Malaria diagnosis in a malaria non-endemic high-resource country: high variation of diagnostic strategy in clinical laboratories in the Netherlands.

BACKGROUND: Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when. METHODS: Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands. RESULTS: The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia. CONCLUSIONS: This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy.

Deep learning-based transformation of H&E stained tissues into special stains.

Pathology is practiced by visual inspection of histochemically stained tissue slides. While the hematoxylin and eosin (H&E) stain is most commonly used, special stains can provide additional contrast to different tissue components. Here, we demonstrate the utility of supervised learning-based computational stain transformation from H&E to special stains (Masson's Trichrome, periodic acid-Schiff and Jones silver stain) using kidney needle core biopsy tissue sections. Based on the evaluation by three renal pathologists, followed by adjudication by a fourth pathologist, we show that the generation of virtual special stains from existing H&E images improves the diagnosis of several non-neoplastic kidney diseases, sampled from 58 unique subjects (P = 0.0095). A second study found that the quality of the computationally generated special stains was statistically equivalent to those which were histochemically stained. This stain-to-stain transformation framework can improve preliminary diagnoses when additional special stains are needed, also providing significant savings in time and cost.

Chemical analysis of saffron by HPLC based crocetin estimation.

Saffron is one of the most expensive and valuable spice having tremendous commercial value in food industry and thus its quality check is of utmost importance. Crocins are unique group of extremely hydrophilic apocarotenoids which are main components of saffron. Crocetin is an aglycone of crocins which do occur naturally in saffron, and is produced in biological system as a bioactive metabolite via hydrolytic cleavage of crocins. Crocins are unstable and tend to undergo isomerisation/ inter-conversions, and therefore their quantitative estimation is difficult. Herein, we have established for the first time, a crocetin-based HPLC method to evaluate the total crocin content of saffron, and thereby analyze the quality of saffron. The present approach comprises alkali-mediated conversion of crocins to crocetin in raw material followed by quantitative estimation of in-situ formed crocetin by HPLC analysis. The unique and efficient protocol for preparation of high purity analytical grade 'crocetin' directly from saffron has also been established. It is simple and efficient way to check the quality of saffron/ saffron-containing products.

Diagnostic utility of touch imprint cytology for intraoperative assessment of surgical margins and sentinel lymph nodes in oral squamous cell carcinoma patients using four different cytological stains.

BACKGROUND: OSCC is most commonly associated with positive surgical margins. The important cause of loco regional recurrence is histologically positive or closed margins. Clear surgical margins might favor the patient with a better prognosis and prevent repetitive surgeries. The present study was designed to the diagnostic utility of touch imprint (TI) smears using H and E, Pap, Giemsa and Feulgen stains, so that they can be used on a routine basis. MATERIALS AND METHODS: A total of 720 smears from 130 margins resected from 32 patients who underwent surgical resection of OSCC were prospectively evaluated. The slides were fixed in alcohol and randomly divided into four different batches for staining with H&E, rapid Pap, Giemsa, Feulgen stain. TI of 30 sentinel lymph node were fixed in 95% alcohol, stained by (H&E) and evaluated by two independent observers. The results were compared with gold standard histopathology. RESULTS: H&E stain showed sensitivity 44%, rapid Pap 35%, Giemsa 29% and Feulgen stain 25%. Positive predictive value-100% for all the four stains. NPV-H&E 70%, Pap 66%, Giemsa 62%, Feulgen 59%. Diagnostic test accuracy of H&E stained smears was higher 72%, compared to Pap 67%. Giemsa 65%, and Feulgen 63%. In lymph nodes, Specificity was 94.74%, PPV 90.91%, NPV 94.74%, diagnostic accuracy 93.33%. CONCLUSION: TIC is effective in identifying an inadequate or severe dysplasia margin comparable to gold standard histopathology. It might be used to intraoperatively identify metastases in sentinel lymph nodes in clinically N0 Patients.

Diagnostic application of patent blue V in sentinel lymph node biopsy for breast cancer - Is it time for a change?

Sentinel lymph node biopsy (SLNB) was introduced in the 1990s, as a minimally invasive procedure for staging the axilla with less morbidity to the traditional axillary lymph node dissection and is now standard management of the axilla in the early breast cancer. SLNB using the combined technique of blue dye and radioisotope is currently the recommended method for lymphatic mapping, and studies have shown high identification rates (IR) (>95%) and low false-negative rates (FNR) 5-10%. However, there are several reports raising awareness regarding patent blue V dye-induced peri-operative anaphylaxis. The main aim of this article is to highlight the emergence of patent blue dye as a new allergen and present evidence regarding the utility of alternative safer methods of evaluation of early breast cancer without compromising IR.

