reported on differentially expressed gene in pregnancies with Obstetric APS were included. Two reviewers independently scrutinized the titles and the abstracts before examining the eligibility of studies that met the inclusion criteria. For each study; diagnostic criteria for APS, method for analysis, and the gene signature were extracted independently by two reviewers. The genes listed were further analysed with the DAVID and the KEGG pathways. Results: Three eligible gene expression studies involving obstetric APS, comprising the datasets on gene expression, were identified. All three studies showed a reduction in transcript expression on PRL, STAT5, TF, DAF, ABCA1, and HBEGF in Obstetric APS. The high enrichment score for functionality in DAVID had been positive regulation of cell proliferation. Meanwhile, pertaining to the KEGG pathway, two pathways were associated with some of the listed genes, which were ErBb signalling pathway and JAK-STAT signalling pathway. Conclusion: Ultimately, studies on a genetic level have the potential to provide new insights into the regulation and to widen the basis for identification of changes in the mechanism of Obstetric APS.

An assessment of three noncommercial DNA extraction methods from dried blood spots for beta-thalassaemia mutation identification.

INTRODUCTION: Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta-thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta-thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta-thalassaemia identification. METHODS: The methods assessed in this study were Tris-EDTA (TE) buffer-based method by Bereczky et al. (American Journal of Tropical Medicine and Hygiene 72, 2005, 249), NaCL/NaOH/Sodium dodecyl sulphate (SDS) method by Huang et al. (Human Genetics 84, 1990, 129) and NaOH method by Zhou et al. (Analytical Biochemistry 354, 2006, 159). Extracted DNA was amplified for three common beta-thalassaemia mutations in Malaysia. RESULTS: Amplicons derived from TE buffer-based method were very faint and almost nonexistent while the NaCl/NaOH/SDS method did not produce any visible amplicons. The extraction using NaOH method produced visible bands that were comparable to the standard method using extraction kit. CONCLUSION: The NaOH method is a simple method that uses minimal equipment and reagents that make it labour- and cost-effective. This method could be adopted by poorer countries to extract DNA for beta-thalassaemia mutation characterization.

Thalassaemia screening among students in a secondary school in Ampang, Malaysia.

Thalassaemia is a common disorder in Malaysia. It is estimated that 4.5% of the population are carriers for beta- or alpha- thalassaemias. We set out to screen Form 4 students aged between 15 and 16 years old in a national school, for thalassaemia in March 2008. Written consent was obtained from 310 students. The carrier rate for the common thalassaemia syndromes was 6.8% (2.9% for beta-thalassaemia, 2.6% for HbE and 1.3% for two-gene deletion for alpha-thalassaemia). Carriers for beta-thalassaemia and two-gene deletion for alpha-thalassaemia were more common in the Chinese (4.3% and 1.4% respectively) while heterozygous HbE was more common in the Malays (3.8%). The laboratory cost of screening one student was RM 45 and the total number of man-hours spent in this screening activity was 600. This screening exercise showed that thalassaemia carriers are common among the Chinese and Malays and it is feasible to carry out a screening programme for secondary school students.

Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled delivery system.

Smooth, highly spherical, crosslinked chitosan microspheres in the size range of 45-300 microns loaded with progesterone were prepared by glutaraldehyde crosslinking of an aqueous acetic acid dispersion of chitosan containing progesterone in a non-aqueous dispersion medium consisting of liquid paraffin and petroleum ether stabilized using sorbitan sesquioleate. In vitro release of the drug into phosphate buffer at 37 degrees C was determined as a function of crosslinking density of the microspheres and particle size. The extent of drug release had a remarkable dependence on the crosslinking density of the microspheres, the highly crosslinked spheres releasing only around 35% of the incorporated steroid in 40 days compared to 70% from spheres lightly crosslinked. Determination of the in vivo bioavailability of the steroid from microsphere formulation by intramuscular injection in rabbits showed that a plasma concentration of 1 to 2 ng/ml was maintained up to 5 months without a high 'burst effect'. Data obtained suggest that the crosslinked chitosan microspheres would be an interesting system for long term delivery of steroids.

Protein release from poly(epsilon-caprolactone) microspheres prepared by melt encapsulation and solvent evaporation techniques: a comparative study.

Poly(epsilon-caprolactone) (PCL) microspheres containing c. 3% bovine serum albumin (BSA) were prepared by melt encapsulation and solvent evaporation techniques. PCL, because of its low Tm, enabled the melt encapsulation of BSA at 75 degrees C thereby avoiding potentially toxic organic solvents such as dichloromethane (DCM). Unlike the solvent evaporation method, melt encapsulation led to 100% incorporation efficiency which is a key factor in the microencapsulation of water-soluble drugs. Examination of the stability of the encapsulated protein by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that protein integrity was unaffected by both methods of encapsulation. In vitro release of the protein into phosphate buffer examined at 37 degrees C from microspheres prepared by both techniques showed that the release rate from melt-encapsulated microspheres was somewhat slower compared to the release from solvent-evaporated spheres. Both released around 20% of the incorporated protein in 2 weeks amounting to approximately 6.5 micrograms mg-1 of microspheres. Although the diffusivity of macromolecules in PCL is rather low, it is shown that PCL microspheres are capable of delivering sufficient quantity of proteins by diffusion for prolonged periods to function as a carrier for many vaccines. Unlike poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) polymers which generate extreme acid environments during their degradation, the delayed degradation characteristics of PCL do not generate an acid environment during protein release and, therefore, may be advantageous for sustained delivery of proteins and polypeptides.

