Automated Capillary Electrophoresis Research Articles

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis.

Neuraminidase inhibitors modulate infections that involve sialic acids, making quantitative analyses of this inhibitory effect important for selecting and designing potential therapeutics. An automated nanogel capillary electrophoresis system is developed that integrates a 5 nL enzyme inhibition reaction in line with a 5 min separation-based assay of the enzymatic product to quantify inhibition as the half maximal inhibitory concentration (IC50) and inhibitor constant (Ki). A neuraminidase enzyme from Clostridium perfringens is non-covalently immobilized in a thermally tunable nanogel positioned in the thermally controlled region of the capillary by increasing the capillary temperature to 37 °C. Aqueous inhibitor solutions are loaded into the capillary during the nanogel patterning step to surround the enzyme zone. The capillary electrophoresis separation provides a means to distinguish the de-sialylated product, enabling the use of sialyllactose which contains the trisaccharide motif observed on serine/threonine-linked (O-linked) glycans. A universal nanogel patterning scheme is developed that does not require pre-mixing of enzymes with inhibitors when an automated capillary electrophoresis instrument is used, thus reducing the consumption of enzymes and enabling adaption of the method to different inhibitors. The universal approach is successfully applied to two classical neuraminidase inhibitors with different electrophoretic mobilities. The IC50 and Ki values obtained for N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA) are 13 ± 3 and 5.0 ± 0.9 μM, respectively, and 28 ± 3 and 11 ± 1 μM, respectively, for Siastatin B. These values agree with literature reports and reflect the weaker inhibition anticipated for Siastatin B in comparison to DANA.

High-Resolution Epitope Mapping and Affinity Binding Analysis Comparing a New Anti-Human LAG3 Rabbit Antibody Clone to the Commonly Used Mouse 17B4 Clone.

Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applications. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare the binding characteristics of a new anti-LAG3 rabbit antibody clone, SP464, with the thirty-year old and extensively used anti-LAG3 mouse 17B4 clone. The rabbit SP464 clone exhibited between 20× to 30× greater binding to LAG3 than did the mouse 17B4 clone. Using these tools, we precisely mapped the relative locations of the epitopes of these two antibodies. The SP464 and 17B4 minimal epitopes were localized to separate, but overlapping, sub-fragments within the amino-terminal fifteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Application of this approach for quantifying the effects of alanine substitutions along the minimal SP464 epitope identified two amino acids essential for binding and four amino acids that likely contribute towards binding. Together, ACE, SPR, and IHC constitute a powerful orthologous approach for comparing antibody-binding characteristics and for fine mapping of linear epitopes within short immunogens. Our results indicate that the rabbit clone SP464 may be useful for assessing LAG3 expression.

Intentional and unintentional nitrite intoxications: A novel diagnostic strategy based on the direct ion determination by capillary electrophoresis

Discussion of analytical and interpretative aspects of nitrite intoxication in 12 forensic cases. In recent years, a significant increase of reports about suicidal cases due to intentional sodium nitrite intake has been described. Also, accidental poisonings can occur by exposure to contaminated food due to the use of sodium nitrite as a coloring agent or preservative in food, as well as an antimicrobial agent in meat and fish. The human toxicity of nitrite is mainly related to its capability to oxidize the Fe2+ of the hemoglobin to Fe3+, thus converting hemoglobin to methemoglobin, which is unable to bind and transport oxygen. The determination of nitrite in blood is challenging since it is rapidly oxidized to nitrate by hemoglobin. Thus, the diagnosis of nitrite intoxication is generally based on methemoglobin quantification. However, methemoglobin is only an indirect non-specific evidence of nitrite intoxication, as increases of this form of oxidized hemoglobin can be observed in different genetic defects and acquired conditions, including treatment with drugs. Consequently, a specific diagnosis of nitrite intoxication can be achieved only by the direct identification and measurement of the toxic compound or its derivatives. Recently, our research group validated a capillary electrophoresis (CE) method specifically devoted to the direct determination of nitrite/nitrate in biological fluids including postmortem blood (Taus F, et al. Forensic Sci Int 2021;325:110855). The aim of the present study was the discussion of analytical and interpretative aspects of 12 forensic cases of intentional and unintentional nitrite intoxication ( n = 4 fatalities; n = 3 suicidal attempts and n = 5 food intoxications). An automated capillary electrophoresis system Capel-205 (Lumex Instruments, Vancouver, Canada) equipped with a spectrophotometric detector was used. The analyses were carried out on a bare fused-silica capillary (75 mm inner diameter) using 100 mM tetraborate (pH 9.24) as background electrolyte and applying a voltage of - 15 kV. The detection was obtained by direct UV absorption recorded at 214 nm wavelength. All the biological samples (blood, urine and vitreous humour) were, after centrifugation, diluted 1:20 with the internal standard solution (Bromide: 250 μmol/L) prior to injection. Nitrate concentrations in blood ranged from 1400 to 6500 μmol/L in fatalities and from 910 and 2590 μmol/L in the suicide attempts. In food intoxication cases nitrate was always below 400 μmol/L. Nitrite was found only in not quantifiable traces due to its rapid conversion to nitrate. No interferences were observed from sample components, including proteins and ions. The proposed CE method allows for the direct and specific determination of nitrite/nitrate in different biological samples (serum/plasma or urine) as well as in postmortem specimens (cadaveric blood and vitreous humor). These characteristics, in addition to the speed and cheapness of the analysis, make this novel approach particularly suitable for the reliable diagnosis of nitrite intoxication in both clinical and forensic cases.

