Annual Scientific Meeting

Theme: Putting Laboratory Diagnosis into Clinical Perspectives: The Molecular Approach
Date: Sunday, 22nd July 2001
Time: 1:45 pm - 5:30 pm
Venue: Salon 6, Level 3, JW Marriott Hotel, Hong Kong





2:00 pm

Opening and Introduction

Mr TT Cheung

HKMTA President

2:05 pm

Consistent Pattern of Allelic Loss in Natural Killer Cell Lymphoma

Ms Lisa LP Siu,
Medical Technologist, QEH

2:25 pm

From Laboratory to Bedside - The Significance of Her2/neu Immuno-histochemistry and In-situ Hybridization in Breast Cancer

Mr Stephen TH Lo
Scientific Officer, CMC

2:45 pm

Micrometastasis and Dissemination of Neuroblastoma to Haematopoietic Stem Cell Autografts

Mr Kent KS Tsang
Scientific Officer, PWH

3:05 pm

Global Situation of Standardization of Urinalysis and its Automation

Mr Atsushi Nakayama
Sysmex Hong Kong Ltd

3:25 pm

Intermission and Coffee Break


3:55 pm

Investigation of Mitochondria DNA (mtDNA) Disorders

Mr KM Au
Scientific Officer, PMH

4:15 pm

Molecular Diagnosis of Chlamydia
And Neisseria Gonorrhoeaeby Duplex PCR

Mr Barry KC Wong
Scientific Officer, UCH

4:35 pm

Detection of Verotoxigenic Escherichia Coli (VTEC) by Multiplex PCR

Ms Polly HM Leung
Lecturer, HKPU

4:55 pm

Laboratory Supports of Innovative Equipment and Services from Biorad

Product Specialist
Biorad (HK) Ltd

5:15 pm

Closing Remarks

Mr TT Cheung
HKMTA President

Dinner will be served after the Annual General Meeting

(HKMTA member at HK$100, Non-member at HK$500)

Please send cheque made payable to HKMTA to GPO Box 2722 Hong Kong

Jointly sponsored by Biorad (HK) Ltd and Sysmex Hong Kong Ltd

Consistent Patterns of Allelic Loss in Natural Killer Cell Lymphoma

Lisa L. P. Siu, Department of Pathology, Queen Elizabeth Hospital, Hong Kong

Natural killer (NK) cell lymphoma is a group of highly aggressive lymphoid malignancies that have been characterized in recent years. Immunophenotypic and genotypic studies have confirmed that the tumour cells are of putative NK cell lineage. The tumour cells characteristically express CD2, cytoplasmic CD3e (but not surface CD3 or the T-cell receptor), and CD56. The T cell receptor gene is in germline configuration, and there is an almost invariable association with monoclonal Epstein-Barr virus infection in the tumour cells. Clinically, NK cell lymphomas can be classified into several categories depending on the initial sites of involvement. In the majority of cases, the tumours initially involve the nasal and upper aerodigestive areas, presenting usually as non-healing necrotic ulcers. These are referred to as nasal NK cell lymphoma. A minority involve primarily non-nasal areas, such as the liver, spleen, gastrointestinal tract, skin, testis and muscle, and are referred to as non-nasal (nasal-type) NK cell lymphoma. Rarely, the lymphoma can be widely disseminated with a leukaemic phase, in which case it is known as NK cell lymphoma / leukaemia. These tumours are very rare diseases but show an interesting geographic predilection. They are reported mostly from Asia, Mexico and South America, but are extremely rare in Western countries. Reported karyotypic data showed chromosomal deletions at 6q and 13q. We have shown by comparative genomic hybridization and loss of heterozygosity analysis that there is consistent pattern of allelic loss at chromosome 6q, 11q, 13q and 17p. Further molecular studies in these regions may help to identify putative tumour suppressor genes or proto-oncogenes that are of pathogenetic importance in this group of rare but clinically aggressive tumour. Finally, these genetic alterations may contribute to defining the positions of NK cell malignancies in current or future lymphoma classification schemes, which are increasingly focusing on phenotypic and molecular features for definitive identification of lymphoma subtypes.

From Laboratory to Bedside - The Significance of HER2/neu Immunohistochemistry and In-situ Hybridization in Treating Breast Cancer Patients

Stephen T.H. Lo, Molecular Pathology Laboratory, Department of Pathology, Caritas Medical Centre, Hong Kong

Breast cancer is the most common cancer in women in the United States, and is the third most common cancer in women in Hong Kong (1998). 1533 new cases of female breast cancer were registered in 1996 and it caused 380 lives in 1998. HER2 is the acronym for Human Epidermal Growth Factor Receptor (EGFR), also known as c-erb-B2 / neu. It is a proto-oncogene located on chromosome 17. HER2 is a member of a family of 4 structurally related cell membrane receptors. The HER2 receptor has no identified ligand, but is able to form a heterodimer with other members of the EGFR family. Ligand binding will activate the intracellular tyrosine kinase activity, which triggers a cascade of events. In in-vitro experiments, transfection of the HER2 gene into human breast and ovarian tumor cell lines produced aggressive growth characteristics, such as increased DNA synthesis, cell growth, growth in soft agar in vitro, tumorigenicity, and metastatic potential in nude mice. HER2 over-expression affects roughly 20% to 25% of women with early-stage breast cancer and roughly 25% to 30% of women with advanced metastatic breast cancer. The discovery of HER2 gene amplification in breast cancer patients led to the development of trastuzumab (Herceptin), a humanized recombinant monoclonal antibody directed against the HER2-receptor protein on breast cancer cells. Preclinical and clinical studies indicated that HER2 overexpression may have prognostic and predictive value. Patients with HER2 gene amplification or protein overexpression had decreased disease-free survival time and lower overall survival rates. Preliminary Phase I and II clinical trials had shown that use of Herceptin on metastatic breast cancer patients significantly extended their disease-free and overall survival times. In the histopathology laboratory, HER2 gene amplification or protein overexpression can be assessed by Fluorescent In-situ Hybridization (FISH) or Chromosomal In-situ Hybridization (CISH) and Immunohistochemical Staining (IHC) techniques respectively. The advantages and disadvantages as well as the pitfalls of these techniques will be discussed. Caution in the interpretation of results from these tests will be highlighted.

Micrometastasis and Dissemination of Neuroblastoma to Haematopoietic Stem Cell Autografts

K.S. Tsang, W.C. Tsoi, C.K. Li*, Y. Leung, S.H. Tang, M.M.K. Shing*, K.W. Chik* and P.M.P. Yuen*, Department of Anatomical & Cellular Pathology and *Department of Paediatrics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong

Neuroblastoma (NB) which originates from the neural crest is the most common solid tumour in children. Not only the evaluation of bone marrow (BM) metastasis is important for clinical staging but also the analysis is vital to implement tumour cell purging of haematopoietic autografts for transplant. Triple-colour flow cytometry (FCM) was applied to enumerate NB cells in 22 diagnostic bone marrow (BM) aspirates, 5 BM autografts and 35 peripheral blood stem cell (PBSC) products of 16 children with NB. Reverse-transcription-polymerase chain reactions (RT-PCR) for tyrosine hydroxylase (TH) transcripts were also run in parallel. FCM detected dual fluorescence in >95% of the NB cell line, SK-N-MC, however BM and PBSC from 6 normal donors showed any positivity. In the cell spiking of SK-N-MC into normal BM and PSBC, the observed incidences of SK-N-MC derived from FCM were well correlated to the expected values up to 1 x 10-5 (BM: R2 = 1, p < 0.0001; PBSC: R2 = 0.9995, p < 0.0001), and RT-PCR was able to detect TH in 1 x 10-6 tumour cell preparation. Conventional BM histology demonstrated micrometastasis in 56.3% (9/16) patients, whereas RT-PCR unveiled an extra 31.2% (5/16) resulting a prevalence of 87.5%. TH transcripts attributed to the median of 0.182% NB cells (range: 0.008% - 20.2%) were unveiled in 9 diagnostic BM aspirates of 7 children, but BM histology hardly uncovered 0.04% (0.008% - 0.182%) tumour cells in TH+ BM of 55.6% (5/9) children. TH positivity was displayed in 80% (4/5) BM and 57.1% (20/35) PBSC autografts, which were harvested shortly after negative BM histology. FCM concomitantly identified tumour cells of the mean numbers of 0.034% (0.018% - 0.049%) and 0.171% (0.019% - 1.128%) in 2 TH+ BM and 12 TH+ PBSC, respectively. Positive selection of CD34+ cells by using the Isolex 300 immunomagnetic separation system enabled the depletion of tumour cells to 3.13 log and rendered 75% (3/4) PBSC autografts TH-free. Data in this study show that micrometastasis to BM and tumour cell dissemination to haematopoietic autografts are common in patients with neuroblastoma. FCM with the quantitative limit up to 1 x 10-5 tumour cells may hold promise to supplement the qualitative RT-PCR in the detection of micro-metastasis and occult tumour cell contamination of haematopoietic autografts.

