7th CLMC

Theme: Advances in Medical Laboratory and Preventive Medicine
Date: 4 - 6 November 2005
Venue: Taipei International Convention Center, Taipei
Presentations by HK Speakers: Public Health Laboratories and Health Protection
    by Dr Wilina Lim

Diagnostic Application of Molecular Cytogenetic Techniques in Leukemia and Cancer
    by Dr Thomas SK Wan

Molecular Diagnosis of Tuberculosis : available technologies, limitations and possibilities
    by Dr Wing-Cheong Yam

The Need for a Multi-disciplinary Approach in Toxicology
    by Dr Albert YW Chan

Sigma Metrics in Quality Control: Myth or Muda?
    by Dr Richard Pang

Induction of Embryonic Stem Cells into Neural Cell Lineage by Stromal Cell-Derived Inducing Activity
    by Dr KS Tsang

Public Health Laboratories and Health Protection
Dr Wilina Lim, Centre for Health Protection, Department of Health, Hong Kong

Although there have been changes in the pattern of infectious diseases in the past 50 years, morbidity associated with infectious diseases continues to be a major burden worldwide. The public health laboratories are essential components of public health infrastructure and have unique role in protecting human health. With technical advances in laboratory methods for the identification of causal pathogens, more precise epidemiology of clinically recognized infections and the impact of each agent could be determined.

The public health laboratories serve unique functions in contributing to effective management of infectious diseases. Data generated by high quality laboratories not only are critical for accurate diagnosis, but also provided coherent baseline information on disease occurrence and trend. The Virology Branch of the Centre for Health Protection of the Department of Health in Hong Kong maintains surveillance on various viral infections and receives clinical samples from sentinel surveillance network and hospital patients. Systematic collection and analysis of data is necessary to get accurate picture of the pattern of disease occurrence as well as the proportion of illness due to specific pathogen.

With large population movement across wide areas, it is crucial to understand the prevalence, incidence, geographic and demographic pattern of different diseases in various places. To enable effective response, one of the critical determination is the timely and systematic information exchange about disease pattern. Communication and collaboration with local and international partners should be strengthened to facilitate the detection, monitoring investigation and control of the diseases.

Strengthening the capacity of public health laboratories to meet the challenges competently should be top priority. Other than developing test system with advanced technology, providing quality service and facilitating the investigation and control of communicable diseases, experiences in recent years testify to the needs of planning and preparing for surge demands during outbreaks and epidemics. Transferring technology to partners could be a means to boost capacity during period of surge demands. Use of standardized protocols and promoting quality assurance should be emphasized to ensure accurate diagnosis for effective management of outbreaks.

Diagnostic Application of Molecular Cytogenetic Techniques in Leukemia and Cancer
Dr Thomas SK Wan, Haematology Division, Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Hong Kong

It is now mandatory to perform cytogenetics study in all new cases of leukemia owing to its usefulness in disease diagnosis, classification and prognostication. Although banding techniques represent the central theme at cancer cytogenetic laboratory, it is sometimes difficult to karyotype the tumor cells from a patient owing to unfavorable factors such as low specimen yield, low mitotic index, poor quality metaphases and other technical difficulties. Recently, interphase fluorescence in-situ hybridization (FISH) study has become more or less routinely utilized for the rapid detection of the genetic alterations in leukemia and cancer. Common translocations such as t(8;21), t(15;17), and inv(16) in acute myeloid leukemia, as well as t(12;21) in acute lymphoblastic leukemia, are associated with a favorable prognosis. In addition, t(9;22) and MLL gene rearrangement at 11q23, which are associated with a poor prognosis, can be readily identified with gene-specific probes. In practical terms, FISH is a reliable method for the analysis of MYCN amplification especially when the rate of cells with MYCN amplification is low or intratumor heterogeneity is present. Furthermore, newer FISH-based. tests such as multicolor karyotyping and comparative genomic hybridization have a clinical potential as they enable resolution of complex karyotypic aberrations and global scanning of genomic imbalances, respectively. The application of FISH techniques in unraveling chromosomal rearrangements will be demonstrated and the potential of the newer FISH developed tests in contributing information on genetic abnormalities will be illustrated.

Molecular Diagnosis of Tuberculosis : available technologies, limitations and possibilities
Dr Wing-Cheong Yam, Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong

Background: Tuberculosis (TB) has reemerged as a global public health concern with an annual mortality of 3 millions. Coincident with the resurgence of tuberculosis, there is also an alarming increase of infections due to multiple drug resistant tuberculosis (MDR-TB) to two or more of the first line anti-tuberculosis drugs including isoniazid and rifampicin.

Methods: Clinical specimens from patients were collected for routine acid fast staining, culture and susceptibility testing to isoniazid and rifampicin. Rapid diagnosis was performed using IS6110 PCR. For rifampicin resistance, a region of rpoB gene (encodes for catalase peroxidase) in Mycobacterium tuberculosis. Clinical isolates were also identified to species using 16s rRNA gene sequencing.

Results: All known mutations of rifampin were detected by PCR-sequencing or DNA microarray using single slide coated with 50 probes. However, katG mutations accounted for only 50% of isoniazid resistant Mycobacterium tuberculosis in Hong Kong. One strain exhibited novel point mutation in inhA at amino acid 194 (Isoleucine to Threonine) which is one of the key amino acids associated with the binding of NADH. The combination of 16s rRNA gene sequencing and phenotypic tests (growth rate and photoreactivity) resolved closely related species or members within a complex or group.