β-globin variants present in Western Sudan

Objective: A variety of observations of the hemoglobin D (Hgb D) phenotype has occurred in association with family studies of patients with sickle cell disease. Very little is known about the occurrence and prevalence of the Hgb D variant and its impact on blood profiles among Sudanese. This study was aimed at determining the percentage of Hb D in North Darfur State, West Sudan, whose population has been shown to have hemoglobin-S (Hb S) disease. Methods: From December 2017 to August 2018, this descriptive community-based investigation was conducted. Six hundred and sixty-six (666) people were randomly selected to participate in this study. With each participant's there was verbal consent. A questionnaire was designed to collect personal details. 5 mL of venous blood was gathered in EDTA containers. The Hb D variant was checked using Sebia Minicap Automated Capillary Electrophoresis System- USA and frequency was calculated using version 21.0 of the software package for social science (SPSS). Result: The prevalence of Hb variants was as follows: AD=0.6%, SS=2%, AS=10.5 %, and AA=86.9%. Conclusion: The prevalence of Hb D variant was 4 (0.6 %) in 666 participants from four western Sudanese tribes, beside reported of Hb SS and Hb AS.

The Unfolded Protein Response As a Predictor of Chemosensitivity in Pediatric Acute Myeloid Leukemia

Background: Survival in newly diagnosed pediatric patients with acute myeloid leukemia (AML) is approximately 65% with a much lower survival in patients with relapsed disease. Leukemia cells are exposed to proteotoxic stress due to their rapid, inefficient metabolism and accumulation of reactive oxygen species. This proteotoxic stress results in the production of misfolded proteins, which leads to activation of cell stress pathways, such as the unfolded protein response (UPR). We hypothesized that UPR induction correlates with a better response to chemotherapy.Methods: Peripheral blood samples from pediatric patients with AML were collected prior to initiation of induction chemotherapy, then at ~6 hours (h) and 24 h post initiation of chemotherapy. Peripheral blood mononuclear cells were frozen as pellets after depletion of non-tumor cells using magnetic bead selection (Miltenyi Biotech). The expression of UPR proteins was determined by chemiluminescence using an automated capillary electrophoresis system, ProteinSimple WesTM. The expressions of 5 UPR proteins were examined including Grp78, IRE1, total and phosphorylated (active) eIF2-α, and ATF6-α. Protein expression was normalized to actin expression and fold differences were compared between samples and within each sample at different time points.Results: Within each group of UPR proteins, there was a wide variability in the level of baseline expression, with 6 of 38 samples expressing >4 fold increase in Grp78, compared to the median level in healthy pediatric and adult controls (Figure 1). Basal IRE1 levels were also significantly higher in pediatric controls vs. adult controls. A trend towards lower overall survival (OS) and event-free survival (EFS) was noted in patients who had a low baseline Grp78 and a low baseline IRE1 (low baseline IRE1 was defined as <2 x below the median level of healthy pediatric controls). Post chemotherapy, UPR induction (defined as >2 x above baseline level) was seen in a subset of patients, most notably for ATF6α. However, post chemotherapy UPR induction was not associated with differences in either event-free survival or overall survival.Conclusion: UPR has a variable expression at baseline in pediatric AML patients, with a trend for lower overall survival in patients with a low basal Grp78 and IRE1 expression, suggesting chemoresistance in this subset of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of Clostridium tyrobutyricum Strains Using Three Different Typing Techniques

Clostridium tyrobutyricum is well known as one of the main causative agents of severe cheese spoilage. The metabolism of this anaerobic bacterium during ripening leads to textural and sensory defects in cheese and consequential loss of product value. The potential to induce cheese spoilage, however, may vary among different strains of the same species. Therefore, a better understanding of the intra-species diversity of C. tyrobutyricum may be of practical relevance for the dairy industry. In the present study, we compared the ability of three typing techniques to differentiate 95 C. tyrobutyricum strains on the subspecies level: (1) repetitive element palindromic PCR (rep-PCR) fingerprinting combined with conventional agarose gel electrophoresis, (2) hexaplex-PCR followed by an automated capillary electrophoresis and (3) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing. MALDI-TOF MS fingerprinting provided only moderate reproducibility and low discriminatory power. Both PCR-based methods were highly reproducible and discriminative, with hexaplex-PCR fingerprinting being slightly more discriminative than rep-PCR typing. Overall, a high intra-species diversity was observed among the tested strains, indicating that further investigations on the strain level may be of interest.

