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Body weight (in Kilogram) and height (in centimetre) were measured and recorded on the tuberculin skin sheet (Appendix 7) 2. The left forearm was chosen by convention to avoid errors in allocating the test site during reading. A small flexible caliper (15 cm length ruler calibrated in millimetre) was used to measure any firm and well circumscribed induration or soft well-defined margin swellings. Evaluating the number of spots obtained provides a measurement of the abundance of M. The test was considered to have failed if the negative control spot count was more than ten spots (> 10 spots) or if there were less than twenty spots (< 20 spots) in the positive control and both panels A and B was nonreactive according to the criteria above. The research investigator was responsible for provincial coordination, fieldwork organization and quality control through coordination of the implementation process, and manipulated and reviewed daily all collected data to ensure legible, complete and consistent recording (Chiang et al. Handling of blood samples and assay components, their use, storage and disposal were also in accordance with defined procedures in appropriate national 144 biohazard safety guidelines or regulations. Care was also taken when working with chemicals considered as potentially hazardous. All recorded questionnaire data were double entered on daily basis by two data entry specialist and revised finally by the researcher checking for discrepancies between the two entries to ensure accuracy. Data were expressed and entered as number for the quantitative variable and as interval number for qualitative variables. Kappa value = (A-E)/ (N-E) A = all positives and negatives common to both techniques E = (positives)/N + (negatives/N) N = the total number of immigrant participants tested by the four techniques A Kappa value can lay between 0 and 1, where 0. A negative Kappa value indicates that the two tests agreement is less than expected by chance (Altman, 1991). Also defined as a range of values has a specified probability of including the true value of the risk factor variable, if the research studies are repeated. This corresponds to hypothesis testing with p-values, with a conventional cut-off for p of less than 0. The p-value is the probability of obtaining an actually observed test results which often rejects the null hypothesis when the pvalue is less than 0. The range of data set is simply a possible limit of spread and is defined as the difference between the highest and lowest values. The researcher and involved project staff companion carried out a pilot study to clarify the field steps using only the project questionnaire and tuberculin skin test followed by ordinary chest X-ray reading (Step 1-4; described in Figure 3. Major limitation in administration and data collection for future facilitations were identified. The search includes only studies published in English and relevant to tuberculosis diagnostic testing. Another limitation is the small sample size which might constrain generalization of the results to a larger population and wider community. This is a research study that can be used to design both qualitative and quantitative mass screening programme, supported with evidence-based statistical findings able to confirm the research hypotheses in strengthening the health system and medical care services. Tuberculosis must be considered as an inter-related risk factors-disease that needs to be addressed through combinations of social, economic, environmental interventions. Similarly, multi-factorial explanation model of the 158 resurgence of tuberculosis, including the interaction between biomedical, political, cultural, and economic factors which point to the analytical challenges achieved by combination of variable disease transmissions with global addressing of environmental conditions such as exposure risk and the socio-cultural surroundings (Dean and Fenton, 2010). Written informed consent was obtained from all participants, which was preceded by adoption pledge signed by the investigator. Analyses of risk factor variables and diagnostic test variants (uni- or multivariate) results were analyzed under 95% confidence intervals 160 using C. The minimum age of participants was 20 years and the oldest immigrant in the study was aged 56 years. Egyptians represented the majority of immigrants coming from non-endemic countries: 14. The majority of participants had graduated from secondary school and higher levels of education 73. For example Filipino participants commonly had diploma and university qualifications 81.

