Noninfectious mixed cryoglobulinaemic glomerulonephritis and monoclonal gammopathy of undetermined significance: a coincidental association?

BACKGROUND: Cryoglobulins are cold-precipitable immunoglobulins that may cause systemic vasculitis including cryoglobulinaemic glomerulonephritis (CGN). Type 1 cryoglobulins consist of isolated monoclonal immunoglobulin (mIg), whereas mixed cryoglobulins are typically immune complexes comprising either monoclonal (type 2) or polyclonal (type 3) Ig with rheumatoid activity against polyclonal IgG. Only CGN related to type 1 cryoglobulins has been clearly associated with monoclonal gammopathy of undetermined significance (MGUS) using the conventional serum-, urine- or tissue-based methods of paraprotein detection. CASE PRESENTATION: We present four patients with noninfectious mixed (type 2 or 3) CGN and MGUS. Two patients had type 2 cryoglobulinaemia, one had type 3 cryoglobulinaemia, and one lacked definitive typing of the serum cryoprecipitate. The serum monoclonal band was IgM-κ in all four cases. Treatments included corticosteroids, cyclophosphamide, plasma exchange, and rituximab. At median 3.5 years' follow-up, no patient had developed a haematological malignancy or advanced chronic kidney disease. Other potential causes of mixed cryoglobulinaemia were also present in our cohort, notably primary Sjögren's syndrome in three cases. CONCLUSION: Our study raises questions regarding the current designation of type 2 CGN as a monoclonal gammopathy of renal significance, and the role of clonally directed therapies for noninfectious mixed CGN outside the setting of haematological malignancy.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part II: limit of detection and follow-up of patients with small M-proteins.

Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part I: factors impacting limit of quantitation of serum protein electrophoresis.

Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%-120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen.

A genome-wide ethyl-N-nitrosourea (ENU) mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

Open-Label Study of Absorption and Clearance of 1% Voriconazole Eye Drops.

Twenty participants undergoing elective cataract surgery received 1% voriconazole eye drops (1 drop per eye) either 20, 40, 60, or 80 min before surgery. Median voriconazole concentrations of 1.9 to 3.2 mg/liter in aqueous humor samples were attained over the first 80 min, which were higher than in vitro MIC90 values for typical fungi that cause keratitis.

A practical limited sampling strategy to predict free prednisolone exposure and glycemia in kidney transplant recipients.

BACKGROUND: Despite significant interindividual variability in prednisolone pharmacokinetics and potentially serious consequences with inadequate or excessive exposure, monitoring of prednisolone levels is not employed to guide therapy. As ultrahigh-performance liquid chromatography-tandem mass spectrometry methods can now measure the active free fraction of prednisolone, this study aimed to evaluate the performance of 15 published limited sampling strategies (LSSs) for predicting free prednisolone exposure in adult kidney transplant recipients and to examine the relationship between free/total prednisolone exposure and plasma glucose. METHODS: The study was performed in 11 subjects without diabetes 3-4 weeks postkidney transplantation. Area under the concentration time curve profiles of total and free prednisolone from 0 to 12 hours postdose (AUC0₋12) were determined and compared with predicted AUC0₋12 values calculated from published LSSs. Venous glucose was measured concurrently with the 13 sampling time points. RESULTS: The mean (±SD) age of subjects was 52 ± 12 years, 5 were men and the median (interquartile range) daily prednisolone dose was 20.0 mg (20.0-22.5). Interindividual variation in dose-adjusted free and total prednisolone exposure was 1.9- and 3.2-fold, respectively. All 15 free prednisolone LSSs exhibited good correlation (r ≥ 0.83), with bias and imprecision less than 15%. An LSS incorporating 1.25- and 3-hour samples had the highest predictive power (r = 0.97, bias 1.2%, imprecision 5.6%). Free prednisolone AUC0₋12 correlated with peak glucose levels (r = 0.65, P = 0.037), as did predicted AUC0₋12 from 14/15 LSSs. CONCLUSIONS: Biologically active free prednisolone exposure can be accurately predicted postkidney transplantation by LSSs incorporating 2-point concentration sampling. Peak plasma glucose concentration correlated well with prednisolone exposure.

Mass or molar? Recommendations for reporting concentrations of therapeutic drugs.

