EDTA Blood Research Articles

Somatic mosaicism in STAG2-associated cohesinopathies: Expansion of the genotypic and phenotypic spectrum.

STAG2 is a component of the large, evolutionarily highly conserved cohesin complex, which has been linked to various cellular processes like genome organization, DNA replication, gene expression, heterochromatin formation, sister chromatid cohesion, and DNA repair. A wide spectrum of germline variants in genes encoding subunits or regulators of the cohesin complex have previously been identified to cause distinct but phenotypically overlapping multisystem developmental disorders belonging to the group of cohesinopathies. Pathogenic variants in STAG2 have rarely been implicated in an X-linked cohesinopathy associated with undergrowth, developmental delay, and dysmorphic features. Here, we describe for the first time a mosaic STAG2 variant in an individual with developmental delay, microcephaly, and hemihypotrophy of the right side. We characterized the grade of mosaicism by deep sequencing analysis on DNA extracted from EDTA blood, urine and buccal swabs. Furthermore, we report an additional female with a novel de novo splice variant in STAG2. Interestingly, both individuals show supernumerary nipples, a feature that has not been reported associated to STAG2 before. Remarkably, additional analysis of STAG2 transcripts in both individuals showed only wildtype transcripts, even after blockage of nonsense-mediated decay using puromycin in blood lymphocytes. As the phenotype of STAG2-associated cohesinopathies is dominated by global developmental delay, severe microcephaly, and brain abnormalities, we investigated the expression of STAG2 and other related components of the cohesin complex during Bioengineered Neuronal Organoids (BENOs) generation by RNA sequencing. Interestingly, we observed a prominent expression of STAG2, especially between culture days 0 and 15, indicating an essential function of STAG2 in early brain development. In summary, we expand the genotypic and phenotypic spectrum of STAG2-associated cohesinopathies and show that BENOs represent a promising model to gain further insights into the critical role of STAG2 in the complex process of nervous system development.

NPM1 Mutation Quantitation Evaluated By a Commercially Available Assay and the Cepheid Prototype‡ Xpert® NPM1 Mutation Assay

Aims The objective of this study was to compare the performance of the novel Cepheid Prototype Xpert® NPM1 Mutation Assay to the Qiagen ipsogen® NPM1 mut A, B&D MutaQuant Kits using samples contrived using cell line-spiked peripheral blood. Our goal was to evaluate the comparative performance of a faster and easier to use assay than commercially available products for use in the monitoring of minimal residual disease (MRD) in NPM1-positive AML patients. Background A common mutation in AML (20-30% of cases) is a four base pair insertion in the NPM1 gene.¹ NPM1 plays a role in ribosome biogenesis, maintaining genomic stability, and modulating growth suppression by binding and stabilizing p53.² NPM1 mutant A consists of about 80% of NPM1 mutations, while mutants B or D are present in a sizeable minority of patients.¹² Monitoring of NPM1 mutations can provide prognostic value and can assist with choice of treatment.³ Methods Three NPM1-positive cell lines, OCI-AML3 MutA, OCI-AML3 MutB, and OCI-AML3 MutD, which express NPM1 MutA, NPM1 Mut B, and NPM1 Mut D, respectively, were grown and isolated. After processing the cells into cell lysate, the cell-lysates were spiked into NPM1-negative EDTA whole blood lysate and serially diluted to target 13 or 14 NPM1/ABL levels from approximately 2500% (log reduction (LR) 1.4) to approximately 0.01% (LR -4.0). A subset of each level underwent hands-off sample preparation, total RNA extraction, and RT-qPCR all within the Cepheid GeneXpert automated cartridge system, and another subset had total RNA extracted using the Zymo Direct-zol™ RNA Miniprep Plus Kit. RNA was qualified and quantified using the Thermo Scientific NanoDrop™ 2000 Spectrophotometer before being tested per the comparator assay vendor's recommendations. The Cepheid Prototype assay utilized 4.5mL of lysate, whereas the Qiagen kits used 100 ng of RNA per test. Specimens spiked with cell lines expressing different NPM1 mutations were tested using the respective ipsogen kit designed for that mutation. The Cepheid Prototype assay was evaluated against the comparator assay for each subtype using Deming regressions. Bland-Altman plots were utilized to evaluate bias between assay quantitation outputs. Results Deming regressions and associated Bland-Altman plots showed a high degree of correlation and agreement between comparator assays and the Cepheid Prototype assay (Figure 1). Conclusions The novel Cepheid Prototype assay showed a high level of agreement with Qiagen kits in this study using whole blood EDTA lysate samples spiked with three NPM1-positive cell-lysates. Agreement tended to decline at low input levels, and a small but acceptable degree of nonlinearity was present in MutA and MutD. Total time to result (TTR) was minimized in the Cepheid Prototype assay to under 170 minutes, while the comparator assay had a TTR of approximately six hours plus RNA extraction. While RNA extraction is performed within Cepheid Prototype assay cartridge and is included in this 170-minute TTR, the Qiagen kit requires additional expensive and labor-intensive extraction steps that can last one-hour to multiple days, which are not included in the six-hour TTR estimate. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic characterization of dengue virus from patients presenting multi-serotypic infections in the Northern West Bengal, India.