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Edentulous patients and poor oral hygiene can be risk factors for cancer of the oral cavity [23,24]. The consumption of the tea beverage, mate (consumed by South Americans), has been associated with an increased risk of cancer of the oral cavity [26]. Epidemiologic studies suggest that the intake of vitamin A, -carotene, and -tocopherol may reduce the risk of developing oral cavity cancers [27-32]. People with this syndrome often have hematologic problems an early age, which can lead to leukemia or aplastic anemia. They also have a risk of developing cancer of the oral cavity, especially tongue cancer [33,34]. Dyskeratosis congenita is a genetic syndrome that can cause aplastic anemia, skin rashes, and nail abnormalities of the hands and feet; they also increase the risk of developing oral cavity cancer [35,36]. Tongue cancers usually cause symptoms related to the upper aero digestive tract, including changes in swallowing, speech, hearing and breathing. During the interrogation the physician must give emphasis to the following symptoms: tongue pain, non-healing ulcer on the tongue, and changes in the ability to form words. The most common presenting complaint of patients with tongue tumors is a sore or lump. Cancer of the tongue mucosa may present as an indurated ulcer with raised edges (Figure 1) or as an exophytic growth. Bleeding from the surface of the lesion is a characteristic of malignancy and immediately raises suspicion for a neoplastic process. Approximately one third of the patients come in to the office with a neck lump [44]. Biopsy of the tongue lesion can often be performed in the office or as an outpatient surgery depending on the anatomic site and patient preference. One can perform the biopsy in the office setting using a punch biopsy or using biopsy forceps (Figure 2). The biopsy should be obtained from the edge of the lesion, away from areas of obvious necrosis or excess keratinization. A fine gauge needle (#23 gauge) makes multiple passes over the lesion while continues suction is applied. However, a negative result should not be interpreted as " absence of disease" when the clinical scenario is highly suspicions for malignancy. A core needle biopsy should not be performed in a lump in the neck, with the exception of an already diagnosed lymphoma. Martin Hayes in a communication to the medical profession in general stated "not only to the needlessness but also to the possible harmfulness of excisional lymph node biopsy as the first or even as an early step in the diagnosis of cancer" [48]. It can help define the extent of disease and the presence and extent of lymph node involvement. It is not very reliable to provide information regarding bone extension, unless, there is full involvement of the medullary cavity. Genetic alterations that are present early in the course of carcinogenesis are mutations or deletions of chromosome 3p and 9p. Mutations or deletions at chromosome 17p (involving the p53 tumor suppressor gene), and chromosome 13q and chromosome 18q generally are seen later in the process. In addition to deletions or mutations of individual genes, evidence exists demonstrating that numeric chromosomal imbalances, known as aneuploidy, may be a cause rather than a consequence of malignant transformation [42]. Aneuploidy may occur as a result of mutations in genes controlling chromosome segregation during mitosis and centrosome abnormalities. Diagnosis the need for a rapid diagnosis and referral of patients to a skilled physician with expertise in the management of tumors of the head and neck is very important because early diagnosis can lead to a reduction in mortality [3]. The risk factors mention on the etiology section of this paper, including history of tobacco and alcohol use should be interrogated. Any adult patient with symptoms attributable to the upper aero digestive tract lasting more than two weeks or an asymptomatic cervical (neck) tumor should undergo a full examination with a high index of suspicion for malignancy [3].

