Advances In Laboratory Methods Research Articles

Abstract A063: What can we learn from a retrospective evaluation of HER2, P53 and P16INK4A protein expression in rare ovarian tumors?

Abstract A063: What can we learn from a retrospective evaluation of HER2, P53 and P16INK4A protein expression in rare ovarian tumors?

Cerebrospinal fluid kappa free light chains for the diagnosis of multiple sclerosis: A consensus statement

Cerebrospinal fluid (CSF) analysis is of utmost importance for diagnosis and differential diagnosis of patients with suspected multiple sclerosis (MS). Evidence of intrathecal immunoglobulin G (IgG) synthesis proves the inflammatory nature of the disease, increases diagnostic certainty and substitutes for dissemination in time according to current diagnostic criteria. The gold standard to determine intrathecal IgG synthesis is the detection of CSF-restricted oligoclonal bands (OCBs). However, advances in laboratory methods brought up κ-free light chains (FLCs) as a new biomarker, which are produced in excess over intact immunoglobulins and accumulate in CSF in the case of central nervous system-derived inflammation. Overwhelming evidence showed a high diagnostic accuracy of intrathecal κ-FLC synthesis in MS with sensitivity and specificity of approximately 90% similar to OCB. κ-FLCs have advantages as its detection is fast, easy, cost-effective, reliable, rater-independent and returning quantitative results which might also improve the value of predicting MS disease activity. An international panel of experts in MS and CSF diagnostics developed a consensus of all participants. Six recommendations are given for establishing standard CSF evaluation in patients suspected of having MS. The panel recommended to include intrathecal κ-FLC synthesis in the next revision of MS diagnostic criteria as an additional tool to measure intrathecal immunoglobulin synthesis.

Abstract 2045: Aneuploidy, immunohistochemical overexpression of MSH-2, P53, P21^WAF1/CIP1, P16^INK4A, and correlation to epithelial ovarian carcinoma patient prognosis

Abstract Aneuploidy, immunohistochemical overexpression of MSH-2, P53, P21WAF1/CIP1, P16INK4A, and correlation to epithelial ovarian carcinoma patient prognosis Overexpression of MSH-2, P53, P21WAF1/CIP1, P16INK4A and DNA status in epithelial ovarian carcinoma (EOC) was determined in paraffin-embedded tissue (PET) to predict prognosis. Immunoreactivity, DNA and % S content were evaluated for their potential role in survival and relapse-free survival (RFS) in 112 patients diagnosed with EOC at Saskatoon Cancer Center, Canada. Immunostaining studies were with the following mouse monoclonal antibodies: MSH-2, FE11; P53, D07; P21WAF1/CIP1, EA 10; except for P16INK4A, rabbit, G175-405, 13251A. DNA and % S content (median, < % 5 = low-risk or > % 5 = high-risk) were assessed by flow-cytometric analysis1. Survival curves were determined by Kaplan-Meier method, mean survival time, and log rank test. P-values < 0.05 were considered significant; X2-test examined association between variables. Patients with aneuploid DNA had 59 months mean survival time, compared to cases with diploid DNA which had 89 months, (p = 0.03). RFS in those with diploid DNA lasted longer (84 months) while aneuploid cases had shorter time to progression (47 months, p = 0.01). % S content in the high-risk group, 47 cases, had worse survival (57 months, p = 0.0008) and RFS (52 months, p = 0.004). Aneuploidy was not related to overall survival within the subsets of early and advanced stage patients. Grade 2 patients with abnormal DNA and P53 expression were adversely associated with survival and RFS. Diploid DNA was observed in all mucinous tumors except in one case with aneuploid DNA. P53 immunoreactivity was related to shorter survival and RFS in 44 cases. P21WAF1/CIP1 overexpression was associated with P53 overexpression in 11 cases (p = 0.04) and shorter median survival (29 months) compared to negative P21WAF1/CIP1 staining (median survival time 62 months, p = 0.03). 11 grade 2 patients with P53 positivity had worse survival (59 months) and RFS (47 months) than the P53 negative tumors (p = 0.05, and p = 0.01 respectively). P16INK4A overexpression (46 cases) had no adverse effect on survival and RFS. No significant prognostic difference was found between MSH-2 positive and negative tumors. We conclude that % S content was a more beneficial prognostic marker than ploidy status. In grade 2 EOC, the effects of DNA content and P53 overexpression were significant variables related to outcome. For pathologists, these variables in grade 2 tumors can be adjunctive aid for reaching an agreement to diagnose the grade of EOC patients.Footnotes1 A. Geissel and J. L. Griffin, “Preparation of nuclei for flow cytometry,” in AFIP Advances in Laboratory Methods in histology and Pathology, ed. Mikel UV (Washington DC: American Registry of Pathology, 1944), 111-121. Citation Format: Kavitha Advikolanu Rao, Antony M. Magliocco, Andrew W. Maksymiuk. Aneuploidy, immunohistochemical overexpression of MSH-2, P53, P21^WAF1/CIP1, P16^INK4A, and correlation to epithelial ovarian carcinoma patient prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2045.

