Richard B. Fulton

Serum free light chain assay reduces the need for serum and urine immunofixation electrophoresis in the evaluation of monoclonal gammopathy

Background The potential for serum free light chain (sFLC) assay measurements to replace urine electrophoresis (uEPG) and to also diminish the need for serum immunofixation (sIFE) in the screening for monoclonal gammopathy was assessed. A testing algorithm for monoclonal protein was developed based on our data and cost analysis. Methods Data from 890 consecutive sFLC requests were retrospectively analysed. These included 549 samples for serum electrophoresis (sEPG), 447 for sIFE, and 318 for uEPG and urine immunofixation (uIFE). A total of 219 samples had sFLC, sEPG, sIFE and uEPG + uIFE performed. The ability of different test combinations to detect the presence of monoclonal proteins was compared. Results The sFLC κ/λ ratio (FLC ratio) indicated monoclonal light chains in 12% more samples than uEPG + uIFE. The combination of sEPG and FLC ratio detected monoclonal proteins in 49% more samples than the combination of sEPG and sIFE. Furthermore, the sEPG + FLC ratio combination detected monoclonal protein in 6% more samples than were detected by the combined performance of sEPG, sIFE, uEPG and uIFE. However, non-linearity of the assay, the expense of repeat determinations due to the narrow measuring ranges, and frequent antigen excess checks were found to be limitations of the sFLC assay in this study. Conclusion The FLC ratio is a more sensitive method than uIFE in the detection of monoclonal light chains and may substantially reduce the need for onerous 24 h urine collections. Our proposed algorithm for the evaluation of monoclonal gammopathy incorporates the sFLC assay, resulting in a reduction in the performance of labour intensive sIFE and uEPG + uIFE while still increasing the detection of monoclonal proteins.

Validation of a rapid test for HLA-B*58:01/57:01 allele screening to detect individuals at risk for drug-induced hypersensitivity

AIM In prevention of allopurinol and abacavir hypersensitivity, screening HLA-B*58:01/57:01 has been highly recommended prior to commencing these therapies. Therefore, we aimed at developing and validating a rapid and robust screening method for HLA-B*58:01/57:01. MATERIALS & METHODS Real-time polymerase chain reaction with TaqMan probes was employed to detect HLA-B*58:01/57:01. RESULTS The newly developed assay has the sensitivity of 100% (95% CI: 79.4-100.0%), the specificity of 98.8% (95% CI: 93.6-99.9%), the positive predictive value of 94.1% (95% CI: 71.3-99.9%) and the negative predictive value of 100.0% (95% CI: 95.7-100.0%). The lowest limit of detection is 0.04 ng/µl of DNA. CONCLUSION The present method is a rapid and robust assay that is appropriate for screening of HLA-B*58:01/*57:01 alleles.

Validation of a novel real-time PCR assay for detection of HLA-B*15:02 allele for prevention of carbamazepine – Induced Stevens-Johnson syndrome/Toxic Epidermal Necrolysis in individuals of Asian ancestry

OBJECTIVES Screening for the HLA-B*15:02 allele has been recommended to prevent carbamazepine (CBZ) - induced Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) in individuals with Asian ancestry. We aimed, therefore, to develop and validate a robust and inexpensive method for detection of the HLA-B*15:02 allele. METHODS Real-time PCR using TaqMan® probes followed by SYBR® Green was used to detect the HLA-B*15:02 allele prior to treatment with CBZ therapy. RESULTS A total of 121 samples were tested. The assay has a sensitivity of 100% (95% CI: 76.84-100.0%), a specificity of 100% (95% CI: 96.61-100%), a positive predictive value of 100% (95% CI: 76.84-100%) and a negative predictive value of 100.0% (95% CI: 96.61-100.0%), respectively. There was 100% agreement between our results and genotyping using Luminex SSO/SBT/SSP. The lowest limit of detection of the TaqMan® probe is 0.05ng/μl and the SYBR® Green is 0.5ng/μl of DNA. The unit cost of using the TaqMan® probe followed by SYBR® Green is only

A polymorphism within the psoriasis susceptibility 1 candidate 1 (PSORS1C1) gene is not linked to HLA-B*58:01 in an Australian cohort

4.7 USD. CONCLUSION We developed a novel assay for the detection of the HLA-B*15:02 allele, which is robust, inexpensive and suitable for screening individuals of Asian ancestry in the prevention of CBZ-induced SJS/TEN.

Providing HIV results via SMS one day after testing: more popular than rapid point-of-care tests.