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A localized form of the disease with involvement of skin, eyes and mouth may also be seen. Although morbidity and mortality is reduced by anti-viral treatment, infants remain at risk for neurological Sequel. Outside the neo-natal period, the most common manifestation is orolabial ulcers which are often extensive. The are 4-5 mm in diameter, painful and may be seen on tongue, lips and mucosal surfaces (Gingivo-stomatitis). The virus can be isolated in culture and detected in tissue culture cells with 1-3 days. Adverse Effects Phlebitis, renal toxicity, nausea, vomiting, rash, neutropenia Remarks Drug of choice for Herpes simplex 1 and 2 valacyclovir, famciclovir are not used in children due to lack of data & availability of pediatric formulations. The vesicles can persist for weeks and coalesce to form large lesions resembling a burn. Giemsa-staining (Tzanck smear) of cell scrapings from lesions may show multinucleated giant cells but is non-specific. Consider treatment failure if lesions continue to develop or fail to heal after 10 days of treatment Useful in acyclovir resistant chickenpox Pediatric Guidelines 2013 91 E6. Clinical Features: In immune-competent adult, herpes zoster typically causes vesicular lesions, usually unilateral in a dermatomal distribution accompanied by pain and fever. Patients may have associated retinitis, pneumonitis, hepatitis and even encephalitis. Pulmonary Causes: Acute bacterial pneumonia and its complications including empyema and / or pneumothorax. Non-pulmonary causes: Children with symptoms due to pulmonary causes have cough as a prominent symptom while cough is not that significant in those who have non-pulmonary diseases or conditions leading to respiratory distress. Complications of pneumonia such as empyema and pneumothorax can easily be identified by clinical and radiological examination. Bronchiectasis is a disease characterized by irreversible abnormal dilatation of the bronchial tree, and represents a common end stage of a number of nonspecific and unrelated antecedent events. Persistent cough and fever with productive purulent sputum or hemoptysis should arouse suspicion of bronchiectasis. Chest examination reveals localized signs which do not easily resolve with usual antibiotic treatment. Chest x ray may show cystic spaces, occasionally with air-fluid levels and honeycombing. Milder cases will show loss of broncho-vascular markings, crowding of bronchi, and loss of lung volume. Acute exacerbations need to be treated with adequate antibiotic therapy (2-4 weeks) and postural drainage of sputum. Antibiotics should be carefully chosen to cover broad spectrum of micro-organism, including Gram ­ve bacteria. Attempt should Pediatric Guidelines 2013 93 be made to isolate the organism by culture of sputum or induced sputum, or secretions obtained through bronchoscopy. Surgical treatment may be necessary if the symptoms are severe or refractory to medical management and the disease is limited to one segment or a lobe. Disease usually has an insidious onset of mild but persistent cough, with or without exertional dyspnoea, and breathing difficulty. The tables below give important differential diagnosis of lung disease in children > 1 year of age and in those beyond 1 year. Severity of diarrhea is influenced by many factors including etiological agent (see Table-39) and host characteristics such as immunodeficiency, nutritional status and age. Table 39: Common etiological agents of persistent and bloody diarrhea Persistent or Chronic Diarrhea Persistent or Chronic Diarrhea Enteropathogenic,& aggregative E. In resource-constrained settings, the available investigations do not often identify specific pathogens and aetiology. Specific organisms such as Giardia lamblia and Entameoba may be identified on routine stool microscopy by demonstrating trophozoites or cysts. Zinc supplementation is recommended for 14 days (10 mg/day for infants under 6 months and 20 mg /day for infants and children over 6 months). If there is blood in the stool, ciprofloxacin at an oral dose of 15 mg/kg /day for 3 days is recommended.

