von willebrand factor ( vwf ) , a multimeric glycoprotein produced by endothelial cells and megakaryocytes , plays important roles in platelet adhesion at the sites of vascular injury and in the coagulation process through stabilization of factor viii [ 13 ] . vwf was first identified in hereditary bleeding disorder known as von willebrand disease ( vwd ) and later it has been associated with other clinical conditions such as cancers , clotting , and vascular and liver disorders . quantitative and functional impairment of vwf
in contrast , elevated levels of vwf predispose to thrombotic complications [ 2 , 3 ] .
epidemiological studies have revealed that an increased level of vwf is significantly associated with cardiovascular diseases and acute vascular events [ 48 ] , making it a potential biomarker in disease progression and prognosis [ 911 ] .
cardiovascular disease is a global health problem and is a leading cause of death in developed countries . in developing countries ,
currently , the available test for measurement of vwf level in plasma is vwf antigen ( vwf  :  ag ) assay that is usually performed by enzyme - linked immunosorbent assay ( elisa ) and automated immunoturbidimetric procedure such as latex - immunoassay ( lia ) , which requires sophisticated instrumentation and well - trained laboratory personnel .
furthermore , elisa test is unsuitable for urgent testing [ 14 , 15 ] . despite the advanced technology of testing vwf levels in a fully equipped laboratory ,
very little attention has been focused on testing the vwf levels in areas with limited laboratory facilities or at the bedside .
thus , the application of rapid , simple , and less expensive test method for the detection of vwf might help in improving clinical management in resource - limited setting . in the future ,
estimation of vwf  :  ag could be possible as a point of care testing in clinical practice routinely .
latex agglutination test has been the method of choice in the development of a rapid test kit in many fields including clinical and veterinary medicines [ 1620 ] . in principle , latex agglutination test is based on agglutination reactions between antigen and antibody .
submicron microspheres or often called  latex beads  are used as a solid support for the antibody ( or antigen ) to be adsorbed onto them .
the latex beads with adsorbed antibody ( or antigen ) are used to detect antigen ( or antibody ) present in biological samples .
polystyrene latex beads are commonly used in the latex agglutination test because of their strong hydrophobic characteristic that is ideal for the adsorption of materials such as proteins by a simple passive adsorption method .
the use of latex beads was first described for the detection of rheumatoid factor by plotz and singer in 1956 . since then
, latex tests have been developed to detect specific infectious diseases , autoimmune diseases , hormones , drugs , and serum proteins .
the original method for attachment of proteins ( antibody ) to latex beads was passive adsorption .
passive adsorption using plain polystyrene latex beads has been used in the development of many latex tests for the detection of antibody or antigen such as group d salmonellae , crp , lactoferrin , and rotavirus .
covalent coupling method using functionalized latex beads such as carboxylated polystyrene latex beads can produce more stabilized protein - latex complex compared to passive adsorption method . it has then become a method of choice in the development of latex tests such as for the detection of anti - cysticercus antibodies , igm quantification in cerebrospinal fluid , and detection of avian influenza virus subtype h5n1 . although covalent coupling methods have many advantages , the passive adsorption method is still widely used until today because of its simplicity and flexibility .
conventional latex agglutination is not a new method , but its application is limited in the field of hematology and hemostasis . the latex method has moved towards automation and currently the antigenic detection has been made easy and specific with this new technology especially using the monoclonal generated antibodies .
however , the conventional method may be useful to be applied as a rapid or screening test before running the usual laboratory investigations .
the present study may provide evidence on the usage of a latex agglutination test for the detection of vwf  :  ag in the plasma sample that could be commercialized in the future . in this study ,
surfactant - free latex beads were selected in order to avoid a variation in protein attachment condition that might occur due to the variation in the surfactant purity .
the aggregation in the latex beads is prevented by the electrical charge that is built onto the surface of the latex beads during their synthesis .
the beads have a high density of chloromethyl groups attached to the styrene monomeric unit , and these functional groups can react directly with amino groups in antibodies .
the beads are of hydrophobic type and can be used at both high and low ph conditions .
the beads are stabilized by negatively charged sulfate groups that provide the colloid stability . in terms of ease of washing and suitability in simple visible test , latex beads with a size of 1  m
this study was conducted with the aims to develop and validate a latex agglutination test for the detection of vwf  :  ag .