Association exists between HLA-B*58:01 allele and allopurinol Stevens-Johnson syndrome (SJS), especially those of an Asian heritage but associations have been also described in Caucasian populations. This creates the need to develop a rapid, robust and inexpensive assay for pre-screening of HLA-B*58:01. A polymorphism within PSORS1C1 gene was recently found in linkage disequilibrium (LD) with HLA-B*58:01 allele in the Japanese population. The aim of this study is to confirm whether this polymorphism can be used as a surrogate biomarker to identify carriers for HLA-B*58:01. No linkage was found between the two in the Australian cohort.

A novel multiplex polymerase chain reaction assay for detection of both HLA-A*31:01/HLA-B*15:02 alleles, which confer susceptibility to carbamazepine-induced severe cutaneous adverse reactions

An inner Sydney sexual health service introduced the option to gay and bisexual men of receiving a negative HIV result by SMS to mobile phone one business day after venipuncture (rapid SMS). Men could also choose one of the other options: a point-of-care-test (POCT), by phone, or in-person (clinicians could also require in-person). We followed-up patients choosing the rapid SMS method to ascertain their satisfaction. During 12 months, 473 men had 591 HIV tests. Of these tests, 5.4% were POCTs, 9.1% were in-person, 24% were by phone, and 62% were rapid SMS. HIV POCTs declined from being 22% of result methods in the pre-study period to 5.4% during the rapid SMS intervention period (odds ratio 0.20, 95% CI 0.13–0.32, P < 0.0001). Phone/in-person results declined from 78% to 33% (odds ratio 0.14, 95% CI 0.10–0.20, P < 0.0001). SMS was sent by the next business day in 95% of cases; 96% of men were satisfied; and 95% would choose this method for their next test. Of 77 men who previously had an HIV POCT, 56 (73%) elected a rapid SMS result rather than having another POCT. The higher accuracy of conventional serology was commonly expressed as the reason for choosing rapid SMS for results.

Validation of a Rapid, Robust, Inexpensive Screening Method for Detecting the HLA-B*58:01 Allele in the Prevention of Allopurinol-Induced Severe Cutaneous Adverse Reactions

HLA‐A*31:01 and HLA‐B*15:02 have been widely reported to confer genetic susceptibility to carbamazepine (CBZ)‐induced severe cutaneous adverse reactions (SCARs). Accordingly, the screening for these alleles has been highly recommended to prevent SCAR prior to introducing CBZ therapy. Although a number of methods are available for screening of HLA‐A*31:01 or HLA‐B*15:02 alleles separately, developing an assay that can detect both these alleles would be more clinically practical, cost‐effective and less time‐consuming. Therefore, in this study, a multiplex polymerase chain reaction (PCR) using TaqMan Probe was designed and validated to be able to detect HLA‐A*31:01 and HLA‐B*15:02. In comparison with Luminex‐SSO/SBT/SSB, the multiplex PCR assay for detection of HLA‐A*31:01 and HLA‐B*15:02 had a perfect agreement in the validation group of 125 samples. The method was able to detect the target genes at the DNA concentration of 0.037 ng/μL. The unit cost of this assay is less than

Validation of a HLA-B*15:02 screening method in the prevention of carbamazepine – Induced severe cutaneous adverse reactions

5 USD with total time of 110 minutes.

Evaluation of testing for specific ige to morphine, pholcodine and chlorhexidine in cases of perioperative anaphylaxis

The HLA B*58:01 allele has been worldwide reported as a pharmacogenetic susceptibility to allopurinol-induced severe cutaneous adverse reactions (SCARs). To prevent these life-threatening conditions, the American College of Rheumatology hingly recommended that the HLA-B*58:01 be screened prior to the initiation of allopurinol therapy. Therefore, we developed a rapid, robust, inexpensive screening method using SYBR® Green real time PCR to detect the HLA-B*58:01 allele. A total of 119 samples were tested. The assay has a sensitivity of 100% (95% CI: 69.15%-100%), a specificity of 100% (95% CI: 96.67%-100%), a positive predictive value of 100% (95% CI: 69.15%-100%) and a negative predictive value of 100% (95% CI: 96.67%-100%). HLA-B*58:01 genotyping results showed 100% agreement with those obtained from Luminex SSO/SBT/SSP. The lowest limit of detection of this method is 0.8 ng/µL of DNA. The unit cost of the test is only

Increased frequency of HIV-1 viral load blips associated with introduction of Roche Ampliprep/TaqMan assay

3.8 USD. This novel screening test using SYBR® real time PCR would be appropriate to identify individuals with the HLA-B*58:01 allele for the prevention of allopurinol-induced SCARs.