A Standardized Method of Applying Toluidine Blue Metachromatic Staining for Assessment of Chondrogenesis.

OBJECTIVES: Staining with toluidine blue is a well-established procedure for the histological assessment of cartilaginous- and chondrogenic-differentiated tissues. Being a cationic dye, toluidine blue staining visualizes proteoglycans in a tissue because of its high affinity for the sulfate groups in proteoglycans. It is generally accepted that metachromatic staining with toluidine blue represents cartilaginous matrix and that the degree of positive staining corresponds with the amount of proteoglycans. DESIGN: Articular cartilage and pellets of chondrocytes or bone marrow stromal cells were analyzed with a standardized staining procedure for toluidine blue. RESULTS: In the present study, we illustrate why such an assumption is invalid unless a detailed description of the procedure and/or reference to a detailed published method are provided. This is because the staining specificity and intensity depend, as we have shown, on the pH of the staining solution, the use of dehydration, and on staining time. CONCLUSIONS: We can, therefore, suggest a well-controlled standardized protocol for toluidine blue staining, which provides an easy and simple selective staining technique for the assessment of cartilage tissue and proteoglycan development in chondrogenic differentiation. If this procedure is not used, then investigators must provide sufficient technical information concerning the staining protocol to allow an assessment of the validity of the staining results.

A new high-throughput method to make a quality control on tattoo inks.

Nowadays, practice of tattooing is very common worldwide and, along with this increasing trend, there is also an increased risk of adverse reactions to tattoo pigments that are well known and described in literature. Previous studies have reported that cutaneous and allergic reactions to a particular pigment can manifest in several ways (allergic contact dermatitis and photo-allergic dermatitis). In this paper, a new high-throughput method is presented, in order to achieve a new system for the quality control on tattoo inks based on chromatographic-spectroscopic approach. The samples, twenty-one tattoo inks and three permanent makeup, comprised the following colors: black inks, yellow, blue, green, white, pink and various shades of red (pigment that gives many allergic responses) were analyzed through the combination of chromatographic and spectroscopic techniques, the HPTLC-Raman. In particular, Raman technique has been chosen because of its high sensitivity towards the inorganic and organic pigments, main constituents of tattoo inks. Moreover, the advantage of this hyphenated technique is to overcome the problem of analysing the complex mixture of tattoo inks, allowing to obtain a Raman spectrum of each single component, isolated by chromatographic separation. This approach aims at developing a powerful instrument to establish the nature of tattoo inks and substances that could be cause adverse reactions in tattooed patients.

Temporal changes in chromium allergy in Denmark between 2002 and 2017.

BACKGROUND: In 2012, Danish authorities submitted a proposal to the European Chemical Agency restricting the content of hexavalent chromium to a maximum of 3 ppm in leather goods. Following its adoption, this proposal was implemented in 2015 as a directive in the EU. OBJECTIVES: To examine the temporal trend of chromium contact allergy in adult dermatitis patients patch tested between 2002 and 2017, and to determine clinical characteristics and causative exposures in these patients. METHODS: All adult dermatitis patients patch tested between 2002 and 2017 were included. Patch test data were reviewed retrospectively. Comparisons were performed with the χ 2 test and logistic regression analysis. RESULTS: A total of 13 379 adults aged 18 to 99 years were patch tested between 2002 and 2017. The overall prevalence of chromium allergy was 2.2%. An overall decreasing trend was found for the prevalence of chromium allergy (Ptrend = 0.00002). Specifically, a significant difference was found for the study periods 2010 to 2013 (Ptrend = 0.002) and 2014 to 2017 (Ptrend < 0.0001) as compared with 2002 to 2005. Leather remained the most important single cause of allergic contact dermatitis caused by chromium. The proportion of clinically relevant leather exposures increased from 42.3% during 2002 to 2009 to 54.8% during 2010 to 2017 (P = 0.04). CONCLUSIONS: The prevalence of chromium allergy is decreasing. The EU Directive restricting the use of hexavalent chromium in leather goods is thought to be playing a central role in this change.

Prospective comparison of cytological specimen adequacy assessment by different rapid staining techniques for rapid on-site evaluation in fine needle aspiration cytology and their cost-effectiveness.