method validation is a process to demonstrate the suitability of the test method for its intended purpose .
method validation is performed to determine the performance characteristics of the test method , for example , reproducibility , stability , linearity , limit of detection , and comparison of method , and also to estimate certain types of analytical errors by performing interference study .
reproducibility study estimates imprecision , stability study determines stability of the sample and reagent , linearity study determines reportable range , detection limit study determines the lowest concentration of analyte , method comparison estimates inaccuracy , and interference study estimates error caused by other materials that may be present in the specimen .
the study protocol was approved by the human research ethics committee from universiti sains malaysia , health campus , kubang kerian , kelantan , malaysia [ reference number : usmkk / ppp / jepem 253.3.(4 ) ] .
blood samples for validation studies were collected into 3.2% buffered trisodium citrate ( becton dickinson and co. , plymouth , uk ) , and platelet - poor plasma was prepared by centrifuging the blood at 2,500  g for 15 minutes at room temperature . following centrifugation , the top 2/3 of the plasma layer was transferred into a plastic tube .
the collected plasma was recentrifuged at 2,500  g for 15 minutes to remove any remaining red cells or platelets .
after centrifugation , the top 2/3 portion of plasma volume was transferred into cryogenic vials , and the plasma samples were frozen at 70c until use . on the day of testing , plasma samples were thawed at 37c for 10 minutes and mixed by gentle inversion prior to analysis , which was performed within two hours after thawing .
chloromethyl latex beads , 1.0  m in diameter , were purchased from life technologies , usa . prior to use , the latex beads were washed according to the manufacturer 's instructions with some modifications .
briefly , 2.5  ml of 4% latex suspension was washed twice in 10  ml imidazole buffer , ph 7.4 ( 120  mm  nacl , 20  mm imidazole , and 5  mm citric acid ) by centrifuging the latex suspension at 3,000  g for 20 minutes at room temperature .
the final pellet was resuspended with 5  ml imidazole buffer to obtain a suspension of 2% .
vwf antibody - latex reagent was prepared by passive adsorption method according to the manufacturer 's protocol and the method described by mina et al . , 2012 , with some modifications .
briefly , 1.0  ml of 2% latex suspension was centrifuged at 3,000  g for 20 minutes at room temperature , and the supernatant was discarded .
the pellet was resuspended with 1  ml phosphate - buffered saline , ph 7.4 ( 135  mm  nacl , 2.6  mm  kcl , 8  mm  na2hpo4 , and 1.5  mm  kh2po4 ) , that was added with 200  l of polyclonal rabbit anti - human von willebrand factor from dako denmark ( 3.1 
the mixture was incubated with gentle mixing on a horizontal rotator ( barnstead thermolyne labquake rotator , usa ) at room temperature for 24 hours .
after incubation , the mixture was centrifuged , and the supernatant was kept for determination of unadsorbed antibody by bca method using micro bca protein determination kit from pierce biotechnology , usa .
the concentration of unadsorbed antibody in the supernatant was determined according to the manufacturer 's instructions .
the concentration of adsorbed antibody was estimated as the difference between the initial concentration of antibody ( i.e. , 620  g ) and the concentration of unadsorbed antibody in the supernatant .
the pellet was then washed twice in 4  ml imidazole buffer by centrifugation as stated earlier and resuspended with 3  ml imidazole buffer containing 1% bovine serum albumin ( sigma - aldrich , usa ) .
after incubation , the mixture was centrifuged , and the pellet was washed twice in 4  ml imidazole buffer by centrifugation as stated earlier .
the final pellet was resuspended with 2  ml imidazole buffer containing 0.1% bovine serum albumin to obtain a suspension of 1% , and the suspension was stored at 4c until use .
the process described was carried out using different concentrations of the vwf antibody to determine the appropriate concentration of antibody for the adsorption procedure . as a control reagent , the process was conducted without the antibody .
normal saline and buffers were used as negative samples while commercial controls were used as positive samples .
vwf antibody - latex reagent was brought to room temperature and mixed gently before use .
the sedimentation of latex beads can occur during storage that can be reversed by pipetting the suspension of latex beads through a fine tip pipette until they are returned to a uniform suspension .