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It obtains cells from the three cell layers of the epithelium of the oral mucosa for a correct analysis (13). The Kit contains a brush cytology sampling tool, a precoded glass slide, two pre-coded forms, two sachets of polyethylene alcohol fixative, a container for the sample holder and an envelope to send the samples (9). For sampling, we followed the steps indicated by the Med Oral Patol Oral Cir Bucal. In the laboratory the sample is stained with Papanicolaou tint (13); then it is scanned and analyzed microscopically using a computer with an image database containing different degrees of abnormal cell morphology (14). The program is capable of detecting two abnormal cells among thousands of normal cells (13). The results are classified as atypical (cellular changes of uncertain diagnosis), positive for dysplasia or carcinoma, negative (normal cells) and inappropriate (incomplete transepithelial sample) (15). With it, we can obtain complete transepithelial samples to perform adequate analysis of the lesion (2,13). No special training is required for its use thus favoring its acceptance among professionals (5,16). Local anesthesia is rarely necessary except in cases of ulcerated lesions in which its use is indicated, since sampling of these areas often results in greater discomfort for the patient (13). Besides the different variables appearing on the form (color, location, symptoms, etc. We decided to consider as low-keratinized lesions, regardless of the oral cavity area in which they were located, those lesions that had a similar color to that of healthy oral mucosa. Moderately keratinized lesions were those having a whitish appearance but that were located in areas of the oral cavity showing little or no keratinization (e. They also had to authorize the performance of a conventional biopsy using a surgical scalpel. We excluded patients who did not sign the consent form for both samples; those who had undergone treatment before the lesion and those whose lesions had a different e7 diagnosis from leukoplakia. The development of this study was approved by the Ethics Committee of the Faculty of Medicine and Dentistry, University of Santiago de Compostela. Results Of the 24 patients included in the study, 12 (50%) were men and 12 (50%) women. The most frequent location of lesions in our study was the lateral border of tongue, appearing in 8 cases (33. Concerning the degree of keratinization, we found 6 cases (25%) of highly keratinized lesions, 14 (58. In the analyses reports we found that the tests were negative (no cellular alterations) in 15 cases (62. Local anesthesia during the sampling procedure was not necessary in any of the cases in our study. The results of surgical scalpel biopsies were classified as negative (without epithelial alteration) in a total of 13 cases (54. Finally, we analyzed the keratinization degree in relation to the difficulty of obtaining a complete sample and we observed that in the total number of cases of low-keratinized lesions (4 samples) we obtained a complete sample; in 4 (28. Meanwhile other authors state that the most common location is the buccal mucosa with rates varying between 31 and 22%, followed by the lateral border of tongue (15-22%). This diffee8 rence may be due to the small size of our sample compared with that of other authors (1,13,16). In our study and in those by other authors we found that samples were taken mainly in predominantly white lesions with percentages ranging from 40-65% (1,13,16,17). This relatively low percentage of incomplete samples was due to the rigid design of the brush for adequate sampling (13,18). Many authors have indicated that a high keratinization degree in some lesions can be contradictory to the use of this technique since it hinders us from securing sufficient cellularity to perform adequate analysis (8,11,13,15). We could then consider, in these cases, taking a sample using a scalpel which would allow for a correct analysis of these lesions.

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Large maxillary cysts are converted to be side cavities of the maxillary sinus, therefore during the Luc-Caldwell operation a bony window is opened towards the lower nasal cavity. The removal of a sialolith in the submandibular gland happens through an intraoral approach, because the salivary gland is easier to approach through the floor of the mouth than through an extraoral incision. Enosseal implants are usually not loaded for 3-4 months after placement, because a good fibrous healing can only be expected this way. The infraorbital nerve is an end branch of the maxillary nerve, therefore it provides the complete sensory innervation of half the face. The lingual nerve is a branch of the inferior alveolar nerve, therefore the lingual nerve is a sensory one. The pH of inflamed tissues may be acidic, therefore in inflamed tissues the concentration of the free unloaded base of the anaesthetic is lower. A cyst does not belong to the group of tumours, because the growth of a cyst is not a result of cell proliferation. The typical site of occurrence of a follicular cyst is the apex of an unerupted tooth, because this cyst develops from the enamel epithelium left behind. The main cause of osteoradionecrosis is the destruction of osteocytes because the small vessels of the bone are occluded due to radiation treatment. Pleomorphic adenoma is a mesenchymal tumour, because histologically it contains mucoid and myxomatous elements as well. Does not be used the following sensory organ by the dentist during the physical examination: A. Role of the panoramic radiography in the examination of apical or magrinal periodontium is: A. Direction of the central beam for lateral upper incisors using bisecting technique: A. Does not cause differenciate diagnostic problem in radiographic appearance of radicular cysts: A. Outline of lingual cortical intersects roots more than 2 mm from the cementum-enamel junction apically B. External oblique line intersects roots more than 2 mm from the cementum-enamel junction apically C. Examining lamina dura and periodontal ligament starting apical toward coronal they disappeare deeper than normal D. The best choice for full mouth radiographic surway regarding the radiation safety is: A. Advantages of the digital radiography