scholarly journals Conventional Rapid Latex Agglutination in Estimation of von Willebrand Factor: Method Revisited and Potential Clinical Applications

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Marianor Mahat ◽  
Wan Zaidah Abdullah ◽  
Che Maraina Che Hussin
Keyword(s):  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Rapid Test ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Latex Agglutination Test ◽  
Test Kit ◽  
Agglutination Method ◽  
Von Willebrand ◽  
Willebrand Factor

Measurement of von Willebrand factor antigen (VWF : Ag) levels is usually performed in a specialised laboratory which limits its application in routine clinical practice. So far, no commercial rapid test kit is available for VWF : Ag estimation. This paper discusses the technical aspect of latex agglutination method which was established to suit the purpose of estimating von Willebrand factor (VWF) levels in the plasma sample. The latex agglutination test can be performed qualitatively and semiquantitatively. Reproducibility, stability, linearity, limit of detection, interference, and method comparison studies were conducted to evaluate the performance of this test. Semiquantitative latex agglutination test was strongly correlated with the reference immunoturbidimetric assay (Spearman’s rho = 0.946,P<0.001,n=132). A substantial agreement (κ=0.77) was found between qualitative latex agglutination test and the reference assay. Using the scoring system for the rapid latex test, no agglutination is with 0% VWF : Ag (control negative), 1+ reaction is equivalent to <20% VWF : Ag, and 4+ reaction indicates >150% VWF : Ag (when comparing with immunoturbidimetric assay). The findings from evaluation studies suggest that latex agglutination method is suitable to be used as a rapid test kit for the estimation of VWF : Ag levels in various clinical conditions associated with high levels and low levels of VWF : Ag.

ADAMTS13 Content of Plasma-Derived Factor VIII-Von Willebrand Factor Concentrates

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3383-3383
Author(s):  
Flora Peyvandi ◽  
Pier Mannuccio Mannucci ◽  
Carla Valsecchi ◽  
Silvia Pontiggia ◽  
Jonathan Bernstein ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Analysis Data ◽  
Current Treatment ◽  
Adamts13 Activity ◽  
Pilot Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3383 Introduction: Anecdotal and published reports on the use of specific plasma-derived Factor VIII (FVIII)-von Willebrand Factor (vWF) concentrates in the treatment of congenital thrombotic thrombocytopenic purpura (TTP) led to the determination of the functional and antigenic content of ADAMTS13 in a number such concentrates commercially available in the US and/or the EU. Deficiency or inhibition of ADAMTS13 is the putative cause of TTP. TTP is a rare disorder causing microvascular thrombosis resulting in low platelet counts. It affects 1 –5 patients per 1,000,000 population. The current treatment of this disorder consists of regular infusions of fresh frozen plasma (FFP) for inherited TTP and exchange plasmapheresis for the acquired version of the disorder. The possibility of utilizing FVIII-vWF concentrates that incorporate robust virucidal treatments in their manufacturing and the possible utilization of lower infusion volumes prompted the evaluation of these concentrates for ADAMTS13 content. Methods: For this analysis, we obtained 5 lots of Koate®-DVI, 2 lots of Human Bioplasma, and 1 lot of each of the following concentrates: Humate® P, Wilate®, Alphanate®, Emoclot® and Fractogel.® Following reconstitution of these concentrates in 10 mL of water for injection, we determined the concentration of ADAMTS13 antigen by an ELISA method previously described by Feys et al. (J Thromb Haemost. 2006; 4: 955–62) with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239). The lower limit of detection was 1% while the lower value of the normal range was 45%. The ADAMTS13 activity was measured using collagen binding (CBA) and FRET assays with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239); in both assays, serially diluted normal human plasma (NHP) and plasma samples were diluted 1:10 in assay buffer and incubated 1:1 with vWF substrate in a final volume of 100 μl. In addition to the determination of ADAMTS13, we evaluated the amount of vWF antigen (vWF:Ag) using a commercial kit (Instrumentation Laboratory, IL US, Bedford, Massachusetts, USA) in these concentrates and the multimeric composition of this protein. Utilizing densitometry, we determined the percentage of ultra-large vWF multimers in each of these concentrate. Results: The analysis showed that among the tested concentrates, Koate®-DVI had the highest ratio of ADAMTS13/FVIII. This concentrated presented ratios of 9.1 ± 0.7% (Mean ± SD) and 8.4 ± 1.1% (Mean ± SD) per IU FVIII for ADAMTS13 activity and antigen respectively. The remainder of the concentrates contained only nominal amounts of ADAMTS13; only Alphanate® reached a ratio of 0.23% ADAMTS13 activity/IU FVIII. These data are in agreement with previous reports (Qorraj et al. Blood 2001, 116: 3677). A summary analysis data are shown in Table 1. Koate®-DVI displayed a ratio of vWF:Ag/FVIII of approximately 2.81, very similar to the ratio of 2.77 included in the single lot of Humate®-P. The ratio of ultra-large multimers to total multimers in Humate® P was higher, at 0.66 vs. 0.39 for Koate-DVI. However, the lower content of ultra-large multimers in Koate®-DVI may be advantageous in its possible utilization in the treatment of TTP. Conclusion: On the basis of these results, Koate-DVI may have a role to play in the management of congenital and, possibly, idiopathic TTP, especially on the basis of observations that FVIII accelerates the breakdown of vWF (Cao et al. PNAS 2008, 105: 7416–7421). Additional characterization, testing in pertinent animal models and pilot studies in humans would be required to determine the extent to which this concentrate can assist in the treatment of TTP. Disclosures: Guazzini: Kedrion S.p.A: Consultancy. Retzios:Kedrion Biopharma, Inc.: Consultancy.

