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.

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.

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.

A universal indicator dye pH assay for crystallization solutions and other high-throughput applications.

In protein crystallization, as well as in many other fields, it is known that the pH at which experiments are performed is often the key factor in the success or failure of the trials. With the trend towards plate-based high-throughput experimental techniques, measuring the pH values of solutions one by one becomes prohibitively time- and reagent-expensive. As part of an HT crystallization facility, a colour-based pH assay that is rapid, uses very little reagent and is suitable for 96-well or higher density plates has been developed.

Ultra-performance liquid chromatographic determination of manufacturing intermediates and subsidiary colors in D&C Red No. 34 and its lakes.

An ultra-performance liquid chromatography (UPLC) method was developed to determine the manufacturing intermediates and subsidiary colors in the monosulfo monoazo color additive D&C Red No. 34 and its lakes. This method is currently used for batch certification of the color additives by the U.S. Food and Drug Administration to ensure that each lot meets published specifications for coloring drugs and cosmetics. The new UPLC method has replaced an HPLC method for determining the intermediates and a TLC method for determining the subsidiary colors. The intermediates are 2-amino-1-naphthalenesulfonic acid (Tobias acid) and 3-hydroxy-2-naphthalenecarboxylic acid (3-hydroxy-2-naphthoic acid). Subsidiary colors are positional isomers of the major dye component or related compounds containing lower numbers of substituent groups. The analytes are identified by comparison of their UPLC retention times and UV or visible absorption spectra with those of standards. Validation studies showed that peak area calibrations for the analytes were generally linear (R > 0.999), and recoveries were 98-103%. The LODs were 0.002-0.02%, and the RSDs at the specification levels were 0.7-2.2%. Survey analyses of 12 samples of certified D&C Red No. 34 straight colors and lakes from six domestic and foreign manufacturers yielded results for the intermediates by UPLC and HPLC that were consistent within experimental error. The UPLC analyses yielded results for the subsidiary colors that were consistently lower than results previously obtained by TLC, which we attribute to limitations of the TLC method. The new UPLC method provides sharper peaks, better peak separation, and faster analysis times than the formerly used HPLC method and is more accurate, much faster, and much less labor-intensive than the formerly used TLC method.

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.

Intuitiveness, quality and utility of intraoperative fluorescence videoangiography: Australian Neurosurgical Experience.

INTRODUCTION: The authors have undertaken a study of their intraoperative experience with indocyanine green fluorescence videoangiography (ICGFV). In particular, the intuitiveness, image quality and clinical utility of this technology have been assessed. METHODS: The records of forty-six consecutive craniotomies utilising ICGFV have been retrospectively reviewed: There were 27 aneurysms, 2 extracranial-intracranial (EC-IC) bypasses, 5 arteriovenous malformations (AVM), 1 dural arteriovenous fistula (DAVF), 3 cavernomas, 5 meningiomas, and 3 gliomas. ICGFV was used in 5 awake-craniotomy patients. ICGFV was performed using a Leica OH4 surgical microscope with integrated near-infrared camera and ICG-PULSION. RESULTS: All attempts of intraoperative ICGFV were intuitive. Image quality and resolution were excellent. Arterial and venous phases were comparable to digital subtraction angiography (DSA) but field of view was relatively limited. In 12 operations (26%) the surgeon was substantially benefited from ICGFV findings. In 22 operations (48%), ICGFV was useful but did not influence surgical management. ICGFV was of no benefit in 11 operations (24%) and was misleading in 1 (2%). In this series, ICGFV was of benefit to 1 of 11 (9%) patients with an intracranial neoplasm or cavernoma. CONCLUSIONS: ICGFV is safe, intuitive and provides neurosurgeons with high quality, valuable, real-time imaging of cerebrovascular anatomy. It can assist in intraoperative surgical management and/or stroke prevention particularly during aneurysm clipping, EC-IC bypass and AVM/DAVF surgery.

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.

Variations in quality of carbol fuchsin stains collected from routine tuberculosis laboratories.

SETTING: In-use carbol fuchsin stains were collected from 10 different routine acid-fast bacilli smear microscopy laboratories. OBJECTIVE: To examine the variations in the composition of carbol fuchsin stains. METHOD: Carbol fuchsin concentrations were first determined spectrophotometrically by measuring absorbance at 547 nm. High-performance liquid chromatography (HPLC) separated and quantified the four basic fuchsin homologues: para-rosaniline, rosaniline, magenta II and new fuchsin, and identity was confirmed by mass spectrometry (MS). RESULTS: Absorbance measurement showed that three of 10 (30%) samples contained insufficient carbol fuchsin (<70%). Wide variations in relative proportions of fuchsin homologues were found. CONCLUSION: The relative abundance of rosaniline + new fuchsin was quite stable among the different laboratories. Spectrophotometry and HPLC/MS are necessary and sensitive tools for monitoring fuchsin quality.

[History of the histological staining survey in the Tokyo Metropolitan Hospital].

The Tokyo metropolitan hospital society of chief medical technologist has carried out a staining survey once a year since 1992. This staining survey is performed to plan improvements of the staining technology at each hospital and reduction of differences among hospitals. This report describes the examination results for PAS stain, Papanicolaou stain, and PAM stain.

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.

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.

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.

Ziehl-Neelsen staining: theory and practice.

The information provided in the guidelines of the World Health Organization and the International Union Against Tuberculosis and Lung Disease for Ziehl-Neelsen staining is not practical on a number of points. The advice given here is meant to supplement the guidelines. It is based on experiments on and field experience of basic fuchsin stain and staining solutions.

Comparison of preparation methods for the evaluation of nerve axons.

We have conducted a study to compare various preparation methods, including a certain number of new methods, to find which ones are best suited to the morphological evaluation of nerve fibers in the pyramidal tract of the human medulla oblongata. Our main concern was to find fixation and staining methods that would minimize errors, especially regarding the tissue shrinkage ratio and the ease of staining. From the two fixation methods we examined, the most satisfactory was the secondary chromic acid fixation (which gave the best overall results when followed by nitrocellulose embedding), as it gives the lowest shrinkage ratio with the narrowest range (10 +/- 0%). Among the ten staining methods we tested, we found that the most suitable for morphological evaluation were the discriminative staining methods (Luxol fast blue-Periodic acid-Schiff-Hematoxylin stain, Masson-Goldner-Goto method and modified Hematoxylin-Eosin stain) and the silver impregnation methods (Luxol fast blue-Silver impregnation and Luxol fast blue-Silver impregnation-Periodic acid-Schiff-Hematoxylin).

Brain histology.

In this article we summarise nervous system histology in health and disease and acquaint the reader with developments in the staining techniques that are in current use, particularly immunostains. Although clinicians do not need to know the details of stain appearances, some familiarity with these aspects of neuropathology is invaluable in interpreting the reports they receive from the laboratory, as well as reminding them of the amazing beauty of the central nervous system's microscopic structure.

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.