Northern West Bengal, popularly known as North Bengal, is a dengue-endemic area, which has been severely affected by Dengue in the past few years resulting in massive hospitalizations and deaths. Genetic characterization of the circulating endemic dengue virus (DENV) serotypes is of paramount importance for the epidemiological understanding of the infection and subsequent vaccine development. The present study was conceived to characterize circulating dengue serotypes and to undertake phylogenetic study. EDTA blood samples of all (N = 83) NS1-positive cases of patients with acute febrile illness referred to different health care facilities were collected and processed for RNA isolation followed by the complementary DNA (cDNA) preparation. Serotype determination of dengue infection was done using conventional PCR by targeting the viral C-prM region. Phylogenetic tree was constructed by implementing the Maximum likelihood method. Out of 83 blood samples 17 were detected to be positive for the presence of dengue viral RNA. DENV3 was found to be the predominant serotype in the single-infection cases; however, we have detected multi-serotypic co-infections throughout the study. Joint pain was found to be the most valuable symptom for the prognosis of dengue. Sequence analyses suggested that both DENV1- and DENV3-circulating genotypes are in the genotype III group and remain closely related to the Indian clade. To the best of our knowledge, this is the first report on the genetic characterization of circulating DENVs in North Bengal, which may contribute to the study of dengue epidemic and pathogenesis.

Clinical presentation, viral kinetics, and management of human monkeypox cases from New Delhi, India 2022.

We describe the clinical and demographic characteristics, virological follow-up, and management of five confirmed monkeypox cases from New Delhi, India without any international travel history. The viral load kinetics and viral clearance were estimated in oropharyngeal swabs (OPS), nasopharyngeal swabs (NPS), EDTA blood, serum, urine, and various lesion specimens on every fourth day of follow-up ranging from 5 to 24 post onset day (POD) of illness. Allfive cases presented with mild to moderate-grade intermittent fever, myalgia, and lesions on the genitals, groins, lower limb, trunk, and upper limb. Four cases had non-tender firm lymphadenopathy. No secondary complications or sexually transmitted infections were recorded in these cases except for the presence of viral hepatitis B infection marker hepatitis B virus surface antigen(HBsAg) in one case. All the cases were mild and had a good recovery. Ahigher viral load was detected in lesion fluid (POD 9), followed by lesion roof (POD 9), urine (POD 5), lesion base (POD 5), and OPS/NPS (POD 5). The monkeypox virus(MPXV) DNA was detected in clinical samples from 5th to 24th POD. These monkeypox cases without international travel history suggest the underdiagnosed monkeypox infection in the community. This emphasizes the need for active surveillance of MPXV in the high-risk population such as men having sex with men and female sex workers.

Comprehensive lipidomic profiling by plasma separation cards.

