Coconut oil as a rehydrant in air dried oral buccal smears: An alternative to routine wet fixation

The routine procedure to prepare a Pap smear is done by fixing the slides with 95% ethanol immediately after the sample is taken. This study was performed to determine the alternative method of air-drying and rehydration prior to alcohol fixation instead of conventional method. Paired buccal smears were collected from 50 patients who participated in the study. One set was labeled as (Wet fixed in 95% ethanol) WF and the other one Air-dried fixed (ARF) rehydrated and fixed with coconut oil. The staining quality of the slides was assessed with respect to nuclear details and cytoplasmic details, cytoplasmic staining and background staining. Single blinded study was done. The results were analyzed by Chi square test to compare the defined parameters between the two groups. Air-dried fixed (ARF) slides with coconut oil was significantly better with regard to clearance of background. There was statistically significant difference between the air dried smear rehydrated with coconut oil and normal wet fixed smear. So air dried smear rehydrated with coconut oil can be used as an alternative to wet fixed smear.

Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.

Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.

Visualisation of GABAergic neurons and synapses in the rat brain using immunohistochemistry for two forms of glutamate decarboxylase

BACKGROUND: Taking into account the importance of GABAergic brain system research and also the opportunity to achieve specific and accurate results in laboratory studies using immunohistochemical approaches, it seems important to have a reliable method of visualization GABA-synthesizing cells, their projections and synapses, for the morphofunctional analysis of GABAergic system both in normal conditions and in the experimental pathology. AIM: The aim of the study was to visualize analyze GABAergic neurons and synapses within rats brain using three different antibody types against glutamate decarboxylase and to identify the optimal conditions for reaction performing. MATERIALS AND METHODS: The study was performed on paraffin brain tissue sections of 5 adult Wistar rats. Immunohistochemical reactions using three antibody types against glutamate decarboxylase isoform 67 (GAD67) and glutamate decarboxylase isoform 65 (GAD65) were performed. Additional controls on C57/Bl6 mice and Chinchilla rabbits brain samples were also carried out. RESULTS: Antibodies used in the research made it possible to achieve high quality of GABAergic structures visualizing without increasing background staining. At the same time different antibody types are distinct in their efficacy to perform immunohistochemistry reaction on laboratory animal brain tissue samples. By performing additional controls, we discovered that there is necessary to adsorb secondary reagents immunoglobulins in order to eliminate nonspecific staining. It was found that GAD67 and GAD65 distribution in rat forebrain structures is different. It was stated that GAD67 immunohistochemistry most completely reveals GABAergic brain structures compared to GAD65 immunhistochemistry. The possibility of determining morphological features of GABAergic neurons and synaptic terminals, as well as performing quantitative analysis, was demonstrated. CONCLUSIONS: The approach proposed makes it possible to specifically visualize GABAergic structures of the central nervous system of different laboratory animals. This could be useful both in fundamental studies and in pathology research.

Diagnostic approach in TFE3-rearranged renal cell carcinoma: a multi-institutional international survey

Transcription factor E3-rearranged renal cell carcinoma (TFE3-RCC) has heterogenous morphologic and immunohistochemical (IHC) features.131 pathologists with genitourinary expertise were invited in an online survey containing 23 questions assessing their experience on TFE3-RCC diagnostic work-up.Fifty (38%) participants completed the survey. 46 of 50 participants reported multiple patterns, most commonly papillary pattern (almost always 9/46, 19.5%; frequently 29/46, 63%). Large epithelioid cells with abundant cytoplasm were the most encountered cytologic feature, with either clear (almost always 10/50, 20%; frequently 34/50, 68%) or eosinophilic (almost always 4/49, 8%; frequently 28/49, 57%) cytology. Strong (3+) or diffuse (>75% of tumour cells) nuclear TFE3 IHC expression was considered diagnostic by 13/46 (28%) and 12/47 (26%) participants, respectively. Main TFE3 IHC issues were the low specificity (16/42, 38%), unreliable staining performance (15/42, 36%) and background staining (12/42, 29%). Most preferred IHC assays other than TFE3, cathepsin K and pancytokeratin were melan A (44/50, 88%), HMB45 (43/50, 86%), carbonic anhydrase IX (41/50, 82%) and CK7 (32/50, 64%). Cut-off for positive TFE3 fluorescent in situ hybridisation (FISH) was preferably 10% (9/50, 18%), although significant variation in cut-off values was present. 23/48 (48%) participants required TFE3 FISH testing to confirm TFE3-RCC regardless of the histomorphologic and IHC assessment. 28/50 (56%) participants would request additional molecular studies other than FISH assay in selected cases, whereas 3/50 participants use additional molecular cases in all cases when TFE3-RCC is in the differential.Optimal diagnostic approach on TFE3-RCC is impacted by IHC and/or FISH assay preferences as well as their conflicting interpretation methods.

The Application of the Immunoperoxidase (IP) Antibody Technique for the Etiological Diagnosis of Infectious Bursal Disease (IBD) in Chickens

The Indirect IP – technique was employed in demonstrating a site where IBD antigen reacted with Its untbody in IBD-affected barya of Fabricius. In the, rabbit - wot-chicken IgG, which was conjugated to borwer dish peroxidase, was used as the Immunochemical tracers of the antigen-antibody la teraction between IBD chicken underum wad wc tions of bursa of Fabricius (BF) from IBD infected chicken. The technical details were examined and compared with the immunofluorescent (IF) and body technique. The IP – technique was simpler to perform, gave permanently stained specimens, and was answer to interpret because it was less subject to background staining than the IF - technique However both the IP and the IF — techniques were of equal diagnostic sensitivity and specificity. Some attempts made to use the IP - technique for detecting IBD teld virus isolates in cell-culture solatoh yarns were unsuccessful. The IP - technique was, however, capable of demonstrating vll-culture adapted IBD viruses lo cell monotavers.