Design of an automated capillary electrophoresis platform for single-cell analysis.

Single-cell analysis of cellular contents by highly sensitive analytical instruments is known as chemical cytometry. A chemical cytometer typically samples one cell at a time, quantifies the cellular contents of interest, and then processes and reports that data. Automation adds the potential to perform this entire sequence of events with minimal intervention, increasing throughput and repeatability. In this chapter, we discuss the design considerations for an automated capillary electrophoresis-based instrument for assay of enzymatic activity within single cells. We describe the key requirements of the microscope base and capillary electrophoresis platforms. We also provide detailed protocols and schematic designs of our cell isolation, lysis, sampling, and detection strategies. Additionally, we describe our signal processing and instrument automation workflows. The described automated system has demonstrated single-cell throughput at rates above 100cells/h and analyte limits of detection as low as 10-20mol.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

Pheno-morphological variation, genetic diversity and population structure of Tunisian Echinus Medic (Medicago ciliaris L.).

Medicago ciliaris L., considered as a valuable genetic resource, is a good candidate for the improvement of marginal or degraded lands with low fertility or high salinity. In this study, the pheno-morphological and genetic diversity were investigated in 14 Tunisian populations of M. ciliaris for the first time. Fourteen morphological traits showed significant differentiation between populations and high levels of diversity. Two amplified fragment length polymorphism primer combinations (E-AGC/M-CAA; E-AAG/M-CTG) were analyzed using an automated capillary electrophoresis system. A total of 528 loci were generated, of which 54% were polymorphic. Allelic polymorphism ranged from 0.02 to 0.5. Significant variation between populations was found for gene diversity, mean number of alleles per locus and Shannon index for which mean values were 0.17, 0.26, and 1.57, respectively. Analysis of molecular variance revealed a high rate of genetic variation within populations. Principal component analysis and genotypic clustering discriminated M. ciliaris populations according to their geographical origin. M. ciliaris clustered into three main groups. The first group was associated with high inland and cold areas, the second was defined by low areas with mild winters while the third described low coastal areas. Similarity of morphological and molecular results indicated that either markers could be used for the study of genetic diversity in this species.

Single-cell sphingosine kinase activity measurements in primary leukemia.

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39 ± 26% per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16 ± 25%, 11 ± 26%, and 13 ± 23% in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87% of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99% of the cell population, 19 ± 36% of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12 ± 0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer.

EGFR mutation detection by microfluidic technology: a validation study

Advanced non-small cell lung cancer samples are tested for epidermal growth factor receptor (EGFR) gene mutations. Their detection by direct sequencing is time-consuming. Conversely, the length analysis of fluorescently labelled...

Cord Blood Analysis for Rapid Prenatal Confirmation of Hb Bart’s Disease Using the Sebia Capillary Electrophoresis System

Prenatal diagnosis of severe α-thalassemia (α-thal) diseases is usually performed by DNA analysis. To establish a simple and rapid method, we evaluated the reliability of cord blood hemoglobin (Hb) analysis using an automated capillary electrophoresis (CE) system. Our results demonstrated that analysis of fetal Hb using the Sebia CapillaryS 2 is an effective, accurate and simple alternative for prenatal diagnosis of Hb Bart’s (γ4) disease.

Multilocus microsatellite typing for Rhizopus oryzae

Rhizopus oryzae is the most frequent causative agent of zygomycosis. Although zygomycosis causes considerable morbidity and mortality in immunocompromised patients, the epidemiology of the disease is not well studied and no standard molecular typing method has been described for any of the causative agents. Here we describe a multilocus microsatellite typing (MLMT) method for R. oryzae. R. oryzae genome sequences were downloaded from the Fungal Genome Initiative database (Broad Institute). The intergenic regions and ORFs of approximately 5.7 Mb were screened for repeat regions with the help of the online repeat search tool Repeat Masker. Of the 30 microsatellite loci identified, 3 microsatellites [RO3, (CCT)(n); RO4, (TA)(n); and RO8, (GAA)(GGA)(n)] were selected after validation of the ability to amplify them and their size variation in 8 randomly selected clinical isolates of R. oryzae. Nucleotide sequence analysis of these loci demonstrated polymorphism in the microsatellite repeat number. The capabilities of these microsatellite loci were assessed for strain differentiation on 30 clinical isolates, based on fragment size determination in an automated capillary electrophoresis using fluorescent labelled primers. These three polymorphic microsatellite loci were found to have good discriminatory power (D) (RO3, D=0.846; RO4, D=0.747; RO8, D=0.742; with a combined D=0.986) and stability for seven subcultures. It was also confirmed that the MLMT method may be applied to both R. oryzae and Rhizopus delemar (a proposed new species), although MLMT analysis could not differentiate them into two clusters. The MLMT system, described here for what is believed to be the first time for a zygomycotic fungus, holds promise as a powerful tool for the strain typing of R. oryzae.