Large-scale lipidomic analyses have been limited by the cost and accessibility of traditional venipuncture sampling. Microsampling techniques offer a less-invasive and more accessible alternative. From a single drop of blood, plasma separation cards (PSC) deliver two volumetric dried plasma samples which are studied here for profiling endogenous blood lipids. Six lots of EDTA-treated human whole blood were used to compare PSC, dried blood spot analyses (DBS), and classic wet plasma extractions. Six replicate extractions were performed for each lot. Nontargeted lipidomics was performed by liquid chromatography-high resolution tandem mass spectrometry. Lipids were annotated by accurate mass/retention time matching and MS/MS spectral library matching using peak intensities for quantitation. Four hundred ninety-eight compounds covering 24 lipid subclasses were annotated. Inter-lot repeatability was evaluated by the percent relative standard deviation (%RSD) for each lot, giving median %RSD values across the lots at 14.6% for PSC, 9.3% for DBS, and 8.6% for wet plasma. Strong correlations of lipid peak intensities between wet plasma and PSCs were observed, but less for DBS. Lipid recovery and stability were comparable between the PSC and DBS samples, with roughly 60% of annotated lipids stable at room temperature after 28 days. Overall, PSCs provide a better alternative for quantitative blood lipidomic analyses compared to dried blood spots. However, problems with lipid stability for samples handled and shipped at room temperature are currently unavoidable outside of a clinical setting. Data transferability and comparability to standard plasma is lipid and lipid class dependent.

Relationship between single nucleotide polymorphism studies in ghrelin gene with obesity subjects

BackgroundObesity is one of the hereditary metabolic conditions that have been recognized in humans which is associated with visceral fat. Ghrelin (GHRL) is mainly linked to obesity as lipid metabolism. The rs626917, rs34911341 and rs20755356 single nucleotide polymorphisms (SNP) were extensively investigated in multiple human diseases including obesity. There are no studies were studied in the Saudi population with GHRL gene and obesity and the current study was aimed to investigate the genetic association between rs626917, rs34911341 and rs20755356 SNPs in GHRL gene in obesity population. MethodologyNinety patients with obesity and ninety healthy individuals served as controls in this case-control study. Genomic DNA was extracted from the collected EDTA blood and genotyping was performed with qPCR analysis using VIC and FAM probes. Clinical and biochemical data was collected from 180 subjects and statistical analysis was performed using SPSS software (25th Version, USA). ResultsAge, weight, BMI, WC, SBP, DBP, HDLC, and TG levels all showed statistical significance with obesity (p < 0.05) when compared with controls. Genotype analysis in rs696217 SNP confirmed no association in allele, genotypes or any other forms of genetic models. A positive association was observed in both rs34911341 and rs2075356 SNPs (p < 0.05). The Anova analysis confirmed the significant association with weight, BMI and SBP in rs696217 SNP, weight and BMI in rs34911341 SNP and SBP in rs2075356 SNP in GHRL gene (p = 0.01). ConclusionThis study confirms the significant association with rs34911341 and rs20755356 SNPs was associated with obesity in the Saudi population.

Stability of Hemoglobin Constant Spring Identified by Capillary Electrophoresis.

Hemoglobin Constant Spring (HbCS) is often missed by routine hemoglobin analysis. The aim of this research was to study HbCS stability as identified by capillary electrophoresis (CE) to determine the specimen storage time limit. The EDTA blood of 29 HbCS samples were kept at 4°C and analyzed every workday until CE could not detect HbCS or until 7 weeks after blood collection. The genotypes were confirmed by multiplex polymerase chain reaction. The median subject age was 27 years and 10 subjects were male. The HbCS levels were stable during the first 7 days but became undetectable in 5 cases (17.2%) after 1 week. All of them were heterozygous HbCS. Longer detection times were correlated with the higher baseline HbCS levels, with a correlation coefficient of 0.582 (P ≤ 0.001). Routine hemoglobin typing and quantitation should be performed within 1 week after blood collection to detect low HbCS levels, especially in heterozygous HbCS.

Platelet transfusion in patients with liver cirrhosis cross-talk with neutrophil: Prospective study

IntroductionImmune dysfunction and thrombocytopenia are common features in liver cirrhosis. Platelet transfusion is the most widely used therapeutic approach for thrombocytopenia when indicated. The transfused platelets are prone to lesions during their storage that empower their interaction with the recipient leucocyte. These interactions modulate the host immune response. The impact of platelet transfusion on the immune system in cirrhotic patients is little understood. Therefore, this study aims to investigate the impact of platelet transfusion on neutrophil function in cirrhotic patients. MethodsThis prospective cohort study was implemented on 30 cirrhotic patients receiving platelet transfusion and 30 healthy individuals as a control group. EDTA blood samples were collected from cirrhotic patients before and after an elective platelet transfusion. Flowcytometric analysis of neutrophil functions (CD11b expression and PCN formation) was performed. ResultsThere was a significant increase in expression of CD11b on neutrophils and Frequency of platelet-complexed neutrophils (PCN) in patients with cirrhosis compared with controls. Platelet transfusion increased level of CD11b and the frequency of PCN even more. There was a significant positive correlation between change in PCN Frequency pefore and after transfusion and the change in expression of CD11b among cirrhotic patients. ConclusionsElective platelet transfusion appears to increase level of PCN in cirrhotic patients, moreover, exacerbate the expression of activation marker CD11b on both neutrophils and PCN. More research and studies are needed to corroborate our preliminary findings.

Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games.

The doping control analyses at the XXXII Olympic Games (July 23 to August 8, 2021) and the XVI Paralympic Games (August 24 to September 5, 2021) held in Tokyo, Japan, after a year of delay due to the COVID-19 pandemic are summarized in this paper. A new satellite facility at the existing World Anti-Doping Agency (WADA)-accredited Tokyo laboratory was established and fully operated by 278 staff, including 33 Tokyo laboratory staff, 49 international experts, and 196 Japanese temporary staff. The numbers of urine samples were 5081 (Olympics) and 1519 (Paralympics), and the numbers of blood samples were 1103 (Olympics) and 500 (Paralympics). The laboratory could prepare for analysis in advance using a paperless chain-of-custody system, allowing for faster turnaround time reporting. For the first time, a new polymerase chain reaction method for detecting erythropoietin (EPO) gene doping was used. The laboratory also analyzed blood samples for detecting steroid esters following the spotting of collected venous EDTA blood onto dried blood spot cards. Moreover, full-scan data acquisition using high-resolution mass spectrometers was performed for all urine samples, allowing for detecting traces of doping substances, which are not currently analyzed in the subsequent data processing. The presence of some prohibited substances was confirmed, resulting in 8 atypical findings (ATFs) and 11 adverse analytical findings (AAFs), including homologous blood transfusion (2 cases) and recombinant EPO in the blood (1 case), at the Olympics, whereas 2 ATFs and 10 AAFs were reported at the Paralympics.

UTILITY OF PLATELET INDICES IN THROMBOCYTOPENIA

Objectives: The objectives of the study were to know the role and utility of platelet indices (MPV and PDW) in differentiating the etiology of thrombocytopenia. Methods: This was a prospective and retrospective study of 400 cases of thrombocytopenia and 100 controls conducted over a period of 17 months from September 2020 to February 2022 at tertiary care hospital, Ahmedabad, Gujarat, India. All patients meeting inclusion criteria were included in the study. Exclusion criteria were radiation/chemotherapy, antiplatelet drugs induced thrombocytopenia, etc. EDTA blood samples were collected and further processed with a Sysmex KX-21 automated hematology analyzer. Platelet count, mean platelet volume (MPV), and platelet distribution width (PDW) were noted and data were statistically analyzed between groups using t-tests. Results: Out of 400 cases, 304 belong to Group A (increased destruction of platelets) and the remaining 96 were in Group B (decreased production of platelets). Among Group A, causes were classified into immune thrombocytopenic purpura and others. Various infections resulting in thrombocytopenia were in the majority among other causes in Group A. Group B was comprised megaloblastic anemia as the leading cause responsible for thrombocytopenia. MPV and PDW values were high in hyperdestructive Group A compared to Group B and were found significant between various groups. Conclusion: MPV and PDW along with platelet count can be used as an adjunct laboratory tool for early screening of the underlying cause of thrombocytopenia, which helps clinicians with targeted therapies in clinical management.

Postmortem Genetic Testing Is an Increasingly Utilized Tool in Death Investigation.

Postmortem genetic testing (PMGT) can provide valuable information about an individual's cause of death and potentially allow at-risk relatives to discern their risks for inherited cardiac disease. Postmortem genetic testing is most often successful with certain specimens. Investigators collected data on postmortem referrals to GeneDx, LLC for PMGT. Orders were reviewed and stratified based on provider, specimen type, and tests ordered. This cohort included 601 deceased individuals referred for PMGT with a total of 673 genetic tests ordered from 247 different providers. The most common test categories ordered were arrhythmia (33.4%) and cardiomyopathy (29.3%). A likely pathogenic or pathogenic genetic variant was identified in approximately 15% of patients. Blood in EDTA was received for 21.6% of patients with a 95% success rate for completion of all test components. Blood samples in EDTA were most successful in completing PMGT, but sequencing was still successful in the majority of suboptimal specimens. The use of PMGT is increasing. Obtaining optimal samples (blood in EDTA) is important for successful completion of genetic testing. Obstacles may still exist for obtaining and storing ideal specimens. Continued efforts are needed for education and awareness around appropriate specimen types, storage and shipping of specimens, DNA banking, and overall availability of PMGT. In addition, access to resources such as supplies, proper storage conditions, DNA banking, and PMGT will allow for more opportunities to complete testing.