latex agglutination test for the detection of vwf in a plasma sample was performed on a slide card ( thermo scientific , uk ) . using a wooden applicator stick ,
20  l of vwf antibody - latex reagent was mixed with 20  l of plasma sample on a black reaction zone of the card .
the card was rotated on a horizontal shaker ( boekel grant microplate shaker , uk ) for three minutes .
after three minutes , the agglutination reaction was observed by visual inspection macroscopically with a naked eye .
the detection and estimation of vwf by latex agglutination test can be performed using a qualitative test and semiquantitative test , respectively .
qualitative test was performed on undiluted plasma sample , and the result was reported as negative or positive .
invisible or hardly visible agglutination was reported as negative and visible agglutination was reported as positive ( figure 1 ) . for a positive result , the degree of agglutination was scored from 1 + to 4 + based on the size of the agglutinated beads , the appearance of the background , and the rapidity of the agglutination development as shown in table 1 .
a positive plasma sample was serially diluted twofold with normal saline . the highest dilution that still shows a positive reaction is the end - titre of a sample .
reproducibility study was carried out by testing vwf antibody - latex reagent on three plasma samples , and the end - titre of each sample was determined three times . between - day
reproducibility study was conducted by testing the reagent in duplicate on two plasma samples , and the end - titre of the samples was determined on days 1 , 7 , 14 , and 21 .
the reagents were also tested on low and normal controls from instrumentation laboratories , italy ( hemosil special test control level 2 and hemosil normal control assayed , resp . ) , on days 2 , 4 , 5 , 7 , 30 , and 50 .
stability of the reagent was checked during storage of vwf antibody - latex reagent at 4c for 21 days .
qualitative test was performed on two plasma samples on days 1 , 2 , 3 , 4 , 5 , 6 , 7 , 14 , and 21 using aliquots of samples that were stored at 70c .
stability of plasma sample during storage was also checked by testing vwf antibody - latex reagent ( stored at 4c ) on aliquots of plasma samples which was stored at 4c .
after 0 ( within two hours after blood collection ) , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 14 , and 21 days , the qualitative test was performed .
the reagents were also tested on hemosil special test control level 2 and hemosil normal control assayed ( instrumentation laboratories , italy ) .
batch to batch variation was tested with the reagents prepared on two different dates , using two different lots of the vwf antibody .
the linearity of latex agglutination test was determined by testing vwf antibody - latex reagent on two patient samples which were serially diluted twofold .
the vwf  :  ag level in diluted samples was measured by immunoturbidimetric assay using hemosil von willebrand factor antigen kit ( instrumentation laboratory , italy ) .
semiquantitative latex agglutination test was performed on diluted samples , and the end - titres were compared with the vwf  :  ag levels .
graphical presentations of the semiquantitative vwf  :  ag by latex agglutination test ( y ) versus the vwf  :  ag by immunoturbidimetric assay ( x ) were made for individual sample and also for a combination of the two samples .
limit of detection of latex agglutination test was determined by testing vwf antibody - latex reagent on low - level samples ( cryosupernatant ) .
seven cryosupernatant samples were serially diluted twofold , and the vwf  :  ag levels in the samples ( neat ) were measured by immunoturbidimetric assay using hemosil von willebrand factor antigen kit ( instrumentation laboratory , italy ) .
vwf  :  ag levels in 1  :  2 , 1  :  4 , and 1  :  8 diluted samples were estimated by dividing the vwf  :  ag levels of neat samples by the dilution factors of 2 , 4 , and 8 , respectively . latex agglutination test was performed to determine the lowest level of vwf  :  ag that shows positive agglutination reaction . 

interference from hemoglobin is common in coagulation testing , and hemolytic specimen is usually not or minimally affected in latex agglutination test .
the interference study was performed by testing vwf antibody - latex reagent on plasma pools and plasma pools spiked at different concentrations of hemolysate .
hemolysate was prepared by freezing and thawing the whole blood followed by the osmotic shock protocol .
briefly , normal sample that was collected in 3.0  ml  k2 edta tube was centrifuged at 1,000  g for 10 minutes .
cells were resuspended , and the suspension was centrifuged again at 1,000  g for 10 minutes .
saline was replaced with distilled water , and the suspension was stored at 20c overnight .
frozen cells were thawed , mixed , and centrifuged at 2,000  g for 30 minutes to remove cell debris .