compared to conventional radiography are: 1. Painful, acute swelling of salivary glands characteristic to the following diseases: 1. A circumscribed periapical transparency on radiographs may include the following pathologies: 1. X-ray picture of odontoma compositum complicatum tumors is featured by the following: 1. Differencial diagnosis means differentiating of similar diseases, therefore it does not used in dental diagnosis. Subjective complaint is the cognition of the patient, therefore subjective complaint always appear before than the objective sign. Performing definitive diagnosis of the general diseases is not the task of the dentist, therefore it is not necessary collecting data on general health during the medical history. The patients age does not considered at the treatment planning, therefore the comprehensive treatment plan is influenced by financial factors only. Performing the treatment plan is solely influenced by the patients aspects, therefore the dentist considers the possible complications only. All the complaints of the patien is marked, therefore data of other examinations should not be marked. Panoramic systems deliver a similar ionization to that of four periapical films, therefore full mouth survay must always be taken by panoramic systems. The aim of excision biopsy is to remove the whole pathological tissue for histological examination and providing deffinitive therapy, therefore in diagnosing the diseases of the oral mucosa the excision biopsy is the only used treatment. Histological examination is important in differenciate diagnosis of salivary gland diseases, therefore parotid biopsy is indicated in case of mumps. In case of fluorosis matt and brownish spots can be seen, therefore teeth having dental fluorosis resistant against caries.

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Mother or caretaker should be given dietary counselling about meeting these increased nutritional needs at home. Table 3 shows the additional energy requirements for children with different nutritional status and examples of dietary modification that would meet these increased needs. Children with no medical complications may be managed at home if they still have a good appetite. Children who are sick and have associated complications like infections, have a poor appetite or are unable to eat, must be referred for inpatient care by trained staff with experience in nutritional rehabilitation. Therapeutic feeding as per National guidelines to provide 150-220 kcal/kg/day based upon the actual weight Poor weight gain or increased Severely malnourished nutritional needs (20 -30 % (50 ­ 100% additional energy) additional energy) Add 2 tsp of edible oil and 2 sugar to porridge,or a medium banana. Extra cup (200ml) of full cream milk with 1 tsp sugar or 2 big idlis or bread butter (2 slice) Therapeutic feeding as per National guidelines to provide 150-220 kcal/kg/day based upon the actual weight * Calories in addition to that recommended for normal children in the same age group Pediatric Guidelines 2013 105 5. Investigate for presence of anemia and give iron supplements if deficiency is confirmed. De-worm every 6 months (albendazole 400 mg single dose orally every 6 months after 1st year of life) Continue co-trimoxazole prophylaxis as indicated In patients with recent history of diarrhoea, give zinc 20 mg daily for 2 weeks Encourage regular play and age appropriate activities: Play helps maintain appetite and build muscles. Parents or caretakers should be encouraged to participate in age appropriate activities with the children. Administer routine childhood immunization F5: Nutritional counselling Nutritional counselling of caretakers is essential for maintenance of a good nutritional status and nutritional rehabilitation. The mother or caretaker should be counselled about need for additional food, dietary modifications required to meet the increased needs, safe food preparation, safe food and water storage methods and hygiene issues. The frequency and interval between visits depends upon the condition and needs of the child (table 4). The child who has chronic increased nutritional needs but investigated and no other active problems. First visit 1-2 weeks Then 1-2 months Tell caregiver to return earlier if problems arise. May require more frequent visits depending on clinical status and support offered or being provided. The child who is unwell and/or showing 2-4 weeks signs of growth faltering or has had recent diarrhoeal illness. Refer for hospitalization Weekly Only if fulfils criteria for management at home and no immediate need of other investigations that require hospitalisation. Sick children need extra drinks and food during illness, for example if they have fever or diarrhoea. In the recovery period it is important to: increase energy and protein consumed in everyday foods by adding one meal per day (refer to Suggestion sheet 1); Feed the child on demand day and night; and Encourage the child in simple and loving ways. Some of the ways to encourage a child to eat include the following: Make the child comfortable. Children may tire easily while eating, making it difficult to eat sufficient food at a sitting. All sick children should be offered appropriate foods unless there is a medical reason. Both the child client and his or her caregivers must be supported through sensitive and caring counselling. The counselling needs are also likely to change as the child grows older and progresses through various stages of child development. Further, the counsellor and other members of the clinical team must judiciously decide whether a particular counselling message is better addressed to the primary client (that is the child) or to her care giver. While the counsellor will carry the major responsibility for counselling both sides, it is also important for other members of the clinical team to develop a child-sensitive attitude and create an atmosphere where children will feel comfortable. Further, efforts should be made to create a child-friendly corner using some portion of the contingency funds. Simple and cost-effective items that are also durable could be purchased such as wall cut-outs of Disney characters or a plastic play house. Simple and cheap chalks or crayons may be purchased for the purpose of counselling and may be placed with the counsellor.