Abstract 3537: Evaluation of genomic instability using microsatellite instability, ploidy status, and p53 expression in archival paraffin embedded tissue from primary ovarian cancer specimens

Abstract A retrospective study of a subset of ovarian cancer cases was evaluated to assess the role of microsatellite instability (MSI) and ploidy status. P53 function is important in the regulation of genomic stability and its expression was examined by immuno-histochemical staining. Genomic instability is also considered a possible indicator of immuno-therapy responsive tumors. Ovarian cancer patients diagnosed between 1983 to 1987 at Saskatoon Cancer Center, Canada, were evaluated for genomic stability. Nearly 400 Epithelial Ovarian Cancer (EOC) cases were reviewed, and Paraffin Embedded Tissue (PET) blocks from 112 cases were retrieved from hospitals in the region. PET blocks containing over 70% neoplastic tissue were chosen for the study. P53 expression was determined in eighteen cases using the mouse monoclonal antibody D07. An assessment of MSI and LOH was performed using the PCR mixes from Perkin Elmer (PE) AB. The replication error repair (RER)/Loss of Heterozygosity (LOH) assays were performed at nine loci using the primers obtained from PE. Fourteen out of eighteen cases were evaluated for ploidy status using a method described in literature[1]. Statistical analyses were performed to determine the significance of MSI/LOH, ploidy status, and p53 expression. In this study, 34 (30%) of 112 EOC cases were found to be aneuploid. Fourteen EOC cases had ploidy status that could be evaluated. Of the eighteen patients analyzed for MSI, at least nine patients showed instability at one or more loci. Of these nine, there were two RER positive phenotypes with aneuploid DNA content which were also p53 immuno-negative. Of the seven (78%) RER negative tumors, 57% (4/7) p53 negative, and the remaining three cases overexpressed p53. In five EOC cases, with an RER negative phenotype, 60% (3/5) were aneuploid and 40% (2/5) were diploid. Furthermore, LOH was a common event at the nine loci. The three events: MSI, lack of p53 overexpression, and aneuploidy, may be characteristics of a subset of ovarian cancer cases. Secondly, the study suggests that RER positivity occurs in ovarian cancer pathogenesis. We conclude that the data indicates general genomic instability due to MSI and DNA ploidy status. [1] A. Geissel and J. L. Griffin, “Preparation of nuclei for flow cytometry,” in AFIP Advances in Laboratory Methods in histology and Pathology, ed. Mikel UV (Washington DC: American Registry of Pathology, 1994), 111-121. Citation Format: Kavitha Advikolanu Rao, Anthony M. Magliocco, Andrew W. Maksymiuk. Evaluation of genomic instability using microsatellite instability, ploidy status, and p53 expression in archival paraffin embedded tissue from primary ovarian cancer specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3537.