hemolysate was transferred to a clean tube and hemoglobin level was measured with the sysmex xe 5000 hematology analyzer ( sysmex corporation , kobe , japan ) .
stock solutions of hemolysate in normal saline were prepared , and each of the stock solutions was used to spike plasma pools with high and low vwf  :  ag levels . in the present study , hemolysate with hemoglobin level of 120 
the hemolysate was diluted 1  :  2 , 1  :  4 , 1  :  8 , 1  :  20 , and 1  :  40 with normal saline to obtain the stock solutions with hemoglobin levels of 60 , 30 , 15 , 6 , and 3 
one hundred microlitres of each stock solution were added to 900  l of plasma pools to obtain plasma pools with a final hemoglobin level of 6 , 3 , 1.5 , 0.6 , and 0.3  mg / ml . 
 ( ii ) interference from rheumatoid factor .
rheumatoid factor ( rf ) is one of the most common interferences in latex - based methods .
it may bind and cross - link antibodies attached to the latex beads , leading to a false - positive result or falsely elevated levels of vwf in a sample .
the effect of rf on agglutination reaction was studied by testing vwf antibody - latex reagent on plasma pools with high and low vwf  :  ag levels .
the plasma pools were spiked at different concentrations of rf using rheumatoid factor control serum from mybiosource , usa ( 1030  iu / ml ) .
the rf control serum was diluted 1  :  8 , 1  :  7 , and 1  :  6 with high plasma pools to obtain plasma pools spiked with rf at a concentration of 129 , 147 , and 172  iu / ml , respectively .
for the preparation of low plasma pools spiked with rf at a concentration of 10 , 15 , 52 , 103 , and 129  iu / ml , the rf control serum was diluted 1  :  100 , 1  :  70 , 1  :  20 , 1  :  10 , and 1  :  8 , respectively , with low plasma pools .
method comparison study between latex agglutination test and the reference method ( immunoturbidimetric assay ) was conducted by parallel analysis of 132 plasma samples of different levels of vwf  :  ag .
the samples were obtained from 40 healthy individuals , 40 patients with cardiovascular diseases , and 32 patients with other diseases including malignant and inflammatory disorders .
the remaining 20 samples were cryosupernatant samples ( i.e. , samples with low levels of vwf  :  ag ) that were obtained from blood transfusion services .
all specimens were tested in ten days , and the testing was spread over ten months .
qualitative and semiquantitative latex agglutination tests were performed using the vwf antibody - latex reagents .
the immunoturbidimetric assay was performed on acl elite pro coagulation analyser ( instrumentation laboratory , italy ) using hemosil von willebrand factor antigen kit from instrumentation laboratory , italy .
the procedure for vwf  :  ag by immunoturbidimetric assay was performed according to the manufacturer 's instructions and adhered to the clsi document h21-a5 on the specimen collection and processing of blood specimens for testing the plasma - based coagulation assays .
normal reference range for vwf  :  ag levels by immunoturbidimetric assay in our laboratory is 50150% .
linear regression analysis was performed to determine the linearity of the semiquantitative latex agglutination test .
the degree of agreement between qualitative latex agglutination test and immunoturbidimetric assay was estimated by calculating the kappa value (  ) : 0.810.99 , almost perfect agreement ; 0.610.80 , substantial agreement ; 0.410.60 , moderate agreement ; 0.210.40 , fair agreement ; 0.010.20 , slight agreement ; and < 0 , less than chance agreement .
spearman 's rho correlation analysis was used to determine the degree of relationship between semiquantitative latex agglutination test and immunoturbidimetric assay .
the strength of the relationship is showed by the correlation coefficient value ( r ) : at least 0.8 , very strong ; 0.6 up to 0.8 , moderately strong ; 0.3 to 0.5 , fair ; and less than 0.3 , poor .
two - tailed probability ( p ) value of less than 0.05 was considered statistically significant .