Antitumour activity of mitoxantrone-loaded chitosan microspheres against Ehrlich ascites carcinoma.

Glutaraldehyde cross-linked chitosan microspheres containing the antineoplastic agent mitoxantrone were prepared and the antitumour activity was evaluated against Ehrlich ascites carcinoma in mice by intraperitoneal injections. The tumour inhibitory effect was followed by monitoring animal survival time and change in body weight for a period of 60 days. While the mean survival time of animals which received 2 mg and 1 mg of free mitoxantrone intraperitoneally was 2.1 and 4.6 days, respectively, animals which received 2 mg mitoxantrone via microspheres showed a mean survival time of 50 days. Five out of 8 animals treated using microspheres lived beyond 60 days. The percentage ratio mean survival time of the treated group divided by the mean survival time of the untreated group for animals treated using mitoxantrone-loaded chitosan microspheres containing 2 mg of the drug was 290 compared with 12.2 for those which received 2 mg of the free drug. The antitumour effect of mitoxantrone-loaded microspheres against Ehrlich ascites carcinoma was much higher than that of doxorubicin-loaded microspheres reported by previous workers. Our data demonstrate the potential of mitoxantrone-loaded chitosan microspheres for sustained drug delivery to minimize drug toxicity and maximize therapeutic efficacy.

Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices.

The use of glutaraldehyde as a fixative in bioprostheses and drug delivery matrices is reviewed. The chemistry of glutaraldehyde cross-linking and its effect on the biological performance of a number of bioprostheses such as tissue heart valves, vascular grafts, pericardial patches, tendon grafts and drug delivery matrices are examined.

Glutaraldehyde cross-linked chitosan microspheres as a long acting biodegradable drug delivery vehicle: studies on the in vitro release of mitoxantrone and in vivo degradation of microspheres in rat muscle.

Chitosan microspheres were prepared from 74% deacetylated chitin by the glutaraldehyde cross-linking of an aqueous acetic acid dispersion of chitosan in a mixture of liquid paraffin and petroleum ether stabilized using sorbitan sesquioleate as the surfactant. Cross-linking and hardening of the spherical particles were achieved by the addition of glutaraldehyde-saturated toluene through the organic phase. A relatively novel antineoplastic agent, mitoxantrone, was incorporated into the microspheres and the drug release was studied in vitro into phosphate buffer for over 4 weeks at 27 degrees C. Drug release was found to be effectively controlled by the extent of cross-linking. Only about 25% of the incorporated drug was released over 36 days from microspheres of high cross-linking density. Implantation of placebo chitosan microspheres in the skeletal muscle of rats was carried out in order to assess the biocompatibility and biodegradability of the microspheres. Histological analysis showed that the microspheres were well tolerated by the living tissue. However, no significant biodegradation of the material was noticed over a period of 3 months in the skeletal muscle of rats. Data obtained indicate the possibility of using cross-linked chitosan microspheres as a drug carrier for sustained drug release for very long periods.

Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs.

Bovine serum albumin (BSA) and diphtheria toxoid (DT) were loaded by passive absorption from aqueous solutions into preformed glutaraldehyde cross-linked chitosan microspheres. In vitro release of BSA under sink conditions at 37 degrees C showed that even though there was a large burst effect, there was a more or less steady increase with time thereafter for several days. Coating the BSA-loaded particles with paraffin oil or with a polymer, such as polylactic acid, modulated drug release. After the initial burst from PLA coated particles, the release rate increased with time for nearly 2 months. Preliminary immunogenicity studies on Wistar rats using DT loaded chitosan spheres showed that the antibody titres were fairly constant over a 5-month period, although very low compared to DT given on alum as control. Histological studies of placebo microspheres intramuscularly injected into rats demonstrated their tissue compatibility. Biodegradation was not complete in 6 months demonstrating the potential of cross-linked chitosan spheres as a long-acting drug delivery vehicle. The study demonstrated the possibility of incorporating biological macromolecules which are very sensitive to organic solvents, pH, temperature, ultrasound, etc. by a passive absorption technique to degradable biopolymer matrices thereby preserving their biological integrity. It is also shown that drugs passively absorbed into such matrices by taking advantage of their swelling behaviour need not necessarily be released completely in the initial 'burst' and a sustained release may be possible for macromolecules thus incorporated.