OBJECTIVE: Rapid on-site evaluation (ROSE) is a technique beneficial in determining the adequacy of the samples, thereby increasing the diagnostic yield, useful in triage of specimens for ancillary studies and can also help determine a preliminary diagnosis in emergency cases. The different rapid stains for on-site evaluation described in the literature are diff quik, toluidine blue (TB), brilliant cresyl blue (BCB), ultra-fast Pap stains, and rapid hematoxylin and eosin (H&E). This study was undertaken as there is sparse literature regarding the best and the most cost-effective rapid stain. METHOD: Fine needle aspiration samples from 200 patients with palpable swellings in easily accessible regions were taken. Smears stained by rapid and routine stains were assessed based on four parameters, with provisional diagnosis on the rapid stained smears. A comparative analysis of the advantages and disadvantages of the rapid stains was carried out with appropriate statistical tests with the routinely stained smears as gold standard. RESULTS: There was adequate material in 100% of ROSE smears. rapid pap stained smears showed well preserved cytoplasmic details, nuclear details, and background details. The time taken was least with TB and BCB being 5 s each. The most cost-effective was found to be TB. CONCLUSIONS: We conclude that TB is the most cost-effective, quick, least labor-intensive, and reliable rapid stain for ROSE especially in resource-poor settings.

Papanicolaou induced fluorescence, Ziehl-Neelsen and Auramine O stains on lymph node fine needle aspiration biopsy specimens from children: A comparative study.

BACKGROUND: The rapid diagnosis of extrapulmonary tuberculosis in children remains challenging. The presence of enlarged lymph nodes provides an opportunity to obtain diagnostic material through fine needle aspiration biopsy (FNAB). Mycobacterial culture, traditionally the reference standard, has a slow turnaround time and PCR-based methods are not widely available in developing countries. Direct visualization of mycobacteria on microscopy can be a rapid method to confirm the diagnosis. This study compared three staining methods to visualize mycobacteria. METHODS: Hundred FNAB specimens from persistently enlarged lymph nodes in children, clinically suspicious for tuberculosis, were evaluated for the presence of mycobacteria by three staining methods: Papanicolaou induced fluorescence (PIF) and Auramine O staining using fluorescence microscopy and Ziehl-Neelsen (ZN) staining using conventional light microscopy. These methods were evaluated against mycobacterial culture. RESULTS: PIF positivity was 30%, with 38% and 48% for Auramine O and ZN respectively. The combined ZN/PIF positivity was 56%. The highest diagnostic accuracy (73%) was demonstrated by ZN alone and in combination with PIF, with PIF alone showing the lowest (49%) accuracy. Although the combined test showed the highest sensitivity, it had the lowest specificity, while ZN was significantly more sensitive than both other staining modalities. No statistical difference in specificity was seen among the tests. CONCLUSION: This study suggests that Auramine O staining on previously ZN stained slides does not significantly improve diagnostic accuracy. While currently widely available methods of direct visualization of mycobacteria suffer from low sensitivity, the ZN stain remains a useful diagnostic test, particularly in resource-constrained countries.

Establishing Standards on Colors from Natural Sources.

Color additives are applied to many food, drug, and cosmetic products. With up to 85% of consumer buying decisions potentially influenced by color, appropriate application of color additives and their safety is critical. Color additives are defined by the U.S. Federal Food, Drug, and Cosmetic Act (FD&C Act) as any dye, pigment, or substance that can impart color to a food, drug, or cosmetic or to the human body. Under current U.S. Food and Drug Administration (FDA) regulations, colors fall into 2 categories as those subject to an FDA certification process and those that are exempt from certification often referred to as "natural" colors by consumers because they are sourced from plants, minerals, and animals. Certified colors have been used for decades in food and beverage products, but consumer interest in natural colors is leading market applications. However, the popularity of natural colors has also opened a door for both unintentional and intentional economic adulteration. Whereas FDA certifications for synthetic dyes and lakes involve strict quality control, natural colors are not evaluated by the FDA and often lack clear definitions and industry accepted quality and safety specifications. A significant risk of adulteration of natural colors exists, ranging from simple misbranding or misuse of the term "natural" on a product label to potentially serious cases of physical, chemical, and/or microbial contamination from raw material sources, improper processing methods, or intentional postproduction adulteration. Consistent industry-wide safety standards are needed to address the manufacturing, processing, application, and international trade of colors from natural sources to ensure quality and safety throughout the supply chain.

Reconsidering the H&E stain as the gold standard in assessing the depth of burn wounds.