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Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Spectra Optia Apheresis System Service Manual 4-37 Troubleshooting Table 4-47: On-screen Instructions Possible Cause Patient hematocrit entered was not correct. Action Steps If the patient hematocrit entered at the start of the procedure is not correct, the system cannot accurately track the current patient hematocrit during the procedure. If the hematocrit entered for the replacement fluid is not correct, the system cannot accurately track the current patient hematocrit during the procedure. You must complete these steps before you restart the pumps and resume the procedure. Go to the fluid data screen and view the hematocrit entered for the replacement fluid: · If the hematocrit entered for the replacement fluid is correct, touch Retry. Reset the system and resume the procedure, or resume the procedure in Semi-Automatic mode: · To reset the system and resume the procedure: A. You may decrease the hematocrit by 3 percentage points up to three times for a maximum decrease of 9 percentage points. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Table 4-68: On-screen Instructions Possible Cause Channel was not correctly loaded. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Spectra Optia Apheresis System Service Manual 4-55 Troubleshooting Table 4-71: On-screen Instructions Possible Cause Channel was not correctly loaded. Remove the connector from the filler and wipe both sides of the connector and the surrounding area, using an alcohol pad. Re-insert the connector in the filler, and confirm that the channel and the lines are correctly loaded. Connect with the service software and verify the optical reference block is in the correct place on the image viewer. Face the centrifuge chamber and move the centrifuge arm to the front right side of the chamber. Run your finger over the groove in the filler to ensure that the channel sits flush with the groove. Clean the glass covers on the lights located on the top and bottom of the centrifuge chamber, using a gauze pad or other lint-free cloth dampened with water. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Spectra Optia Apheresis System Service Manual 4-61 Troubleshooting Table 4-77: On-screen Instructions Possible Cause Channel was not correctly loaded. Clean the glass covers on the lights located on the top and bottom of the centrifuge chamber using a gauze pad or other lint-free cloth dampened with water. Spectra Optia Apheresis System Service Manual 4-63 Troubleshooting Air may not have been removed from return line. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Table 4-80: On-screen Instructions Possible Cause Saline container was empty. Possible Causes Spectra Optia Apheresis System Service Manual 4-65 Troubleshooting Air removal is complete. Table 4-84: Service Troubleshooting Occurs During Detection Possible Causes Suggested Actions Run mode this is an operator prompt only. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Table 4-85: On-screen Instructions Possible Cause Air entered return line. Do one of the following: Action Steps · If Remove Air is available, touch Remove Air to start the air removal. Spectra Optia Apheresis System Service Manual 4-67 Troubleshooting Table 4-87: Service Troubleshooting Occurs During Detection Possible Causes Run mode the Return Line Air Detector detected air. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Table 4-88: On-screen Instructions Possible Cause System components malfunctioned. Table 4-90: Service Troubleshooting Occurs During Detection Continuously the Control system detected that the A/D conversion did not complete or has an error. Possible Causes Suggested Actions Spectra Optia Apheresis System Service Manual 4-69 Troubleshooting Bolus volume caused fluid balance to exceed limits. Machine Start-up Tests Patient Not Connected Patient Connected Patient Disconnected Table 4-91: On-screen Instructions Possible Cause Bolus volume entered was not correct. The system will deliver the bolus, and the procedure will resume in Caution status. Table 4-92: Alarm Information Alarm Identification Layer System BolusFluidBalanceLimitsExceeded Protocol Control 4-70 Spectra Optia Apheresis System Service Manual Spectra Optia Alarms Table 4-92: Alarm Information (continued) Protocol Alarm Name Alarm Explanation All Exchange Protocols Bolus volume caused fluid balance to exceed limits.