Panleukopenia Outbreak Diagnosed by Real Time QPCR in a Catterry in Romania

Abstract Feline parvovirus is a very often found cat infection, especially if we have unvaccinated animals and sometimes the infection can occur even on vaccinated animals, as we will describe in our paper. Feline Panleukopenia have been diagnosed in a cattery using a very sensitive test for this disease- Real Time qPcr. Twenty-one Edta blood samples have been tested, from British Shorthair cats from a cattery. Most of the tested cats had mild enteric symptoms and no death was registered. In our paper we prove that even on vaccinated animals we can found Parvovirus outbreaks and a very rapid and sensitive way to establish the diagnostic is a Real Time qPCR detection.

Metagenomic Sequencing for the Diagnosis of Plasmodium spp. with Different Levels of Parasitemia in EDTA Blood of Malaria Patients-A Proof-of-Principle Assessment.

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = −0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR.

P452 Estimation of the detection limit of extracted Candida DNA from spiked EDTA blood sample

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundCandidemia caused by genus Candida, are opportunistic pathogens with life-threatening infection in immunocompromised individuals with a mortality rate being 40%–54% in patients. Blood culture is not a reliable and rapid method for the diagnosis of candidemia as its sensitivity is as low as 50%. Molecular diagnosis is an alternative to conventional culture for early detection of candidemia. An appropriate DNA isolation technique is needed to obtain high purity DNA from blood specimens and improves the sensitivity of the polymerase chain reaction (PCR). In this study, two different methods were used to extract Candida DNA from spiked EDTA blood specimens, the In-house phenol-chloroform and potassium acetate method.ObjectivesDNA extraction from the spiked blood samplesQuantification of the Candida DNA extracted from the spiked sampleComparison of the extracted DNA by phenol-chloroform and potassium acetate methodMethodsA total of 5 ml of EDTA blood samples from healthy volunteers were spiked with 104-108 Candida albicans cells (ATCC 90028) to determine the detection limit of our extraction method. DNA was extracted from whole blood using phenol-chloroform and the potassium acetate methods which involves pretreatment with erythrocytes, leukocytes and fungal lysis buffer. DNA from C. albicans were amplified using ITS1 and ITS4 based primers. PCR products were visualized by agarose gel electrophoresis.ResultsThe spiked Candida cells taken were from 104-108 yeast cells. The total extracted DNA by phenol chloroform extraction method ranged from 18.29 to 51.64 ng/μl respectively. Whereas the total extracted DNA from potassium acetate extraction method ranged from 15.58 to 32.30 ng/μl respectively. The presence of a 535 base pair (bp) product was considered positive (Figs. 1 and 2). The lowest detection limit of PCR has been determined as 106-108 Candida cells for our spiked samples.ConclusionIn our study, till date, the extracted quantity of DNA was found to be higher by phenol-chloroform method as compared to the potassium acetate method. Greater numbers and processing are required to obtain conclusive data.

Performance Comparison of Dymind DH-76 and Sysmex Xn-1000 Automated Hematology Analyzers

An automatic hematology analyzer is an essential instrument for the modern laboratory. A new hematology analyzer must undergo comparability testing with a currently used hematology analyzer. This study aimed to compare the performance of the Dymind DH-76 and Sysmex XN-1000 hematology analyzers. This study involved 96 EDTA blood samples from patients aged 18 to 85 years old at a hospital in Jakarta. A complete blood count was performed on each blood sample using two above-mentioned analyzers Dymind DH-76 and Sysmex XN-1000. The results from both instruments were compared by using the Pearson correlation test and the Passing-Bablok regression analysis to determine the agreement in performance between the two instruments. Between the Dymind DH-76 and Sysmex XN-1000, there was a high degree of agreement and correlation concerning the investigated parameters with r &gt; 0.900 and p&lt;0.001 for the parameters RBC, HGB, WBC, HCT, MCV, MCH, and neutrophil, monocyte, and eosinophil counts. The MCHC parameter had the values of r=0.797; p=0.0001, while the lymphocytes parameter had r=0.734 and p=0.0001. Only the basophils parameter showed a different result between the two aforementioned analyzers, with r=-0.179 and p=0.102. The majority of complete blood count parameters showed an excellent correlation and a high degree of agreement between the two instruments. The Dymind DH-76 hematology analyzer meets international standards (National Committee for Clinical Laboratory Standards/NCCLS) and can be used for hematological assay in the laboratory.