scholarly journals DEVELOPMENT AND IMPROVEMENT OF ASF SEROLOGICAL DIAGNOSTIC TECHNIQUES

2019 ◽  
pp. 30-34
Author(s):  
D. V. Sharypova ◽  
O. V. Kapustina ◽  
I. Yu. Zhukov ◽  
N. N. Vlasova ◽  
A. S. Igolkin
Keyword(s):  
Cell Line ◽  
Rapid Test ◽  
Laboratory Diagnosis ◽  
Diagnostic Techniques ◽  
Test Methods ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Latex Agglutination Test ◽  
Rapid Tests ◽  
Fixed Cell

Due to the lack of effective tools of ASF specific prevention it is evident that early diagnosis is one of the most important and resultative ways of the disease control. However, contemporary diagnosis is a complex component of any effective surveillance system. Latest scientific achievements facilitated not only highly specific and sensitive but also rapid methods of laboratory diagnosis. Nevertheless, further development, improvement and expansion of ASF diagnosis techniques including rapid tests is a topical task of a great concern. The research is devoted to development of rapid test methods for rapid detection of antibodies to ASFV in blood sera of infected animals as well as to analysis of their use effectiveness. The following methods were suggested: immunoperoxidase monolayer assay using fixed cell line (ASFV permissive CV-1 cell-line infected with the virus strain ASF/ARRIAH/CV-1) and latex agglutination test using ASFV p30 recombinant protein. The performed research demonstrated the effectiveness of the applied techniques for ASF serological diagnosis. Latex agglutination test and immunoperoxidase monolayer assay give rapid and high quality test results (within 1–2 hours). The advantage of the specified methods as compared to ELISA is their simplicity and the possibility of use in conditions of limited technical support.  

Abstract P102: Elevated Von Willebrand Factor in Patients Presenting With Large-Vessel Occlusion Stroke as First Symptom of COVID-19 Mirrors Levels in Patients With COVID-19 Requiring ICU-Level Care

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Lyra B Olson ◽  
Ibtehaj A Naqvi ◽  
LINGYE CHEN ◽  
Loretta G Que ◽  
BRYAN D KRAFT ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Large Vessel ◽  
Minimal Risk ◽  
Healthy Controls ◽  
Stroke Patients ◽  
Vessel Occlusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Large Vessel Stroke

Introduction: COVID-19 is a coagulopathic disease marked by elevated d-dimers, fibrinogen, and von Willebrand factor (vWF) levels accompanying arterial and venous thrombosis. While the majority of thrombotic events associated with COVID-19 occur in hospitalized patients, a subset of patients with minimal risk factors for CVA but with positive SARS-CoV-2 testing present with stroke as presumed first manifestation of infection. It is unclear if the pro-coagulant milieu present in patients requiring hospitalization for the respiratory complications of COVID-19 is the same as that of patients who present with stroke as first symptom of disease. Methods: Following emergent revascularization, clinical vWF levels were measured in patients presenting with stroke who tested positive for COVID-19. In parallel, plasma vWF levels from 28 patients with COVID-19 requiring ICU-level care and 8 healthy volunteers were measured via ELISA. Results: Three otherwise healthy patients between the ages of 45-55 years with positive test for SARS-CoV-2 presented with large-vessel stroke. By comparison, the average age of non-COVID stroke patients was 66 years. The consistency of the clots extracted through the aspirating catheter was dark, gelatinous throughout, without evidence of calcification, and distal thrombosis was noted minutes after revascularization. The vWF level for one patient was 345%, while the other two patients had vWF levels >400% of normal, exceeding the upper limit of detection of clinical assays. In the ICU cohort, 12 of 28 had thrombotic events during hospitalization. vWF levels were elevated by a mean of 800% over healthy controls with a range of 230-1670%. Conclusions: vWF levels were markedly elevated in both ICU patients and stroke patients with COVID-19 with an overlapping range of elevation over healthy controls. This suggests that widespread endothelial inflammation accompanies infection with SARS-CoV-2 even in the absence of respiratory symptoms.