Conclusions: DNA sequencing and microarray exhibited highly sensitive and specific results for direct detection of MDR-TB in clinical specimens. Turnaround time for laboratory diagnosis of MDR-TB was shortened from 6 weeks to 3 days. A strategy of initial screening with species-specific PCR and direct sequencing of the strains reveal that 16s rRNA sequencing is more affordable for diagnostic application. Genotypic detection of Mycobacterium tuberculosis provides rapid diagnosis and PCR based methods are especially suitable for routine laboratories in high endemic area. Our findings offer new insights towards future public health control measures for tuberculosis.

The Need for a Multi-disciplinary Approach in Toxicology
Dr Albert YW Chan, Hospital Authority Toxicology Reference Laboratory, Princess Margaret Hospital, Hong Kong

The importance of multi-disciplinary approach in clinical practice can never be over-emphasized. Neither should it be taken for granted. In the day to day practice of clinical toxicology, we require input from clinical, scientific and technical staff, as well as front-line clinicians. In addition, for difficult cases, there is a need for additional expertise from other professionals, such as clinical pharmacologist, pharmacist, TCM practitioners, TCM pharmacists, public health physicians, experts in molecular biology and other basic sciences. This has been our experience before and after the commissioning of the HA Toxicology Reference Laboratory in March 04. The presentation will be a summary of our experience especially of the last 18 months.

Sigma Metrics in Quality Control: Myth or Muda?
Dr Richard Pang, Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong

Quality of laboratory service is difficult to define. The definition of quality requires definition of the needs which a medical laboratory has to satisfy. The concept of Six Sigma quality management is rather vague. Generally, its goal has been defined as "business excellence". The term, sigma-metric, however, needs some explanation if applied to medical laboratories. The laboratory must audit the quality of service. The internal Quality Control (IQC) program primarily is designed to measure test precision and control analytical process. The program must include tolerance limits and corrective action procedures when limits are exceeded. The international standards ISO 15189 and corresponding checklist documents for accreditation of medical laboratories have been published. Although these new standards are found useful in certain areas particularly in the pre- and post-examination phases they are considered to some extent to be insufficient in the assessment of IQC procedures. The sigma metric may provide new insight into the QC needed for a measurement process in a laboratory. What are the unique challenges that can arise in applying the sigma metric in defining acceptable laboratory quality: Myth or Muda? That is the question.

Induction of Embryonic Stem Cells into Neural Cell Lineage by Stromal Cell-Derived Inducing Activity
KS Tsang, SP Fong, JCS Pang, HK Ng
Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong.

Embryonic stem (ES) cells can be induced into neural cell lineage but the efficacy varies in terms of cell yield and purity. The applicability of stromal cell-derived inducing activity (SDIA) of ancillary cells, mimicking the in-vivo micro-environment of the central nervous system, in the induction of ES cells into a hierarchical population of neural cells was elucidated in-vitro and in-vivo. The SDIA of neural precursor C17.2 cells, areola-derived L cells and Wnt-3a-secreting L-Wnt-3a cells were studied by non-contact co-cultures and conditioned medium (CM)-supplemented cultures of ES cells, D3 and E14TG2a. Stromal cells were molecularly analyzed for neurotrophic and neutroprotective factors, whereas the derived products were immunologically and molecularly characterized for neural commitment. In-vivo studies of SDIA-induced products were conducted in the mouse model of brain ischaemia by transient bilateral common carotid artery occlusion and reperfusion. Behavioural assessment of ischaemic mice post-transplant into the striatum was performed in the water maze system. Tracking of BrdU-labelled products was made on brain sections and teratoma was examined. BDNF, GDNF, CNTF, NGF, NT-3, IGF-1, IGF-2, bFGF, VEGF and EPO were detected molecularly in C17.2, L and L-Wnt-3a cells suggesting the neurotrophic and neuroprotective potential. The immunoreactivity of nestin, Beta-TuJ III, MAP-2, TH, GDNF and MBP and gene expression of Pax6, Otx1 and Nurr1 indicated a neural lineage of neuroectodermal precursors, neural stem cells, mature neurons, dopaminergic neurons, astrocytes and oligodendrocytes. The products were likely free of mesodermal and endodermal cells as no gene products of Brachyury and alpha-fetoprotein were amplified, respectively. Nevertheless, Oct4 was weakly expressed. SDIA were remarkable in both co-cultures and CM-supplemented cultures, despite readouts of CM was lesser! . There were 75% nestin+ colonies in co-cultures of D3 and C17.2. In-vivo studies of SDIA-induced products displayed a significant improvement in spatial learning and memory ability in ischaemic mice post-transplant. Migration of BrdU+ cells into brain tissues contralateral to the site of injection was evident. Nevertheless, teratoma was noted in one of the transplanted mice. A hierarchical population of neural lineage could be in-vivo derived from ES cells through SDIA of neural precursor cell C17.2. The clinical relevance was evident, yet the hurdle of teratoma post-transplant had to be tackled. The finding of this study may help translate to human setting.