Quantifying and reducing spurious alignments for the analysis of ultra-short ancient DNA sequences

BackgroundThe study of ancient DNA is hampered by degradation, resulting in short DNA fragments. Advances in laboratory methods have made it possible to retrieve short DNA fragments, thereby improving access to DNA preserved in highly degraded, ancient material. However, such material contains large amounts of microbial contamination in addition to DNA fragments from the ancient organism. The resulting mixture of sequences constitutes a challenge for computational analysis, since microbial sequences are hard to distinguish from the ancient sequences of interest, especially when they are short.ResultsHere, we develop a method to quantify spurious alignments based on the presence or absence of rare variants. We find that spurious alignments are enriched for mismatches and insertion/deletion differences and lack substitution patterns typical of ancient DNA. The impact of spurious alignments can be reduced by filtering on these features and by imposing a sample-specific minimum length cutoff. We apply this approach to sequences from four ~ 430,000-year-old Sima de los Huesos hominin remains, which contain particularly short DNA fragments, and increase the amount of usable sequence data by 17–150%. This allows us to place a third specimen from the site on the Neandertal lineage.ConclusionsOur method maximizes the sequence data amenable to genetic analysis from highly degraded ancient material and avoids pitfalls that are associated with the analysis of ultra-short DNA sequences.

Peroxisomal disorders: Improved laboratory diagnosis, new defects and the complicated route to treatment

Peroxisomes catalyze a number of essential metabolic functions of which fatty acid alpha- and beta-oxidation, ether phospholipid biosynthesis, glyoxylate detoxification and bile acid synthesis are the most important. The key role of peroxisomes in humans is exemplified by the existence of a group of peroxisomal disorders, caused by mutations in > 30 different genes which code for proteins with a role in either peroxisome biogenesis or one of the metabolic pathways in peroxisomes. Technological advances in laboratory methods at the metabolite-, enzyme-, and molecular level have not only allowed the identification of new peroxisomal disorders but also new phenotypes associated with already identified genetic defects thus extending the clinical spectrum. Unfortunately, progress in the field of pathogenesis and treatment has lagged behind although there are certainly new and hopeful developments with respect to X-linked adrenoleukodystrophy and hyperoxaluria type 1.

Rationale for Specific Allergen Testing of Patients With Asthma in the Clinical Pulmonary Office Setting

Asthma is a chronic inflammatory disease that affects an estimated 25 million people in the United States. In 70% to 90% of cases, asthma is associated with IgE-mediated mechanisms, which have proved central to allergen-induced inflammation in preclinical and clinical models. The importance of IgE levels in patients with moderate to severe asthma has been confirmed in randomized controlled studies with a targeted IgE blocker. Advances in laboratory methods to detect and quantify allergen-specific IgE antibodies have allowed for a quick-and-easy diagnosis of allergic IgE-mediated sensitivities in the office. Pulmonologists tend to order in vitro tests to measure allergen-specific IgE rather than to perform allergen skin testing, which is seen as the purview of allergists. This article reviews the importance of allergen testing in patients with asthma—whether by skin testing or by in vitro methods—and highlights the advantages, limitations, and interpretation of results derived from each method. Additionally, this article includes suggested documentation and administrative details for physician reporting in the office setting.

Descubrimientos y técnicas que han contribuido a mejorar el tratamiento de las enfermedades pancreáticas

Due to its retroperitoneal location, the pancreas has historically been a mysterious organ that is difficult to examine and which complicates treatment. The discovery of anesthesia and asepsis in the mid-19th century allowed laparotomic diagnosis, which was previously only possible at autopsy. The expectations of surgery were improved by the detection of blood groups, vitamin K synthesis, and the development of intensive care units. In addition, high levels of presurgical diagnosis and an unquestionable improvement of its results were enabled by advances in laboratory methods (serum quantification of amylase and lipase, tumoral markers, genetics, and techniques for measuring exocrine pancreatic function), imaging and endoscopic modalities, and fine tuning of surgical techniques. In this article, we review the history of the main milestones that have allowed progress in all these aspects.

Advances in laboratory methods for detection and typing of norovirus.