A Rapid Sensitive Assay for Measuring ADAMTS13 Activity in Plasma Utilizing a Novel Recombinant Alexa488-vWF86 (Q1599\P1611C) FRET Substrate.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4040-4040
Author(s):  
Enriqueta R. Guinto ◽  
Nicholas Grafos ◽  
John Owen ◽  
Heather L. Lawson ◽  
Robert S. Greenfield
Keyword(s):  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Resonance Energy ◽  
Cysteine Residues ◽  
Activity Levels ◽  
Analytical Sensitivity ◽  
Adamts13 Activity ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract ADAMTS13 (von Willebrand Factor cleaving protease) is a zinc metalloprotease that cleaves the Tyr(1605)/Met(1606) bond within the A2 domain of von Willebrand Factor (vWF). Low levels of ADAMTS13 activity (&lt;5%) are strongly associated with the life threatening hemostatic condition thrombotic thrombocytopenic purpura (TTP). A rapid clinical assay for ADAMTS13 is necessary to provide critical information for the early diagnosis of TTP and to guide and monitor the treatment of TTP. Present methods for measuring ADAMTS13 activity (e.g. collagen binding, vWF multimer analysis and ELISA) are cumbersome, take a long time to complete and lack sensitivity. A fluorescence resonance energy transfer (FRET) assay for ADAMTS13 has been described, which uses the costly and difficult to synthesize truncated A2 domain VWF73aa peptide as substrate (1). However, the lower limit of detection of ADAMTS13 assays using VWF73 FRET substrate is only about 5–10% of normal ADAMTS13 activity levels (1,2). Recently, Zhang et al (3) reported a novel recombinant VWF A2 FRET substrate of 86 amino acids in which cysteine residues were substituted for residues Q1599 and P1611, respectively. The cysteine residues were chemically labeled with fluorescein maleimide to produce a FRET substrate whereby the two fluorescein dyes flanking the ADAMTS13 Tyr/Met cleavage site interact sufficiently such that auto-quenching of fluorescence occurs. In this study, we developed a rapid more sensitive fluorescent assay for ADAMTS13 using a novel recombinant VWF86(Q1599C/P1611C) FRET substrate. VWF86(Q1599C/P1611C) peptide was chemically coupled via the cysteine residues to the brighter, more photostable Alexa488 fluorochrome. Assay conditions were optimized to obtain higher analytical sensitivity &lt; 5% normal ADAMTS13 activity (0.5 ng/ml) and shorter assay times (20–30 minutes). ADAMTS13 activity in 50 normal individuals as well as subjects with TTP, thrombosis and lupus anticoagulant will be presented. The rapid FRET assay using the novel recombinant Alexa488-vWF86 (Q1599\P1611C) substrate should prove clinically useful for quantitation of ADAMTS13 activity and the rapid diagnosis of TTP.

VON WILLEBRAND FACTOR mRNA IS SEVERELY REDUCED IN PIGS WITH HOMOZYGOUS VON WILLEBRAND DISEASE

1987 ◽  
Author(s):  
Q Y Wu ◽  
B R Bahnak ◽  
L Coulombel ◽  
J P Caen ◽  
G Pietu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Cdna Probe ◽  
Von Willebrand Disease ◽  
Northern Hybridization ◽  
Transcriptional Level ◽  
Total Rna ◽  
Von Willebrand ◽  
Willebrand Factor

Porcine von Willebrand disease (vWD), an autosomal recessive disorder, is similar to some of the severe forms of vWD in humans and is characterized by a prolonged bleeding time and very low or undetectable amounts of von Willebrand factor (vWF) antigen and activity in plasma, platelets and endothelial cells. The molecular events that control the lack of expression of vWF in the vWD pigs is not known and could be at the transcriptional or post-transcriptional level. Lungs from normal and two homozygous vWD pigs were extracted immediately after harvesting of the animals and placed on dry ice. Tissues were homogenized in 6 M guanidinium thiocyanate and RNA isolated by centrifugation through cesium chloride. Total RNA was analyzed by Northern hybridization including dénaturation in glyoxal, electrophoresis in 1.0 % agarose-2.2 M formaldehyde gels and transfer onto nitrocellulose. Messenger RNA was detected with a nick-translated human vWF cDNA probe or a human actin control probe. The vWF probe, cloned from a human lung library, was 2,280 bp in length and spanned nucleotides 960 to 3,240 of the human cDNA. These human probes were considered valid to detect levels of porcine vWF and actin mRNA because they hybridized with restriction enzyme digested genomic DNA from normal and vWD pig leucocytes under conditions of high stringency. The size of the vWF mRNA in the normal pigs after Northern hybridization was approximately 9.0 kb, similar to that of human vWF mRNA, and was easily detectable at the lowest concentration of RNA blotted (5 ug). In contrast, vWF mRNA from vWD pigs was at the lower limit of detection even at 10 ug of total RNA blotted. Nevertheless, although at extremely low levels, vWF mRNA from vWD pigs appeared to be the same size as the normal mRNA. These results agree with observations on the relationship of vWF secreted from 24 hr. cultures of endothelial cells from the pulmonary artery of normal and vWD pigs where the vWF levels were 0.90 and 0.06 U/108 cells, respectively. Therefore, it appears that the very low expression of vWF in the vWD pigs is due to a lack of transcription of the vWF gene. At this time, however, turnover of unstable transcripts in the vWD pigs can not be ruled out.