 table 2 shows the estimated concentration of adsorbed antibody on the latex beads in the vwf antibody - latex reagents that were prepared using different concentrations of the vwf antibody .
the results showed that the concentration of adsorbed antibody increased as the initial concentration of the vwf antibody increased .
however , the percentage of adsorbed antibody , that is , 70% and 74% , was not much different when adsorption method was performed using antibody concentration of 620  g and 700  g , respectively .
visual observation on the agglutination reaction showed that less agglutination was observed from latex beads adsorbed with less than 400  g of antibody ( i.e. , 118  g , 276  g , and 328  g ) compared to agglutination reaction from latex beads adsorbed with about 400  g of antibody .
agglutination reactions from latex beads coated with 520  g antibody were slightly more compared to agglutination reactions from latex beads coated with 436  g antibody ( data not shown ) . in other words , latex beads with ~400  g adsorbed antibody were sufficient to produce strong agglutination reaction when mixed with plasma sample .
thus , 200  l of the vwf antibody with a concentration of 620  g has been chosen to be the most appropriate concentration for the adsorption procedure .
autoagglutination was not seen when vwf antibody - latex reagent alone was rotated for three minutes . extending
the time beyond three minutes resulted in reagent drying that might give false - positive result .
no agglutination was seen when the control reagent ( i.e. , reagent without adsorbed antibody ) was tested on plasma sample and commercial controls .
negative reaction was consistently observed when the vwf antibody - latex reagent was tested on normal saline and buffers throughout this study .
validation studies were conducted using vwf antibody - latex reagents that were prepared in seven batches .
the concentration of adsorbed antibody on the latex beads ranged from 403 to 447  g ( i.e. , 6572% from the initial concentration of the vwf antibody used in the adsorption procedure ) which showed that the percentage of the vwf antibody adsorbed on the latex beads was consistent throughout this study . in the reproducibility study , the end - titre of three samples ( i.e. , 1  :  32 , 1  :  64 , and 1  :  128 ) was consistent when tested three times within a day . similarly , the end - titre of two samples ( i.e. , 1  :  16 and 1  :  128 ) did not change from day 1 to day 21 ( between days ) . testing on the controls showed that the end - titre of low and normal controls ( 1  :  4 and 1  :  16 , resp . ) was reproducible from day 1 to day 50 .
in the stability study , vwf antibody - latex reagent and plasma sample were found to be stable for qualitative latex agglutination test when stored at 4c for at least 21 days .
semiquantitative test on controls showed that the vwf antibody - latex reagent was stable for at least 50 days .
all these findings showed that the semiquantitative latex agglutination test results were reproducible within a day and between days and the reagent was found to be stable for at least 50 days when stored at 4c .
batch to batch variation was not detected . in the linearity study , a series of known concentrations of vwf  :  ag was established by dilution with five equally spaced concentrations as shown in table 3 .
the table shows the latex agglutination test results ( titre ) and the levels of vwf  :  ag in undiluted ( tube 1 ) and diluted samples ( tubes 26 ) .
it was shown in the table that the semiquantitative latex agglutination test results ( titre ) were directly proportional to the levels of vwf  :  ag in the plasma sample .
linearity of the semiquantitative vwf  :  ag latex agglutination test was demonstrated in sample 1 ( r = 0.9985 ) and sample 2 ( r = 0.9999 ) .
a regression line that was fit to the points was observed in both samples ( graphs not shown ) .
the linearity was also demonstrated at the titre ranging from 1  :  2 to 1  :  128 when the results of both samples were combined and analyzed ( r = 0.9361 ) .
all these findings have demonstrated a linear relationship between the observed results ( vwf  :  ag titre ) and the true concentrations of vwf  :  ag in the plasma samples . in the limit of detection study , plasma samples with low levels of vwf  :  ag ( in the range of 20.2% to 30.6% ) were obtained from seven cryosupernatant samples ( table 4 ) .
table 4 shows the agglutination reaction observed when latex agglutination test was performed on undiluted ( neat ) samples and diluted samples ( at 1  :  2 , 1  :  4 , and 1  :  8 dilutions ) . as shown in the table , the lowest level of vwf  :  ag that still showed positive reaction is 5.1% ( sample number 2 at 1  :  4 dilution ) , and the highest level of vwf  :  ag that showed negative reaction is 3.8% ( sample number 7 at 1  :  8 dilution ) .
these results showed that the lowest level of vwf  :  ag that the latex agglutination test can detect to determine the presence ( positive result ) or absence ( negative result ) of vwf  :  ag was 5.1% .
thus , the limit of detection of vwf  :  ag by latex agglutination test is approximately 5% . besides that
, reproducibility of the agglutination reaction at vwf  :  ag levels of 5.1 and 3.8% was also checked by testing six times the diluted samples of number 2 ( at 1  :  4 dilution ) and number 7 ( at 1  :  8 dilution ) , respectively .