Human noroviruses are the leading cause of epidemic and sporadic gastroenteritis across all age groups. Although the disease is usually self-limiting, in the United States norovirus gastroenteritis causes an estimated 56,000 to 71,000 hospitalizations and 570 to 800 deaths each year. This minireview describes the latest data on laboratory methods (molecular, immunological) for norovirus detection, including real-time reverse transcription-quantitative PCR (RT-qPCR) and commercially available immunological assays as well as the latest FDA-cleared multi-gastrointestinal-pathogen platforms. In addition, an overview is provided on the latest nomenclature and molecular epidemiology of human noroviruses.

Top 10 Research Questions Related to Body Composition

An understanding of body composition is crucial to understanding human health, disease, and function. Research in body composition has focused on the development of assessment methods, description of normal changes in body composition with growth and development and aging, and the changes that occur in body composition in response to challenges ranging from illness to planned interventions. Each focus is significant, and in a sense, they are interdependent, because technological advances allow more sophisticated questions to be addressed, which in turn drives the development of better methods. Significant advances have been made in each area, although perhaps surprisingly basic questions remain. For example, growth trajectories are often estimated from cross-sectional data, given the resources needed for long-term observational studies, and thus, longitudinal descriptive data are still needed. Along with advances in laboratory methods, development of field methods remains relevant for screening and clinical practice. Despite recognition of wide interindividual differences in intervention response, average outcomes continue to be emphasized. With technological advances, it is now possible to examine genetic along with nongenetic factors that underlie changes in body composition, and these techniques need to be applied in long-term, well-controlled trials. In this article, we review 10 key questions in related areas in which research is needed to continue to advance the field.

High-Throughput Genomic Data in Systematics and Phylogenetics

High-throughput genomic sequencing is rapidly changing the field of phylogenetics by decreasing the cost and increasing the quantity and rate of data collection by several orders of magnitude. This deluge of data is exerting tremendous pressure on downstream data-analysis methods providing new opportunities for method development. In this review, we present (a) recent advances in laboratory methods for collection of high-throughput phylogenetic data and (b) challenges and constraints for phylogenetic analysis of these data. We compare the merits of multiple laboratory approaches, compare methods of data analysis, and offer recommendations for the most promising protocols and data-analysis workflows currently available for phylogenetics. We also discuss several strategies for increasing accuracy, with an emphasis on locus selection and proper model choice.

Development and Application of Laboratory Tools To Predict Particle Properties upon Scale-Up in Agitated Filter-Dryers

Agitated filter-dryers (AFDs) are commonly used for performing both filtration and drying operations in the manufacture of active pharmaceutical ingredients (APIs) and intermediates. Successful scale-up from the laboratory to manufacturing AFD equipment requires that physical properties specifications such as particle size be consistently met in addition to chemical purity specifications. Depending on the API–solvent system and equipment operational parameters, undesired attrition or agglomeration may occur, so an improved understanding of these phenomena upon scale-up is of key importance. In this paper, we describe recent advances in laboratory methods, based on material characterization methods common to drug product formulation development, to better assess the risk of agglomeration and attrition potential upon scale-up. These methods provide data to evaluate solid behavior, in both wet and dry states, associated with processing in an AFD. For agglomeration prediction, the application of mixer torque ...

Analytic methods for colorectal cancer

Colorectal cancer (CRC) is a major cause of morbidity and mortality from cancers in the United States. The field of molecular epidemiology is in the forefront in the discovery of genes associated with the development and progression of complex diseases such as CRC, including hereditary CRC, and polyposis syndromes such as familial adenomatous polyposis and hereditary nonpolyposis colon cancer. Recent advances in laboratory methods (ie, high-throughput genotyping and high-density single nucleotide polymorphism chips, etc.) allow the molecular epidemiologist to elucidate biological determinates of disease. However, with these advancements in the lab, advances in modeling and testing of such data are needed as well. Herein, we review the current analytical methods that have been applied to molecular epidemiologic studies for CRC and hereditary nonpolyposis colon cancer within the past 2 years, comment on the application of these methods, and examine new and promising tools that will likely influence the discovery of gene-gene and gene-environment interactions and development of pathway-based analytical approaches for CRC risk and progression.