An automated capillary electrophoresis system for high‐speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.

Cover Picture: Electrophoresis 19'2010

AbstractIssue no. 19 is a special issue on “CE and CEC Innovations” comprising 1 Fast Track manuscript and 20 manuscripts distributed over 5 separate parts. Part I has 5 research articles on novel trends in CEC. Part II has 4 research articles dealing with innovative methodologies. Part III reports a variety of interactive CE systems described in 5 research articles. Proteins and biomarkers are treated in 3 research articles making up part IV. The last 3 articles in this issue (Part V) are on detection approaches. The FAST TRACK article describes “An automated capillary electrophoresis system for high speed separation of DNA fragments based on a short capillary”.

A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis

Population screening has been proposed for Fragile X syndrome to identify premutation carrier females and affected newborns. We developed a PCR-based assay capable of quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity and specificity. This assay combines a triplet repeat primed PCR with high-throughput automated capillary electrophoresis. We evaluated assay performance using archived samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat expansions. Two hundred five previously genotyped samples were tested with the new assay. Data were analyzed for the presence of a trinucleotide "ladder" extending beyond 55 repeats, which was set as a cut-off to identify expanded FMR1 alleles. We identified expanded FMR1 alleles in 132 samples (59 premutation, 71 full mutation, 2 mosaics) and normal FMR1 alleles in 73 samples. We found 100% concordance with previous results from PCR and Southern blot analyses. In addition, we show feasibility of using this assay with DNA extracted from dried-blood spots. Using a single PCR combined with high-throughput fragment analysis on the automated capillary electrophoresis instrument, we developed a rapid and reproducible PCR-based laboratory assay that meets many of the requirements for a first-tier test for population screening.

Potential of the Sebia Capillarys® neonat fast automated system for neonatal screening of sickle cell disease

Most screening programs for sickle cell disease (SCD) utilize isoelectric focusing (IEF) or high performance liquid chromatography (HPLC) to detect haemoglobin (Hb) variants. The first method is not automated and becomes too tedious when many samples have to be investigated. The aim of this work is to explore the capacity of an automated capillary electrophoresis (CE) system, with full traceability, as a tool for newborn screening of SCD. The Capillarys neonat fast automated system has been developed by Sebia for newborn screening. We performed separate studies using different types of samples to evaluate the utility of the Capillarys for (i) separating Hb S and other variants, and (ii) for performing the routine activity of our laboratory for 20 working days. A throughput of 48 samples per hour with a loading capacity of 192 samples was achieved. Migration times of the major Hb variants were distinct. There were few variants showing similar migration times to Hb S and Hb C and thalassaemia could be detected. In addition, late screening, screening of premature or transfused babies and screening performed using poor quality Guthrie's cards did not interfere with reporting of accurate phenotypes. Sebia Capillarys neonat fast automated system is a reliable tool for haemoglobinopathy neonatal screening.

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.

Mitochondrial DNA as a Cancer Biomarker

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Cosegregation of a Factor VIII Microsatellite Marker with Mild Hemophilia A in Golden Retriever Dogs

Mild hemophilia A (factor VIII deficiency) was diagnosed in Golden Retrievers and pedigree studies were undertaken to test the cosegregation of an intragenic factor VIII marker with the disease phenotype. The study population consisted of 30 client-owned dogs (22 males and 8 females). Hemophilic males (n = 12) typically demonstrated prolonged bleeding after trauma or surgery rather than spontaneous hemorrhagic events. The affected males had a proportionate reduction in factor VIII coagulant activity (mean FVIII:C = 4%) and factor VIII protein concentration (mean FVIII:Ag = 3%). Twenty-five dogs (10 affected males, 8 clear males, 2 obligate carrier dams, and 5 suspect carrier daughters) were genotyped for a factor VIII microsatellite marker, with allele size assigned by an automated capillary electrophoresis system. Five distinct marker alleles were present in the study pedigree and a 300-base pair allele was found to segregate with the hemophilia A phenotype. The inheritance of the hemophilia-associated allele defined carrier status for 5 suspect daughters of obligate carrier dams. The limitations inherent to linkage analyses (ie, lack of access to key family members and homozygosity at the marker locus) did not preclude carrier detection in this pedigree. We conclude that genotype analysis for the intragenic factor VIII marker can aid in control of canine hemophilia A through enhanced carrier detection.