sample number 2 showed positive reactions in all six replicates , while sample number 7 showed negative reaction in all six replicates .
these findings showed that the latex agglutination test reliably produced consistent results near the cutoff concentration .
visible agglutination was seen when latex agglutination test was performed on undiluted plasma pools and undiluted plasma pools with a final hemoglobin level of 0.3 , 0.6 , 1.5 , 3 , 6 , and 12  mg / ml . the end - titre of 1  :  32 was obtained when semiquantitative latex agglutination test was performed on high plasma pool . the same end - titre ( 1  :  32 ) was observed when the test was performed on high plasma pools with hemoglobin level of 0.3 , 0.6 , 1.5 , 3 , 6 , and 12  mg / ml . similarly , the end - titre of low plasma pools with hemoglobin level of 0.3 , 0.6 , 1.5 , 3 , 6 , and 12  mg / ml was the same as the end - titre of low plasma pool without hemoglobin interference ( 1  :  4 ) . these results showed that hemoglobin did not cause interference in latex agglutination test and semiquantitative test result was not affected by hemoglobin level up to 12  mg / ml . 
 table 5 shows the end - titre of high and low plasma pools spiked at different concentrations of rf . as shown in the table , interference from rf on latex agglutination test was not detected in high plasma pools spiked with rf at concentrations up to 129  iu / ml .
nevertheless , the interference from rf was observed in low plasma pools spiked with rf at concentrations of 26 , 52 , 103 , and 129  iu / ml .
the end - titre of high and low plasma pools without interference from rf was 1  :  64 and 1  :  2 , respectively .
these results showed that rf interfered with the latex agglutination test result when the test was performed on the plasma sample containing low levels of vwf  :  ag and high levels of rf ( > 26  iu / ml ) .
however , latex agglutination test result of plasma sample containing high levels of vwf  :  ag was not affected by rf at concentrations of < 130  iu / ml .
comparison study between qualitative latex agglutination test and immunoturbidimetric assay showed a kappa value of 0.77 which indicates a substantial agreement between the two tests .  
semiquantitative latex agglutination test was strongly correlated with immunoturbidimetric assay ( spearman 's rho , r = 0.946 , p < 0.001 ) across a range of vwf  :  ag levels from 34.5% to 870% .
based on the results from kappa statistic and correlation study , the scoring system for agglutination reactions and correlation with the vwf  :  ag levels ( using immunoturbidimetric assay ) is shown in table 7 . the proposed interpretation guide is also put in a footnote .
to the best of our knowledge , there has been no reported study or technical description available on the rapid test for vwf  :  ag utilising latex agglutination method . at present , the application of this method has not been used in clinical practice for monitoring of patients with low and high plasma levels of vwf  :  ag .
screening of vwd patients before confirmation tests and monitoring of patients at high risk of thrombotic event in the area with low - resource settings or at the bedside can be performed if rapid test for vwf  :  ag is available . hence , there is a need to develop a rapid and simple latex agglutination test that can reliably detect and estimate vwf  :  ag in the plasma sample .
latex agglutination test is based on the observation of visible clumps that are formed from the reaction of antigen - antibody complexes .
the antigen - antibody complexes can be prepared by passive adsorption or covalent coupling method . in the early stage of the present study
, attempts had been made to couple vwf antibody with the carboxylated latex beads by covalent coupling method .
the coupling procedure was performed in mes buffer at ph 6.0 according to the protocol from the manufacturer .
unfortunately , there was no antibody coated on the latex beads ( data not shown ) .
however , the vwf antibody was successfully adsorbed onto the surface of chloromethyl latex beads by passive adsorption method .
these findings were similar to the reported study by garcia et al . , 2015 .
in the present study , a maximal physical adsorption of the vwf antibody onto chloromethyl latex beads was obtained at ph 7.4 , a ph that is close to the isoelectric point of polyclonal immunoglobulin ( igg ) antibody [ 36 , 37 ] .
interference by rf in the latex agglutination test for the detection of vwf  :  ag was expected .
rf is known to cause analytical error in automated immunoturbidimetry assay that presented the main limitation of this method . despite this limitation
, the findings do nevertheless show that the effect of the interference was less prominent in the sample with high levels of vwf  :  ag compared to the sample with low levels of vwf  :  ag .