PEDIATRIC HIV INFECTION: IMMUNE AND VIRAL EVALUATION

Advances in laboratory methods have driven improvements in the management and treatment of HIV infection. The methods to accurately and rapidly diagnose HIV infection in infants and children have been outlined in the previous article. In this review, the laboratory evaluation of infected children is described and methods to monitor progression of disease and response to therapy outlined.

Coccidioidomycosis Pulmonary Infection.

Disease caused by Coccidioides immitis has increased in frequency in recent years because of marked population shifts into highly endemic areas, as well as perturbations of the environment caused by construction, agricultural, and natural activities. Because disease may occur in persons in locations outside of the endemic regions, practitioners must be aware of the possibility of infection in those who may have traveled or resided in areas of risk. Recently, advances in laboratory methods have facilitated diagnosis of coccidioidomycosis, and information about therapies that are easier to administer has become available. Challenges in the management of this infection still remain, particularly with regard to more reliable antifungal drugs and protective vaccines.

Cushing's syndrome--1988.

Despite major advances in laboratory methods, radiology, and neurosurgery, the evaluation and therapy of Cushing’s syndrome remains a major challenge to clinicians. In a substantial percentage of the patients with Cushing’s syndrome, laboratory and radiologic studies yield data that are confusing or misleading. This paper reviews the various disorders that comprise Cushing’s syndrome and addresses some of the problems in diagnosis and therapy.

Effect of Medication During Labor and Delivery on Infant Outcome

The following statement is made because of the concern of the Committee on Drugs of the American Academy of Pediatrics and the Committee on Obstetrics: Maternal and Fetal Medicine of the American College of Obstetricians and Gynecologists about known and unknown effects on the fetus and the newborn of drugs given during labor and delivery. In the past, the effects on infant welfare of various intrapartum events, obstetrical procedures, and pharmacological agents administered to women during labor and delivery were measured by mortality, Apgar scores, acid-base studies, and specific types of neonatal morbidity.1 With advancing medical knowledge and improved technology for monitoring the well-being of the infant in utero, safer methods for delivery became available. Along with this advancement in technical skills came a decrease in the use of centrally depressant analgesic, sedative, and anesthetic drugs and an increased use of local or regional anesthesia so that the mother could remain alert and aware during labor and delivery. Recent advances in laboratory methods for drug analysis2 have given physicians a greater insight into maternal-fetal pharmacokinetics, although there is still a great void in medical knowledge related to specific drug transfer, routes of metabolism, and effects of these drugs and drug metabolites on the infant. Prenatally the mother plays the major role in metabolizing and excreting a drug, but once delivery is accomplished the infant is required to perform these functions. Adverse effects on later intelligence, neuromuscular physiology, learning ability, and behavior have not been reported beyond infancy in children whose mothers received acceptable doses of analgesic, sedative, or anesthetic drugs during labor and delivery.

Renaissance of the retinal pigment epithelium.

THE APPLICATION of recent advances in laboratory methods to the study of individual intraocular structures has greatly enhanced our ability to evaluate their function in health and disease. Nowhere is this more apparent than in the investigation of the retinal pigment epithelium where the utilization of light and electron microscopy coupled with histochemistry and autoradiography has recently revealed unsuspected functions. Long associated with the storage of vitamin A and its conversion to a form that can be utilized by the photoreceptors for the synthesis of rhodopsin, the retinal pigment epithelium has now been found to play an important role in the phagocytosis and digestion of the rod outer segment discs as well as other retinal substances. In addition, the ability of the pigment epithelium to produce and excrete a portion of the mucoprotein which surrounds the outer segments of the photoreceptors is now generally accepted. The ultrastructural pathways for the