scholarly journals Recombinant leptospiral immunoglobulin like B protein based latex agglutination test for serodiagnosis of human leptospirosis

2020 ◽  
Vol 6 (2) ◽  
pp. 229-236
Author(s):  
Yosef Deneke ◽  
Rajib Deb ◽  
SM Lutful Kabir
Keyword(s):  
Acute Infection ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
The Body ◽  
Latex Agglutination Test ◽  
City Hospital ◽  
Indian Veterinary Research Institute ◽  
Test Kit ◽  
Human Sera ◽  
Sera Samples

Humans get leptospirosis by contact with fresh water, damp soil, or vegetation contaminated by the urine of infected animals, swallowing contaminated food or water or while working in contaminated flood plains or at wet agricultural settings. The bacteria enter the body through abrasions in the skin and mucous membranes. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against leptospirosis suspected human sera. A total of 28 human sera samples were received from Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh India, which tested positive by IgM ELISA test kit were subjected to both rLigB based LAT and MAT. All the 28 sera showed seropositivity by both the tests. Icterohaemorrahigae was the predominant serovar followed by Javanica and Grippotyphosa. Six out seven sera samples received from Indian Veterinary research Institute, Human Hospital and City Hospital, Bareilly were tested positive both by rLAT and MAT. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, reliable diagnostic tool at resource poor and remote diagnostic centers with high sensitivity and specificity, under laboratory and field conditions, for the detection of leptospirosis. Asian J. Med. Biol. Res. June 2020, 6(2): 229-236

scholarly journals Evaluation of a Commercial Latex Agglutination Test Kit for Cryptococcal Antigen

1974 ◽  
Vol 27 (3) ◽  
pp. 620-621
Author(s):  
Leo Kaufman ◽  
Glenda Cowart ◽  
Sharon Blumer ◽  
Amy Stine ◽  
Ross Wood
Keyword(s):  
Latex Agglutination ◽  
Agglutination Test ◽  
Latex Agglutination Test ◽  
Cryptococcal Antigen ◽  
Test Kit

Development and Performance of the GTI ADAMTS-13 Activity Assay.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4097-4097
Author(s):  
Melissa A. Pressman ◽  
Elizabeth Wuitschick ◽  
Suzette C. Chance
Keyword(s):  
Enzyme Activity ◽  
Von Willebrand Factor ◽  
Clinical Symptoms ◽  
Limit Of Detection ◽  
Fluorescent Detection ◽  
Activity Assay ◽  
Assay Sensitivity ◽  
Extensive Evaluation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. It was recently discovered that ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor; deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of vWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. Currently there are no commercially available diagnostic assays for TTP. Several laboratories have developed “in house” assays that measure the activity of the ADAMTS-13 protease. However, these assays are cumbersome, complex, difficult to perform, and there is a long turn around time, as they require long incubations. The results are difficult to interpret and are not easily quantitated. In addition, there is no standardization of results from laboratory to laboratory, mainly because the assays use various technologies. Determining the extent to which the ADAMTS-13 enzyme activity is decreased is believed to be important in distinguishing a patient with TTP from those presenting with similar clinical symptoms. Until ADAMTS-13 assays are more quantitative, there is not a good method for studying patients with severe decreases in ADAMTS-13 levels versus those who have some intermediate but less than normal level of enzyme activity. Furthermore, the lack of standardized results makes the assays less useful in monitoring the effectiveness of treatment of the TTP patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a substrate which consists of a peptide fragment of von Willebrand Factor and subsequent fluorescent detection. An initial study using plasma (obtained from the Blood Center of Wisconsin) from 5 TTP patients demonstrated good correlation between the GTI assay and the collagen binding assay, thus demonstrating feasibility of the assay. A more extensive evaluation using 40 normal samples and 55 samples from suspected TTP patients was initiated, the results of which will be reported. Assay sensitivity/limit of detection (based on dilution studies) was 3% normal ADAMTS-13 activity. Assay precision using 3 samples (mean values of 96%, 55%, and 9% normal ADAMTS-13 activity) run in duplicate over 8 days was evaluated. Assay imprecision was less than 10% cv for all samples tested.

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