Global Situation of Standardization of Urinalysis and its Automation

Atsushi Nakayama, Sysmex Scientific Division, Sysmex Corporation, Japan

Urine sediment analysis is a fundamental clinical examination and its clinical significance is highly valued. It is described as "Renal biopsy without needles." This significance is clearly mentioned in the NCCLS and ECLM urinalysis guidelines, which are published consecutively. In Japan, "Urine Sediment Analysis JCCLS guideline (2000)" was released last year. In the Guideline, it is described that; when using automated instrument such as flow cytometry, the characteristics of the instrument as a urine formed element analyzer should be understood. Especially for RBC and WBC, these instruments give a high degree of accuracy and precision. In the year 2000, ECLM-European urinalysis group published European Urinalysis Guideline. In the guideline, optimized urine screening procedures were proposed for general patients, UTI patients or patients with kidney diseases respectively and this leads cost effective examination. And especially for the screening procedure for general patients, fully automated urine formed element analyzer contributes to the streamline in the routine urinalysis.

Investigation of Mitochondrial DNA (mtDNA) Disorders

K. M. Au, Chemical Pathology, Department of Pathology, Princess Margaret Hospital, Hong Kong

Each human cell contains thousands of copies of maternally inherited 16.5 kb circular double stranded DNA that accounts for 1% of total cellular DNA content. It encodes 13 of over 100 polypeptides of oxidative phosphorylation complexes, 22 transfer RNAs and 2 ribosomal RNAs. Mitochondrial disorder refers to a group of heterogeneous diseases of the common final pathway of mitochondrial energy metabolism caused by nuclear DNA or mtDNA mutations, i.e. oxidative phosphorylation (OXPHOS), many of which are associated with lactic acidosis. Random segregation of mitochondria results in heteroplasmy and tissue mosaicism. Characteristics of mtDNA disorders are maternal inheritance, high mutation rate, heteroplasmy, threshold effect and heterogeneous clinical presentations. Phenotypic expression depends on the mutant load and threshold of affected tissues. Clinical manifestation is diversified and multisystemic and is insufficient to provide a systematic classification. Organs with highest aerobic demand; such as skeletal muscle, brain, heart and eyes are most severely affected. In paediatric cases, lactic acidosis (blood & CSF) and neurological abnormalities (i.e. Leigh's syndromes) are important clues. In adult population, muscle weakness, ophthalmoplegia with ragged-red fibres, retinitis pigmentosa, progressive myoclonal ataxia and early on-set stroke-like episodes are frequently combined in complex syndromes with lactic acidosis an inconsistent finding. A few common mtDNA point mutations accounts for the majority of MELAS, MERRF, NARP, MERRF/MELAS overlaps, LHON and MMC. Sporadic mtDNA rearrangements (deletions, duplications) are found in Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia and Pearson syndrome. For mtDNA point mutation screens, traditional PCR/RFLP tests based on loss or gain of a restriction site on blood/serum/plasma may be sufficient; while skeletal muscle biopsy gives a better yield if clinical suspicion is strong. For verification of low % mutant, Amplification Refractory Mutation System (ARMS) allows reliable detection at 1% mutant. As for mtDNA rearrangement mutation detection, long range PCR and enzyme digestion followed by Southern hybridisation on muscle biopsy are used. Currently, our laboratory provides molecular diagnostic tests on MELAS (A3243G, A3252G, T3271C), MERRF-A8344G and NARP-T8993G/C; as well as ARMS for MELAS-A3243G & MERRF-A8344G.