Hemogram Findings in Cats from an Area Endemic for Leishmania infantum and Feline Immunodeficiency Virus Infections.

Simple SummarySome cats positive for Leishmania infantum (Li) show clinical signs and clinicopathological changes, including hemogram abnormalities. However, co-infections or co-morbidities are often detected in cats with leishmaniosis, and they can have a role in the clinical abnormalities observed. In feline Leishmania infantum infections and in clinical cases of feline leishmaniosis, a significant association with feline immunodeficiency virus (FIV) has been detected, but the role of this co-infection is controversial. The aims of this study were to retrospectively evaluate hemogram changes in cats from areas endemic for Leishmania infantum and feline immunodeficiency virus infections (Sicily and Calabria regions, Southern Italy) and to analyze the role of both pathogens in the abnormalities detected. A retrospective cross-sectional study was carried out on 496 cats. Hematological changes in cats positive for Leishmania infantum were found, particularly abnormalities compatible with chronic inflammation including moderate anemia, monocytosis, and morphologically activated monocytes. Moreover, some abnormalities, such as thrombocytosis, seem to arise when cats are co-infected with FIV. Feline leishmaniosis should be considered when some hemogram abnormalities compatible with chronic inflammation are detected.In feline Leishmania infantum (Li) infection and in clinical cases of feline leishmaniosis, co-infection with feline immunodeficiency virus (FIV) has been reported. However, the role of the retroviral co-infection in the impairment of feline clinical health is still controversial. The aim of this study was to evaluate hemogram changes in cats from regions endemic for both Li and FIV infection. Four hundred and ninety-six cats tested for Li (EDTA blood polymerase chain reaction and immunofluorescence antibody test) and for FIV infection (enzyme-linked immune assay) were retrospectively evaluated. Hemogram results including blood smear morphological evaluation were statistically compared considering four infection patterns: Li+FIV+, Li+FIV−, Li−FIV+, and Li−FIV−. Significantly lower values of erythrocytes (Li+FIV−: p = 0.0248; Li−FIV+: p = 0.0392) and hemoglobin (Li+FIV: p = 0.0086; Li−FIV+: p = 0.0249) were found in both infections when compared to Li−FIV− cats, and severity of anemia was more frequently moderate in Li-positive cats (p = 0.0206) and severe in FIV infection (p = 0.024). Li infection was associated with monocytosis (p = 0.0013) and morphologically activated monocytes (p = 0.0209). Moreover, FIV infection was associated with the presence of inflammatory leukogram (p = 0.023), and an association between thrombocytosis and the co-infection was found (p = 0.0347). Li infection in cats induces hematological changes compatible with chronic inflammation, some of which are due to co-infection with FIV.

Single-exon approach to non-invasive fetal RHD screening in early pregnancy: An update after 10 years' experience.

Anti-D prophylaxis, administered to RhD-negative women, has significantly reduced the incidence of RhD immunization. Non-invasive fetal RHD screening has been used in Stockholm for more than 10 years to identify women who will benefit from prophylaxis. The method is based on a single-exon approach and is used in early pregnancy. The aim of this study was to update the performance of the method. The single exon assay from Devyser AB is a multiplex kit detecting both exon 4 of the RHD gene and the housekeeping gene GAPDH. Cell-free DNA was extracted from 1 ml of plasma from EDTA blood taken during early pregnancy, weeks 10-12. The genetic RHD results were compared with serological typing of newborns for a determination of sensitivity and specificity. In total, 4337 pregnancies were included in the study; 44 samples (1%) were inconclusive either due to maternal RHD gene variants (n= 34) or technical reasons (n= 10). Of the remaining 4293 pregnancies, a total number of nine discrepant results were found. False positive results (n= 7) were mainly (n= 4) due to RHD gene variants in the child. False-negative results were found in two cases, of which one was caused by a technical error. None of the false-negative cases was due to RHD gene variants. Overall, the sensitivity of the method was 99.93% and specificity 99.56%. The single-exon assay used in this study is correlated with high sensitivity and specificity.