Determining Community Immunization Level with Examples for Poliomyelitis

T HE IMMUNIZATION level of a community may be defined as the percentage of the population protected by natural and artificially induced immunity. The level is ultimately measured by the community's disease experience as evidenced by cases reported and otherwise discovered. This method of measurement is applicable to all communities, but it may be of little value when cases of disea,se are rare, as with smallpox, or infrequent, as with poliomyelitis. In Pennsylvania there were few cases of poliomyelitis in Berks, Lehigh, and Northampton Counties for 5 years before the mass vaccination programs of 1963, and only one case since (see table). However, there was a considerable potential as shown by sizable 1963 outbreaks in nearby Philadelphia and Cumberland Counties. Case investigations necessary to validate the diagnosis include clinical, serologic, and virological studies on the patients. Such studies in Cincinnati were reported by Sabin (1) and in Czechoslovakia by Skovranek (s), as ruling out the diagnosis of poliomyelitis during 1960 and 1961. Case attack rates will show whether differences exist between vaccinated and unvaccinated groups at one particular time and may show the influence of vaccination over a period of time. An example of a study at one point in time is Fendall's report on the epidemic in Kenya in 1960, with more than twice as many cases in the unvaccinated as in the vaccinated (3). Chin showed the decrease in case rates in Des Moines, Iowa, and Kansas City, Mo., from 1954 to 1959, concurrent with increased Salk vaccination (4). The immunization level may also be measured by detection of antibodies in a reipresentative sample of individuals as reported from South Africa (5). The most recent report describes a survey of children 1 through 4 years of age in Cleveland, Ohio, following an oral vaccination program in 1962. Titers were determined in vaccinated and unvaccinated children. Advances in laboratory methods may bring periodic sero-immunity surveys within the, capabilities of many health departments (6). Intestinal infection occurring naturally or produced by ingestion of vaccine may be demonstrated by identification of virus in patients and in sample surveys. Both were, done in Israel before and during an outbreak of poliomyelitis in 1961 (7). Spread of virus may be shown by its recovery from sewage as was done in Hillsborough County, Fla. (8).

Teaching Physical Diagnosis of the Chest

Physical diagnosis of the chest is a dying ar t in this country. The information provided by skilled, available roentgenologic examination has insinuously replaced observation, palpation, percussion and auscultation as the basis for decisions in diagnosis, therapy and prognosis of many chest diseases. Perhaps this is truest for those of us who have been trained during the recent, exciting advances in laboratory methods. And yet, in our haste to employ a definitive procedure, we are vulnerable through neglect of this fundamental discipline-competent, painstaking, time consuming physical diagnosis. All of us have studied cases in which physical diagnosis was the only accurate or available means of determining the cause of illness. Pneumonia, atelectasis, pulmonary edema, vascular anomalies and bronchial obstruction syndromes may be found by physical examination when chest x-ray films are not helpful. House officers frequently discard a diagnosis based on their physical diagnosis because roentgenograms fail to confirm their findings. Either their methods are faulty or they lack confidence in them. It can be a revealing experience to ask a student or house officer to demonstrate his concept of a complete physical examination of the chest, explaining the physiology and significance of his findings. Many of us might fare poorly also under such scrutiny. The need then, is for sounder instruction and more diligent practice of the art. Two remedies for our fading physical diagnostic acuity are (1) performance of comfortable, careful, complete examination in all chest cases as an example to junior members of the medical echelon and (2) reorientation of our methods in teaching physical diagnosis. It is axiomatic that medical students learn most by observing their house staff and attending physicians. Despite the number of formal presentations, the third and fourth year students are more impressed by watching internes, residents and attendings. The interne also learns from his resident and from the exceptional physician who demonstrates to him findings that he has missed and the method whereby the findings are elicited. The entire echelon profits by contact with the clinician who continually demonstrates his proficiency with physical diagnosis. But the a r t suffers by lack of example and by the attitude propagated a t ward rounds and chest conferences where examination of the patient is replaced by perusal of x-ray films. We cannot preach the theory of physical diagnosis and practice diagnosis by x-ray films without profoundly affecting .our students. It might be better to replace formal lectures by more demonstrative lessons of everyday practice.