While histological examination is considered by most as the gold standard for burn depth assessment, it has no practical use in the clinical setting. It has, however, been used in the research setting, as a mean for evaluating emerging techniques of depth measurement. Due to the limitations of the H&E stain, other stains have also been explored, such as lactate dehydrogenase (LDH), as presented in this issue, in "Improving the Histologic Characterization of Burn Depth." As the determination of burn depth is not a typical subject in dermatopathology, a summary of selected techniques and the possible role for the LDH stain in future research, is described herein.

Improving the histologic characterization of burn depth.

BACKGROUND: Visual assessment of burn wound appearance is the standard of care to determine the depth of thermal injury but often incorrectly predicts wound healing potential. Histologic evaluation of hematoxylin and eosin (H&E) stained burn tissue is prone to subjectivity and is challenging for the novice. Lactate dehydrogenase (LDH) staining may offer a simplified and consistent technique to identify burn tissue viability. METHODS: Thirty tissue samples were obtained from 6 patients undergoing surgical excision for clinically determined deep partial thickness or full thickness burns. Tissues were stained with H&E or LDH. Each specimen was scored by 3 individuals with varying levels of skill in histologic interpretation using a standardized checklist at 2 distinct time points. RESULTS: Agreement within raters was highest for the expert rater and lowest for the novice; however, the LDH stained tissue method had improved agreement for an experienced burn surgeon and novice. Agreement between raters was greater for the LDH stained samples which were determined to have greater viability than the corresponding H&E section in 100% of samples scored by the expert and in 80% for the novice clinician. CONCLUSION: LDH staining offers a more consistent measure of tissue viability that can be used by experienced and novice clinicians.

Application of a colorimetric technique in quality control for printed pediatric orodispersible drug delivery systems containing propranolol hydrochloride.

The feasibility of a colorimetric technique was investigated in CIELAB color space as an analytical quality control method for content uniformity of printed orodispersible pediatric delivery systems. Inkjet printing was utilized to fabricate orodispersibe film formulations containing propranolol hydrochloride in a colored ink base using three different edible substrates. A thin sweetener coating layer of saccharin was successfully included in the final dosage forms for palatability purposes using a casting knife. Optical microscopy, scanning electron microscopy and scanning white light interferometry analyses were conducted to study the effect of printing on the surface morphology and topography of the substrates. Differential scanning calorimetry and attenuated total reflectance infrared spectroscopy were used to study the solid state properties and possible interactions between the drug and the excipients. The inkjet printing technique deposited precise and uniform escalating doses (0.08-3.16mg) of the active pharmaceutical ingredient onto the substrates (R(2)≥0.9934). A disintegration test with clear end-point detection confirmed that all the substrates meet the requirements of the Ph. Eur. to disintegrate within 180s. The colorimetric technique proved to be a reliable method to distinguish the small color differences between formulations containing an escalating dose of propranolol hydrochloride.

Manufacturing of tattoo ink products today and in future: Europe.

The article describes the European market situation and the legal framework in Europe. It shows the state-of-the-art production under ISO 9001:2008 quality management and describes the future of tattoo ink production based on good manufacturing practice guidelines for tattoo inks.

The realistic variables underlying true safety in tattoo pigment manufacturing.

The regulation of the manufacture of tattoo ink products in the USA and the rest of the world is the focus of this article, which outlines the historical relationships between official and unofficial manufacturing and associated regulations, self-regulating movements within the industry and the impacts of over-regulation on the economics of ink manufacturing markets. The author, Mario Barth, highlights that changes in industry standards of production that are too rapid can cause the system to deteriorate, leading to an essentially negative shift to the underground markets. In addition, these regulations would not lead to a healthier end product because the currently considered health problems associated with tattoos (affecting 6% of tattoos performed in Germany) could be caused by multiple additional factors, such as the tattooing technique and aftercare. The pigment itself (which causes health issues in only 0.6% of tattoos) could in this equation not be optimized within an overregulated market. Further, aspects of price and revenue are analyzed in detail, showing that high quality suppliers are spending most of their efforts on passing regulations and that these regulations are not decreasing the amount of low-quality products in the general market. Finally, the notion of tattooing as 'an adult decision' is explained as another variable that has to be considered in creating regulations because the decision-making process for a tattoo (considering the price, quality and definitely the permanency) has and will have a self-regulating impact driven by the clients.

Making innovative tattoo ink products with improved safety: possible and impossible ingredients in practical usage.