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Normal tissue that fulfills these criteria Window of Visual Staining Intensity ce ren refe al P son Per Window of Visual Staining Intensity Variation of Antigen Expression A Variation in Pre-Analytic Factors B Few protocol controls with limited variation Figure 5. Many samples with differing expression levels from different sources in optimal signal transfer incubation reactions are tested. B) When only a few samples with little variation and poor signal transfer reactions are used to optimize a staining protocol, the window in which the protocol generates visual signals is reduced and some samples may fall outside the window, increasing the risk of false negatives and false positives results. However, evaluation of various normal tissues may identify cases where antigen expression varies too much to be defined as stable. Using the same Anti-Actin antibody as an example, normal liver tissue shows staining of perisinusoidal smooth muscle cells ­ but only in some samples (Figure 5. This finding serves to underline that all normal tissues may not, by default, be suitable as control tissue. Detailed analysis should be carried out when selecting a normal tissue that is optimal as control tissue, by using a validated protocol that is able to identify variations in antigen expression. Validation Validation is performed on production lots and validates the intended use of the product. The validation testing is performed according to the instructions for use of the antibody on an extensive set of positive and negative clinical specimens related to the intended use of the product. The test is performed as a method comparison study, and the positive and negative agreement is compared to a second antibody of similar specificity and documented performance, if obtainable. This test is evaluated by an external, qualified pathologist and the results are included in the instructions for use for the antibody (package insert). The antibody undergoes extensive precision testing to ensure reproducibility and repeatability of antibody performance. The test is performed as a Gauge R&R study and includes intra-run reproducibility, inter-run reproducibility and inter-instrument reproducibility, to confirm that the antibody performance is the same on the same day, between days, and when used on different (Dako) platforms. Normal control tissue with well-defined and false positive results by ensuring that the protocol transfers all available targets into a visual signal without introducing background staining. To reach the core protocol the antibody has been tested using a protocol decision tree. If the staining is not optimal the protocol is adjusted in the next step and so forth (Figure 5. In the case that the percentages differ too much from the average, both the assay and the protocol are scrutinized to identify possible issues with the test. Amplification steps should only be considered when adjustment of parameters outside the core protocol (Figure 5. Therefore, amplification should not to be used to create a more intense staining when the problem of weak signal may be due to a non-optimal protocol. Amplification may produce a very intense staining of structures in the control tissues, which some pathologists find undesirable ("over-stained"). It also forces the standardization of reagents, dilutions, detection systems and staining protocols among those different laboratories using the same system. Optimal protocols minimize the impact of pre-analytical factors, due to optimal signal transfer incubations, and thus increase the diagnostic confidence, by detection of cells and structures with both low and high expression of the antigen. Neutral buffered formalin has become the standard choice of fixative in most laboratories worldwide but many laboratories still do not have standard procedures for the preparation of formalin, the source (vendor/home-made), or even the fixation time. Practical daily procedures in the hospital can cause fluctuations in fixation time, which can influence efficacy of antigen retrieval, and ultimately the staining result. Standardization starts in the operating room, where prolonged duration of pre-fixation time (ischemic time) may permit degradation of tissue resulting in loss of epitopes. Not even the best protocol can restore the primary sequences of an epitope lost in this manner. Similarly, the effect of other steps in the pre-analytical phase, including processing, dehydration, paraffin embedding, storage before staining, de-paraffinization and rehydration are not fully understood (12). Something as simple as the water, or type of paraffin, used in the laboratory may have an impact on the parameters in the optimal staining protocol.