Molecular Diagnosis of Chlamydia Trachomatis and Neisseria Gonorrhoeae by Duplex PCR

Barry K.C. Wong, Department of Pathology, United Christian Hospital, Hong Kong

A duplex polymerase chain reaction (PCR) assay for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical samples was developed and evaluated using genital swab specimens collected from sexually transmitted diseases clinic in China and social hygiene clinic in Hong Kong. N. gonorrhoeae and C. trachomatis were detected in each specimen with a number of tests; including enzyme immunoassays (Gonozyme for N. gonorrhoeae and IDEIA for C. trachomatis respectively) and PCR assays using both single primer pair and mixed primer pairs. For N. gonorrhoeae, test results were also compared to that obtained by culture and Gram's smear of the genital discharges. Primer pair for N. gonorrhoeae was derived from the cppB gene on its cryptic plasmid and the PCR product was of size 390 bp. For C. trachomatis, the primer pair was derived from a common 7.4 kb plasmid which was present in all serovars of C. trachomatis. The corresponding chlamydial PCR product was of length 473 bp. Urine samples were also collected from patients attending the social hygiene clinic in Hong Kong and the use of urine deposits as an alternative specimen to genital swabs for the diagnosis of gonococcal and chlamydial urethritis was evaluated. In the present study using genital swab specimens, the duplex PCR assay showed identical results compared with single primer pair PCR for both gonococcal and C. trachomatis detection. All these PCR assays (both duplex and single primer pair) were more sensitive than culture and Gonozyme in detecting N. gonorrhoeae and IDEIA in detecting C. trachomatis. With male patients, the use of urine as an alternative specimens to urethral swabs appears to have promising results for the detection of both N. gonorrhoeae and C. trachomatis.

Detection of Verotoxigenic Escherichia Coli (VTEC) by Multiplex PCR

Polly HM Leung, Department of Nursing and Health Sciences, Hong Kong Polytechnic University, Hong Kong

We have screened for the presence of VTEC in faecal and carcass samples from 986 cows and 487 pigs collected from Government abattoir during the period 8/96 to 12/98. At the same time, we also screened faecal samples from 1003 patients suffering from diarrhoea. The samples were inoculated onto sorbitol MacConkey agar. For animal carcasses, an additional enrichment medium was used. Cultures were screened for VTEC by multiplex PCR. The two pairs of primers allow simultaneous amplification of verotoxin-1 (vt-1) and vt-2 genes. For each PCR positive sample, verocytotoxicity assays were done on 10 single colonies randomly selected for identity confirmation. By using multiplex PCR, VTEC was detected in 62.5% and 11.4% of the faecal and carcass samples from cattle, respectively. VTEC was also detected in 5.1% and 2.3% of the faecal and carcass samples from pigs, respectively. For the diarrhoea patients, VTEC was detected in 0.4% samples. By using conventional culture and verocytotoxicity assays, lower percentages of samples were tested positive for VTEC. Our results show that the multiplex PCR is a sensitive technique and that the simplicity of the PCR system makes it a valuable tool for rapid VTEC detection. VTEC carriage rate is high in cows but extremely low in pigs. Contamination of carcasses occurred at a very low level and this is attributed to the hygienic evisceration practised in the abattoir. Detection rate of VTEC in human is also very low in Hong Kong, this may be attributed to the dietary habits of the local population.

Copyright 2001 Hong Kong Medical Technology Association .
All rights reserved.