Today's tattoo inks are no longer just simple solids in liquid suspension. Nowadays, these inks are high-tech dispersions made from finely spread pigments in a binder-solvent mixture. These so-called colour dispersions must follow the modern standards of tattooing, which are increasing every year. They must be rich in chromophoric pigments and yet fluid, they must not dry rapidly, and there should be no occurrence of any sedimentation, even during longer tattoo seasons. An innovative tattoo ink should enable long-lasting, brilliant tattoos without a negative impact on the artist's workflow and of course without endangering the consumer. The high standard in tattoos, regarding the motives and techniques, that is witnessed today could not be achieved by the artists without quality tools and modern tattoo ink. This article will give the reader a brief overview of the different ingredients of tattoo ink and of the function of binding agents and solvents in modern tattoo ink as well as describe what additives are used to achieve the desired behaviour during application. Furthermore, the article will take a look into the pigments that are used in tattoo ink and show why certain pigments are not suited for tattoo ink. The differences, advantages and disadvantages of organic and inorganic pigments will be explained.

Chemical purity and toxicology of pigments used in tattoo inks.

The safety of tattoo inks has obviously increased in Europe since the existence of European Union Resolution ResAP(2008)1, which resulted in the improved quality control of pigment raw materials due to the definition of impurity limits that manufacturers can refer to. High-performance pigments are mostly used in tattoo inks, and these pigments are supposed to be chemically inert and offer high light fastness and low migration in solvents. However, these pigments were not developed or produced for applications involving long-term stay in the dermis or contact with bodily fluids. Therefore, these pigments often do not comply with the purity limits of the resolution; however, it is required that every distributed tattoo ink does not contain aromatic amines and not exceed the limits of heavy metals or polycyclic aromatic hydrocarbons. Current toxicity studies of pigments underline that no ecotoxicological threat to human health or to the environment should be expected. However, the pigment as well as its impurities and coating materials must be considered. In order to evaluate the safety of pigments according to their impurities, two different validated sample preparation methods are necessary: (1) simulation of their long-term stay in the bodily fluid of the dermis and (2) simulation of cleavage due to laser removal or ultraviolet exposure. The development of standardized, validated and well-adapted methods for this application has to be part of prospective efforts. Concerning legislation, it might be appropriate that the first regulative approaches be based on those of cosmetics.

Tattoo inks: legislation, pigments, metals and chemical analysis.

Legal limits for chemical substances require that they are linked to clearly defined analytical methods. Present limits for certain chemicals in tattoo and permanent make-up inks do not mention analytical methods for the detection of metals, polycyclic aromatic hydrocarbons or forbidden colourants. There is, therefore, no established method for the determination of the quantities of these chemicals in tattoo and permanent make-up inks. Failing to provide an appropriate method may lead to unqualified and questionable results which often cause legal disputes that are ultimately resolved by a judge with regard to the method that should have been applied. Analytical methods are tuned to exactly what is to be found and what causes the health problems. They are extremely specific. Irrespective of which is the correct method for detecting metals in tattoo inks, the focus should be on the actual amounts of ink in the skin. CTL® has conducted experiments to determine these amounts and these experiments are crucial for toxicological evaluations and for setting legal limits. When setting legal limits, it is essential to also incorporate factors such as daily consumption, total uptake and frequency of use. A tattoo lasts for several decades; therefore, the limits that have been established for heavy metals used in drinking water or soap are not relevant. Drinking water is consumed on a daily basis and soap is used several times per week, while tattooing only occurs once.

Towards the limiting of health risks associated with tattooing: whitelists for tattoo pigments and preservatives.

The number of pigments that could potentially be used in tattoo inks is vast. However, pigments are generally not manufactured for the purpose of being injected into subepidermal layers of the skin. Assuming 100% bioavailability after injection means that pigments can be imminently hazardous to human health. Given the ever-increasing number of pigments being circulated on the market or through the internet, a 'negative list' ('black' list) containing pigments with known adverse effects will never be finalised. If incriminated, substances could easily be replaced by structurally similar pigments that might be even more deleterious to human health. Therefore, we and others suggest the establishment of a whitelist ('positive list') that would only contain pigments that had undergone a risk assessment specifically for their application into the dermis. Some of the problems associated with such a 'positive list' are discussed. Another important issue with regard to tattoo safety is related to the preservatives used in ink preparations. Notwithstanding the demand for sterile tattoo inks, a whitelist for these compounds would be beneficial. At present, many technical preservatives are being used, despite their known detrimental effects to human health. Criteria for the inclusion of preservatives in a 'positive list' are also discussed.