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These biopsies are superior to punch biopsies for revealing or ruling out microinvasive cancer (Prendiville et al. If endo-cervical material is required for histological examination, the colposcopist should take this using an endo-cervical curette or an endo-cervical brush (see 6. The diagnostic quality of the histological examination of a biopsy may suffer from a number of imperfections. On the one hand, sampling error is a major cause for underreporting of lesions; on the other hand subjectivity of histological interpretation adds to the limitations in reliability (Ismail et al. For more precise instructions on biopsy taking, storage, transport, processsing and examination, see Chapter 5. Presence or absence of an invasive lesion cannot be confirmed because the specimen is often superficial. It is used less frequently in Europe where more often a diagnostic conisation is preferred when an endo-cervical lesion has to be excluded (Gath et al. There is no obviously superior conservative surgical technique for treating and eradicating cervical intra-epithelial neoplasia (Martin-Hirsch et al. Excisional techniques are preferred in the majority of circumstances because of their clear superiority over ablation in terms of histological evaluation of the transformation zone. Histological examination of the excised tissue allows the pathologist to recognize or rule out microinvasive cancer, glandular disease, margin involvement and depth of excision. The sample can only be planned safely by colposcopic assessment of the lesion by an experienced colposcopist. It should be performed without delay in the presence of high-grade intra-epithelial neoplasia or suspicion of early stromal invasion or microinvasion. In cold knife conisation cervical tissue is removed using a knife, and the excised product has the shape of a cone. Surgeons should avoid damage of the ecto-cervical epithelium or of the endo-cervical canal. The size and shape of the excised specimen will be determined by the colposcopic delineation of the lesion. If the lesion involves the endo-cervical canal, endo-cervical sampling should be considered after the excision. Thorough histological assessment by a pathologist skilled in gynaecological pathology is essential. Cold knife conisation gives excision margins that are not affected by thermal artefact, whereas the margins of laser excisional cone or diathermy loop excision cone may be damaged. Excision of the transformation zone in multiple fragments can complicate histopathological assessment. Furthermore, if microinvasive disease is present, it may be impossible to allocate a substage or define completeness of excision in fragmented excisional specimens. If cold knife conisation is performed great care must be taken to minimise side effects such as haemorrhage and cervical stenosis. Haemorrhage can be minimised by injecting the cervix pre-operatively with adrenalin 1 in 200,000. Radical diathermy (or electrocoagulation) uses a straight electrodiathermy needle and aims to destroy tissue to a depth of approximately 1 cm. Diathermocoagulation is a technique which uses heat to destroy cervical epithelium only to a depth of 2-3 mm. Cryotherapy (or cryocautery) employs a probe which is applied directly to the tissue to be destroyed by freezing: the depth of destruction is 3-4 mm. Cold coagulation uses a probe similar to a cryocautery probe, but destroys the tissue by heating it to 100°C. However, provided that certain selection criteria are adhered to, the various techniques can be safe and very effective. One or more biopsies should be taken from the area or areas that colposcopically show the most severe change. The result of the biopsy or biopsies should be available prior to the destructive therapy. Cryotherapy should not be offered to women with large lesions, occupying more than 75% of the ectocervix, extending to the vaginal wall or extending more then 2 mm beyond the cryoprobe (Gaffikin et al. There should be no evidence of invasive disease on cytology, colposcopy, or biopsy.


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