Evaluation of blood thiamine concentration in hospitalized dogs with and without critical illness.

To evaluate blood thiamine concentration in healthy and critically ill hospitalized dogs over 72hours of hospitalization and to identify any association of thiamine concentration with patient morbidity and mortality. Prospective, observational, clinical study. University veterinary teaching hospital. Thirty-one hospitalized healthy dogs and 37 dogs with critical illness. Dogs with critical illness had nonseptic (n=24) or septic (n=13) etiologies. None. Whole blood EDTA samples were collected within 24hours of admission and 72hours later. Samples were stored at -80°C and batch analyzed. Total thiamine (tB1) and thiamine diphosphate (TDP) concentrations were measured using high-performance liquid chromatography, and free thiamine concentration was determined using liquid chromatography with tandem mass spectrometry. Collected patient data included CBC, biochemistry profile, plasma lactate concentration, shock index, Acute Patient Physiologic and Laboratory Evaluation Score (APPLEfast ) score, comorbid conditions, and patient outcome. Approximately 92% of tB1 present in whole blood is measured as TDP. TDP concentration was strongly correlated with tB1 (R=0.97, P<0.0001). Thiamine concentration, represented by TDP, was not different at admission or at 72hours in all dogs. TDP concentration was lower in septic dogs requiring surgery at admission (P=0.044) and 72hours later (P=0.008), compared to dogs not requiring surgery. TDP concentration was not different between dogs with and without stable chronic diseases at any time point. Older dogs had lower TDP concentration. Positive correlations were seen between TDP and body weight, APPLEfast score, and WBC count. Critically ill dogs with lower admission plasma lactate concentration were more likely to have an increase in thiamine over time (P=0.0142). Differences in thiamine concentration were not identified in hospitalized healthy or critically ill dogs. Further investigation into the clinical relevance of thiamine deficiency is indicated in septic dogs undergoing surgery.

TOTAL AND DIFFERENTIAL LEUKOCYTE COUNT IN HEALTHY NEWBORNS

Objectives: The objectives of the study were to establish normal reference values of total and differential leukocyte count (DLC) in Indian neonatal population. Methods: The study was conducted on 200 healthy newborns admitted to neonatology section of Department of Paediatricsa. Maternal and Neonatal data was recorded on a pretested pro forma. Thin-layer chromatography (TLC) was done by manual method (hemocytometer method) using improved Neubauer Chamber. For calculating DLC smear was made from the EDTA blood and was stained by Leishman stain (Romanowsky group). DLC was done in body and tail part of the stained smears by following Z pattern. Statistical Analysis: Descriptive statistical analysis was carried out in the present study. Mean, standard deviation, t-test, and analysis of variance tests were used to test the significance of the differences among sample means. Results: Mean TLC at various period of gestations were 4500/cumm at 31 weeks, 7627±2320/cumm at 33 weeks, 8325±1943/cumm at 34 weeks, 8141±1538/cumm at 35 weeks, 8455±904/cumm at 36 weeks, 8840±658/cumm at37 weeks, 9380±896/cumm at 38 weeks, 10143±1369/cumm at 39 weeks, 9155±2444/cumm at 40 weeks, and 11750±353/cumm at 41 weeks. Conclusion: TLC showed increase with gestation except at 35 weeks and 40 weeks. Neutrophils shows increase with gestation except at 35 weeks and 40 weeks lymphocytes shows increase with gestation except at 34 weeks, 39 weeks, and 40 weeks.

Megakaryocyte cytoplasmic fragments and ragocytes in the peripheral blood of a domestic cat infected with Anaplasma

In this case report, the author reports three strong and highly unusual changes in the peripheral blood of a domestic cat infected with Anaplasma. A freshly made blood smear from the cat Anaplasma had a shower of about 100 variably large to huge cytoplasmic fragments of megakaryocyte cytoplasm along the feathered edge. These had budding of platelets at their margins. Neutrophils with red cytoplasmic inclusions resembling ragocytes and neutrophils with ingested round inclusions of nuclear material were seen in a 1-day-old EDTA blood sample but not the freshly made blood smear. The cells with nuclear material resembled LE cells or alternatively Tart cells.