Staining Cells with Extracts Prepared from Flowers of Bougainvillea X Buttiana.

Background: Staining is the application of dyes to specimens to impart colour to cells through a chemical reaction. The study aimed at finding plant extracts to stain human blood cells, stem sections of Amaranthus species, Gram-negative organisms such as Escherichia coli, and Gram-positive organisms such as Staphylococcus aureus. Methodology: Healthy mature flowers of Bougainvillea X buttiana and Amaranthus species plants were picked from gardens around the University of Kisubi. Bracts of Bougainvillea X buttiana were separated from other flower parts and air-dried. Both negative and positive controls for cells were prepared. Results: White blood cells, platelets, and cells of Escherichia Coli and Staphylococcus aureus did not stain under all treatments with the extracts while human red blood cells and stem sections of Amaranthus species stained under certain treatments with the extracts. The extracts were more successful in staining stem sections of Amaranthus species as compared to human red blood cells where staining occurred in very few circumstances. Stem sections of Amaranthus species required shorter to stain effectively while human red blood cells required longer to stain effectively. Conclusion: Extracts of the bracts of Bougainvillea X can be experimented with various cells when their pH is neutral and alkaline.

Staining Cells with Extracts Prepared from Flowers of Bougainvillea X Buttiana.

Background: Staining is the application of dyes to specimens to impart colour to cells through a chemical reaction. The study aimed at finding plant extracts to stain human blood cells, stem sections of Amaranthus species, Gram-negative organisms such as Escherichia coli, and Gram-positive organisms such as Staphylococcus aureus. Methodology: Healthy mature flowers of Bougainvillea X buttiana and Amaranthus species plants were picked from gardens around the University of Kisubi. Bracts of Bougainvillea X buttiana were separated from other flower parts and air-dried. Both negative and positive controls for cells were prepared. Results: White blood cells, platelets, and cells of Escherichia Coli and Staphylococcus aureus did not stain under all treatments with the extracts while human red blood cells and stem sections of Amaranthus species stained under certain treatments with the extracts. The extracts were more successful in staining stem sections of Amaranthus species as compared to human red blood cells where staining occurred in very few circumstances. Stem sections of Amaranthus species required shorter to stain effectively while human red blood cells required longer to stain effectively. Conclusion: Extracts of the bracts of Bougainvillea X can be experimented with various cells when their pH is neutral and alkaline.

Levamisole usage for the block of intestinal alkaline phosphatase in immunohistochemical staining

Introduction. Immunohistochemical staining by an indirect method with a chromogenic label requires en-zymes, among which is alkaline phosphatase (AP). AP is also present in human tissues. It can break down the molecules of the immunohistochemical substrate, which leads to significant background staining. It is necessary to block endogenous enzymes prior to immunohistochemical staining to reduce this effect. One of the ways to block endogenous alkaline phosphatase is to use levamisole solutions. The aim of the study was to describe the technique of levamisole use to block the alkaline phosphatase intestinal form during immunohistochemical assays. Materials and methods. This article provides the calculations for 0.001M working levamisole solution preparation from a 10% officinal veterinary levamisole hydrochloride produced by Livisto Invesa Industrial Veterinaria S. A., Spain. The inactivation of AP intestinal form was checked by a reaction with one marker (PDGFRb) and two markers (FAP and SMA) on the colon cancer specimens. We used the Abcam ab210061 DoubleStain IHC Kit: M&R on human tissue (HRP/Green&AP/Red, Great Britain) according to the method recommended by the manufacturer with some changes to identify two markers on the same slide. Results. During the immunohistochemical assay, a complete absence of background staining and a bright contrast reaction with antibodies (both with one and two in the same section) using 1mМ levamisole was achieved. It indicates sufficient inactivation of the AP intestinal isoform in the colon cancer specimens. A comparative cost-benefit analysis for one slide using ready-to-use commercial blocking reagents and officinal levamisole solution shows a significant economic advantage of the latter. Conclusion. The high reaction quality and the palpable economic profits open up opportunities for using 1mM levamisole solution for immunohistochemical studies in laboratory practice and research work. Keywords: levamisole, immunochistochemistry, alkaline phosphatase

Life After Death

Objectives To test the performance characteristics of 69 primary immunohistochemistry antibodies after expiration and compare with fresh primary antibodies wherever possible. Methods A total of 69 expired primary antibodies were evaluated for specificity, background staining, and intensity. An optimal staining result corresponded to a semiquantitatively scored 2+ or 3+ intensity, with intact specificity devoid of moderate or strong background staining. Any deviation from a normal staining pattern was also considered to be a suboptimal result. Results Nearly half of the antibodies studied showed an optimally positive staining result after expiration (34/69, 49.2%). Overall, 10.1% (7/69) of antibodies could be compared with fresh primary antibodies of the same clone with equivalent results. Eight of 69 (11.6%) expired antibodies showed splotchy or granular staining. Conclusions Evidence from this study and previous work point to maintained functionality of a fair number of primary immunohistochemical antibodies after expiration. Decisions about the use of such reagents should be guided by a thorough assessment of functionality by the pathologist rather than a manufacturer-specified deadline. Quality maintenance should imply a sensible balance between histopathologic performance and economics.