the expected interference is without any doubt due to the usage of whole igg in the passive adsorption method .
it is well known that rheumatoid factor can bind with the fc region of igg , causing false agglutination that may alter the result of the test . in the literature , the usage of antibody fragments such as fab or f(ab)2 was recommended to eliminate the interference from rheumatoid factor .
antibody fragment is suggested in future study , and optimization is required as the antibody fragment is more acidic than the polyclonal antibody . in the present study ,
the agglutination observed from the reaction between vwf antibody - latex reagent and positive sample may not be optimum as expected .
experiments using higher initial concentration of vwf antibody with a more intensive procedure to ensure maximal bead coating could be performed in the future study
. not only that , but the orientation of adsorbed antibody could also be studied to ensure a proper alignment of antibody on latex beads .
clarizia et al . , 2009 , have demonstrated a method for detecting the aligned and misaligned antibodies on the surface on latex beads . due to some technical constraint
, maximal bead coating can not be ensured , and a study on orientation of adsorbed antibody could not be performed .
another limitation of this study is that the size of the clumping was only estimated by visual observation . in future study , it is recommended to measure the clumping or macroscopic clusters using microscope or other methods .
there is a potential clinical application of this rapid latex agglutination test as a routine test in various premises including small laboratory and clinic and at the bedside .
the clinical aspect of low levels of vwf  :  ag is well recognized , but not many medical personnel realized the implications of high vwf  :  ag levels in medical practice . an inherited bleeding disorder known as von willebrand disease ( vwd ) is associated with an abnormality in vwf , and type 1 vwd is the commonest form that is associated with low vwf  :  ag . on the other hand , high levels of vwf  :  ag are associated with thrombotic risks and had been extensively studied in many medical conditions particularly coronary heart disease ( chd ) and stroke .
it has been shown that chd patients with high baseline of vwf  :  ag levels are at risk of acute vascular events related to thrombosis .
the current clinical guidelines do not incorporate the measurement of vwf  :  ag in the routine assessment of chd patients but , from various studies , there is a role in monitoring the levels of vwf  :  ag .
detection of persistent high levels or increasing trends of vwf  :  ag levels would alert the treating doctor for certain clinical measures to improve the patient care .
latex agglutination test for the detection of vwf  :  ag was found to be simple to perform , and the results can be obtained within three minutes , making it suitable to be used in areas with limited laboratory facilities .
the result obtained by the latex agglutination test is reliable as shown by the findings from reproducibility , stability , linearity , limit of detection , and method comparison studies .
prozone effect was not detected at vwf  :  ag levels up to 870% .
the stability of the reagents used for this method was up to 50 days and in this study no stabilizer or preservatives were used .
the plasma for vwf  :  ag estimation was stable for up to 21 days which may be related to the protein structure ; however , more tests are needed to confirm the suitable plasma storage recommendation for this method .
all these findings showed the usefulness of this method in detecting and estimating high and low levels of vwf  :  ag .
this method might be useful in the monitoring of vwf  :  ag in patients that are at a high risk of vascular event related to endothelial dysfunction .
similarly , this method could be used to estimate vwf  :  ag levels in patients with vwd before confirmation with standard test panels in the specialized coagulation laboratory .
however , we only evaluate the latex agglutination test in quantitative terms by using plasma samples containing low and high levels of vwf  :  ag
. this application mainly addressed the vwd type 1 which is more common in clinical practice . in future study , plasma samples with qualitative vwf abnormalities ( e.g. , type ii vwd or acquired vwd patients demonstrating abnormal vwf function with variables vwf  :  ag levels ) could be included in the comparison study to confirm the application of the latex agglutination test for assessing vwf  :  ag levels in various clinical situations . in summary , this test method needs to be refined and compared with other latex agglutination tests available in the market before it is used on patient samples .
latex agglutination test for vwf  :  ag is simple , rapid , and reproducible , correlates well with the reference method , and is suitable to be used in areas with limited laboratory facilities .
the test has commercial potential as a low - cost alternative method for the detection and estimation of low levels and high levels of vwf  :  ag that could help in the management of vwd type 1 patients and patients at risk of thrombotic events , respectively .
however , the test may not be suitable for patients with a high level of rheumatoid 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 (  = 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 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion