Displaying publications 1 - 20 of 29 in total

  1. Ithoi I, Ahmad AF, Mak JW, Nissapatorn V, Lau YL, Mahmud R
    PMID: 22299400
    Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.
    Matched MeSH terms: Staining and Labeling/methods*
  2. Salleh FM, Anuar TS, Yasin AM, Moktar N
    J. Microbiol. Methods, 2012 Oct;91(1):174-8.
    PMID: 22986100 DOI: 10.1016/j.mimet.2012.08.004
    Permanent staining of faecal smears by Wheatley's trichrome technique has been used by many scientists for the detection of parasites in the past and it was found to be highly sensitive. This study was conducted to evaluate the use of Wintergreen oil in comparison with xylene in Wheatley's trichrome staining technique, as the reference technique. In a blind comparison study, 500 collected faecal samples from aboriginal communities were examined. Wintergreen oil was found to be more superior than xylene as a clearing agent in the Wheatley's trichrome staining of polyvinyl alcohol-fixed faecal smears for the identification of intestinal protozoa. Elimination of toxic, carcinogenic, and fire hazards makes Wintergreen oil the preferred choice in routine parasitology examinations.
    Matched MeSH terms: Staining and Labeling/methods*
  3. Tan ZN, Wong WK, Nik Zairi Z, Abdullah B, Rahmah N, Zeehaida M, et al.
    Trop Biomed, 2010 Apr;27(1):79-88.
    PMID: 20562817 MyJurnal
    Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.
    Matched MeSH terms: Staining and Labeling/methods*
  4. Pirehma M, Suresh K, Sivanandam S, Anuar AK, Ramakrishnan K, Kumar GS
    Parasitol. Res., 1999 Oct;85(10):791-3.
    PMID: 10494803
    Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.
    Matched MeSH terms: Staining and Labeling/methods*
  5. Abu Bakar N
    Trop Biomed, 2015 Sep;32(3):485-93.
    PMID: 26695209 MyJurnal
    Studies show that the pH of the malaria parasite's digestive vacuole (DV) plays a key role in the physiological functions of this organelle and antimalarial drug accumulation, and yet is technically difficult to measure. In this study, a flow cytometry-based technique was developed to measure the DV pH using a ratiometric pH indicator, FITC-dextran loaded into the DV of saponin-permeabilized parasites. To calculate the DV pH, a standard pH calibration curve was generated by incubating the saponin-permeabilized cells in buffers with different pH in the presence of an ionophore, CCCP. The measured average pH of the DV was 5.27 ± 0.03 that is approximately the same in the parasites observed microscopically by Hayward et al. (2006) (5.50 ± 0.14) using the same probe. The removal of glucose from the medium, causing a rapid depletion of parasite ATP, resulted in an alkalization of the DV. The DV was reacidified upon restoration of glucose to the medium. This technique provides a rapid, simple and quantitative measurement of the DV pH on a large number of cells. It will also be useful in future attempts to evaluate the effect of antimalarial drugs (i.e. chloroquine and artemisinin-based drugs) in pH changes of the DV.
    Matched MeSH terms: Staining and Labeling/methods
  6. Kimura M, Teramoto I, Chan CW, Idris ZM, Kongere J, Kagaya W, et al.
    Malar. J., 2018 Feb 07;17(1):72.
    PMID: 29415724 DOI: 10.1186/s12936-018-2214-8
    BACKGROUND: Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent.

    METHODS: The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya.

    RESULTS: Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard.

    CONCLUSIONS: Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

    Matched MeSH terms: Staining and Labeling/methods*
  7. Anuar TS, Al-Mekhlafi HM, Abdul Ghani MK, Abu Bakar E, Azreen SN, Salleh FM, et al.
    J. Microbiol. Methods, 2013 Mar;92(3):344-8.
    PMID: 23361047 DOI: 10.1016/j.mimet.2013.01.010
    This study was conducted to evaluate two routinely microscopic diagnostic methods in comparison with single-round PCR assay as the reference technique to detect Entamoeba histolytica/dispar/moshkovskii. Examination was performed on 500 stool samples obtained from Orang Asli communities in different states of Malaysia using formalin-ether sedimentation, trichrome staining and single-round PCR techniques. Ninety-three stool samples were detected E. histolytica/dispar/moshkovskii positive by routine microscopy, while single-round PCR detected 106 positive samples. Additional positives detected by PCR assay were eventually confirmed to be negative by both microscopic techniques. Detection rate of E. histolytica/dispar/moshkovskii was highest in combination techniques (18.6%), followed by trichrome staining (13.4%) and formalin-ether sedimentation (11.2%) techniques. Single-round PCR detected 21.2% of the stool samples. The sensitivity and specificity of formalin-ether sedimentation and trichrome staining techniques compared to the reference technique were 31.1% (95% CI: 29.0-36.0) and 94.2% (95% CI: 89.8-98.9), and 53.8% (95% CI: 46.0-76.2) and 97.5% (95% CI: 92.8-99.1), respectively. However, the sensitivity [59.4% (95% CI: 48.9-78.5)] of the method increased when both techniques were performed together, but the specificity decreased to 92.4% (95% CI: 81.0-98.0). The agreement between the reference technique, trichrome staining and combination techniques were statistically significant by Kappa statistics (trichrome staining: K = 0.592, p < 0.05; combination techniques: K = 0.543, p < 0.05). Hence, the combination technique is recommended to be used as a screening method in the diagnosis of E. histolytica/dispar/moshkovskii infections either for clinical or epidemiological study.
    Matched MeSH terms: Staining and Labeling/methods
  8. Ning TZ, Kin WW, Mustafa S, Ahmed A, Noordin R, Cheong TG, et al.
    Asian Pac J Trop Biomed, 2012 Jan;2(1):61-5.
    PMID: 23569836 DOI: 10.1016/S2221-1691(11)60191-3
    To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster.
    Matched MeSH terms: Staining and Labeling/methods*
  9. Ude CC, Shamsul BS, Ng MH, Chen HC, Norhamdan MY, Aminuddin BS, et al.
    Tissue Cell, 2012 Jun;44(3):156-63.
    PMID: 22402173 DOI: 10.1016/j.tice.2012.02.001
    Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 μmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 μmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 μmol. On dye retention comparison at 8 μmol and 4 μmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 μmol and BMSCs is 8 μmol, and they can be tracked up to 49 days.
    Matched MeSH terms: Staining and Labeling/methods*
  10. Afzan MY, Suresh K
    Parasitol. Res., 2012 Jul;111(1):371-81.
    PMID: 22398830 DOI: 10.1007/s00436-012-2848-3
    Trichomonas vaginalis, a flagellated protozoan parasite causes a variety of adverse health consequences in both men and women. The parasite exists in the trophozoite and the pseudocystic stage. The study reports for the first time that pseudocyst forms of T. vaginalis isolated from cervical neoplasia (CN) patients demonstrated distinct, different and significant in vitro growth profiles when grown in vitro cultures from day 1 up to day 5 (p<0.05, Mann-Whitney test) when compared with the same life cycle stages isolated from non-cervical neoplasia but symptomatic patients (NCN). Pseudocysts from CN and NCN isolates remained viable in distilled water until 3 h 10 min and 2 h 10 min, respectively. The nucleus of pseudocysts in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of pseudocysts in CN isolates showing more rough and creased surface with higher numbers of deep micropores with larger numbers of chromatin masses, vacuoles, and hydrogenosomes. The study confirms that pseudocystic stage from CN, despite the uniformity in appearance of being rounded and showing no motility without a true cyst wall under light microscopy, demonstrated different biochemical, surface, and ultrastructural properties. The study provides evidence that phenotypic variant forms of pseudocysts does exist and possibly does play a role in exacerbating cervical cancer.
    Matched MeSH terms: Staining and Labeling/methods
  11. Al-Mekhlafi MA, Fatmah MS, Anisah N, Azlin M, Al-Mekhlafi HM, Norhayati M
    PMID: 21323160
    Abstract. The species identification of Enterocytozoon bieneusi and Encephalitozoon intestinalis is only possible using transmission electron microscopy (TEM), mo lecular techniques and immunofluorescence antibody assays (IFA). In this study, 50 positive and 50 negative fecal specimens for microsporidial spores using the Weber modified trichrome (WMT) staining technique were examined using IFA-MAbs. Of the 100 specimens examined, the microsporidial spores identified by IFA-MAbs were Enterocytozoon Bieneusi 42 (75%) Encephalitozoon intestinalis 7 (12.5%) and mixed infections 7 (12.5%). The sensitivity and specificity of IFA-MAbs in detecting microsporidial spores were 98% and 86%, respectively. The agreement between the WMT staining technique and IFA-MAbs was statistically significant by Kappa statistics (K = 0.840; p < 0.001). E. bieneusi was the commonest Microsporidia species isolated from the studied population; the presence of microsporidial spores detected by IFA-MAbs should be confirmed by other methods.
    Matched MeSH terms: Staining and Labeling/methods
  12. Ragavan AD, Govind SK
    Parasitol. Res., 2015 Mar;114(3):1163-6.
    PMID: 25614298 DOI: 10.1007/s00436-014-4296-8
    Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.
    Matched MeSH terms: Staining and Labeling/methods*
  13. Koh SF, Tay ST, Puthucheary SD
    Trop Biomed, 2013 Sep;30(3):428-33.
    PMID: 24189672 MyJurnal
    Burkholderia pseudomallei the causative agent of melioidosis, is being increasingly recognized as an important cause of morbidity and mortality in South East Asia. Biofilm formation of B. pseudomallei may be responsible for dormancy, latency and relapse of melioidosis. Based on the colonial morphology of the bacteria on B. pseudomallei selective agar medium, seven distinct morphotypes were identified. This study was conducted to assess the in vitro biofilm produced by B. pseudomallei and to investigate possible correlation between B. pseudomallei morphotypes with biofilm forming abilities of the isolates. Using a standard biofilm crystal violet staining assay, comparison was made between the biofilm forming ability of 76 isolates of B. pseudomallei and Burkholderia thailandensis ATCC 700388. Amongst the blood isolates, 30.2% were considered as high biofilm producers and 27.9% were low producers, 33.3% of the pus isolates were considered as high and 16% low biofilm producers. Most of the isolates were identified as morphotype group 1 which displayed a rough centre with irregular circumference on the agar medium. However, we did not find any correlation of B. pseudomallei morphotypes with biofilm forming abilities (p > 0.05). Additional studies are needed to identify internal and external factors which contribute to the high and low biofilm formation of B. pseudomallei.
    Matched MeSH terms: Staining and Labeling/methods
  14. Lono A, Kumar GS, Chye TT
    Trans. R. Soc. Trop. Med. Hyg., 2010 Mar;104(3):214-8.
    PMID: 19716577 DOI: 10.1016/j.trstmh.2009.07.006
    Microsporidia are ubiquitous parasites thought to be closely related to fungi. Their presence in the environment means that humans are frequently exposed to infection. Stool samples were collected from 151 indigenous villagers from the eastern state of Pahang in 2005. The samples were concentrated with water-ether sedimentation, stained with modified trichrome stain and examined under oil-immersion microscopy. Thirty-two specimens (21.2%) were positive for microsporidia. Microsporidia were observed as ovoid or rounded ovoid shapes measuring approximately 1mum, with a bright pink outline containing a central or posterior vacuole. PCR amplification with specific primers on microscopy-positive specimens amplified Encephalitozoon intestinalis DNA from five of the ten specimens used.
    Matched MeSH terms: Staining and Labeling/methods
  15. Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S
    Trop Biomed, 2010 Dec;27(3):611-23.
    PMID: 21399603 MyJurnal
    This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.
    Matched MeSH terms: Staining and Labeling/methods
  16. Salleh FM, Al-Mekhlafi AM, Nordin A, Yasin 'M, Al-Mekhlafi HM, Moktar N
    Diagn. Microbiol. Infect. Dis., 2011 Jan;69(1):82-5.
    PMID: 21146718 DOI: 10.1016/j.diagmicrobio.2010.08.028
    This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics (K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis.
    Matched MeSH terms: Staining and Labeling/methods*
  17. Sirskyj D, Weltzin R, Golshani A, Anderson D, Bozic J, Diaz-Mitoma F, et al.
    J. Virol. Methods, 2010 Feb;163(2):459-64.
    PMID: 19913054 DOI: 10.1016/j.jviromet.2009.11.014
    Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.
    Matched MeSH terms: Staining and Labeling/methods
  18. Afzan MY, Sivanandam S, Kumar GS
    Diagn. Microbiol. Infect. Dis., 2010 Oct;68(2):159-62.
    PMID: 20846588 DOI: 10.1016/j.diagmicrobio.2010.06.005
    Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa.
    Matched MeSH terms: Staining and Labeling/methods*
  19. Wong YP, Shah SA, Shaari N, Mohamad Esa MS, Sagap I, Isa NM
    Asian Pac. J. Cancer Prev., 2014;15(4):1725-30.
    PMID: 24641399
    Management of patients with stage II colorectal carcinomas remains challenging as 20 - 30% of them will develop recurrence. It is postulated that these patients may harbour nodal micrometastases which are imperceptible by routine histopathological evaluation. The aims of our study were to evaluate (1) the feasibility of multilevel sectioning method utilizing haematoxylin and eosin stain and immunohistochemistry technique with cytokeratin AE1/AE3, in detecting micrometastases in histologically-negative lymph nodes, and (2) correlation between nodal micrometastases with clinicopathological parameters. Sixty two stage I and II cases with a total of 635 lymph nodes were reviewed. Five-level haematoxylin and eosin staining and one-level cytokeratin AE1/AE3 immunostaining were performed on all lymph nodes retrieved. The findings were correlated with clinicopathological parameters. Two (3.2%) lymph nodes in two patients (one in each) were found to harbour micrometastases detected by both methods. With cytokeratin AE1/AE3, we successfully identified four (6.5%) patients with isolated tumour cells, but none through the multilevel sectioning method. Nodal micrometastases detected by both multilevel sectioning and immunohistochemistry methods were not associated with larger tumour size, higher depth of invasion, poorer tumour grade, disease recurrence or distant metastasis. We conclude that there is no difference between the two methods in detecting nodal micrometastases. Therefore it is opined that multilevel sectioning is a feasible and yet inexpensive method that may be incorporated into routine practice to detect nodal micrometastases in centres with limited resources.
    Matched MeSH terms: Staining and Labeling/methods*
  20. Tan SW, Ideris A, Omar AR, Yusoff K, Hair-Bejo M
    J. Virol. Methods, 2009 Sep;160(1-2):149-56.
    PMID: 19447142 DOI: 10.1016/j.jviromet.2009.05.006
    SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C(t)) 18.19+/-3.63 and a melting temperature (T(m)) 86.0+/-0.28 degrees C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C(t) value 14.70+/-2.32 and T(m) 87.4+/-0.21 degrees C. The assay had a dynamic detection range which spans over a 5log(10) concentration range, 10(9)-10(5) copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45+/-0.31% and 1.30+/-0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.
    Matched MeSH terms: Staining and Labeling/methods
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