A working party (WP) from the Australasian Association of Clinical Biochemists, Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, Royal College of Pathologists of Australasia and Royal Australasian College of Physicians recommends the following: *mass units should be used for reporting therapeutic drug concentrations in Australia and New Zealand; and the litre (L) should be used as the denominator when expressing concentration. Examples of these units are mg/L and µg/L Exceptions to these principles include: *drugs for which there is current uniformity of reporting and supporting information using molar units, notably lithium (mmol/L) and methotrexate (µmol/L); *drugs that are also present as endogenous substances, where the units used routinely should continue to be used. This applies to many substances, including minerals (eg, iron; µmol/L), vitamins (eg, vitamin D; nmol/L) and hormones (eg, thyroxine; pmol/L). *drugs for which the denominator is not a 198 of fluid and there is international uniformity of reporting (eg, thiopurine metabolites; per 109 red blood cells). These recommendations relate to drugs that are used therapeutically, whether measured for therapeutic drug monitoring purposes or for assessment of overdose. Other substances, such as drugs of misuse, heavy metals or environmental toxins, were not considered by the WP and are thus not covered by this document. These recommendations should also be applied to other supporting documentation such as published guidelines, journal articles and websites. The implementation of these recommendations in New Zealand is subject to local confirmation.

Penetration of topically administered 0.5-percent caspofungin eye drops into human aqueous humor.

Ten participants attending elective anterior segment eye surgery received 0.5% caspofungin eye drops either 1 drop hourly for 4 h or 1 drop an hour before surgery. The eye drops were generally well tolerated. In the absence of inflammation or corneal abrasion, topical caspofungin does not achieve clinically relevant concentrations.

Current status of therapeutic drug monitoring in Australia and New Zealand: a need for improved assay evaluation, best practice guidelines, and professional development.

The measurement of drug concentrations, for clinical purposes, occurs in many diagnostic laboratories throughout Australia and New Zealand. However, the provision of a comprehensive therapeutic drug monitoring (TDM) service requires the additional elements of pre- and postanalytical advice to ensure that concentrations reported are meaningful, interpretable, and clinically applicable to the individual patient. The aim of this project was to assess the status of TDM services in Australia and New Zealand. A range of professions involved in key aspects of TDM was surveyed by questionnaire in late 2007. Information gathered included: the list of drugs assayed; analytical methods used; interpretation services offered; interpretative methods used; and further monitoring advice provided. Fifty-seven responses were received, of which 42% were from hospitals (public and/or private); 11% a hospital (public and/or private) and pathology provider; and 47% a pathology provider only (public and/or private). Results showed that TDM is applied to a large number of different drugs. Poorly performing assay methods were used in some cases, even when published guidelines recommended alternative practices. Although there was a wide array of assays available, the evidence suggested a need for better selection of assay methods. In addition, only limited advice and/or interpretation of results was offered. Of concern, less than 50% of those providing advice on aminoglycoside dosing in adults used pharmacokinetic tools with six of 37 (16.2%) respondents using Bayesian pharmacokinetic tools, the method recommended in the Australian Therapeutic Guidelines: Antibiotic. In conclusion, the survey highlighted deficiencies in the provision of TDM services, in particular assay method selection and both quality and quantity of postanalytical advice. A range of recommendations, some of which may have international implications, are discussed. There is a need to include measures of impact on clinical decision-making when assessing assay methodologies. Best practice guidelines and professional standards of practice in TDM are needed, supported by an active program of professional development to ensure the benefits of TDM are realized. This will require significant partnerships between the various professions involved.

Rapid and sensitive liquid chromatography/mass spectrometry assay for caspofungin in human aqueous humor.

A rapid, precise, and sensitive liquid chromatography/mass spectrometry (LC/MS) method to quantify the caspofungin concentration in human aqueous humor was developed and validated. Sample preparation involved simple dilution of aqueous humor samples with acetonitrile. Azithromycin was the internal standard. Good linearity over 10 to 5,000 ng/ml was observed. The lower limit of quantification was 10 ng/ml. The intra- and interday accuracies (percent bias) were within 11%, while the intra- and interday precisions were within 6%.

Penetration of 1% voriconazole eye drops into human vitreous humour: a prospective, open-label study.

PURPOSE: Although there have been reports describing the use of 1% voriconazole eye drops in the treatment of fungal infections, little is known about the penetration of voriconazole eye drops into the vitreous humour. The aim of this study was to elucidate if topical application of 1% voriconazole eye drops could reach therapeutic levels in the vitreous humour. METHODS: A prospective, open-label trial involving 10 participants selected from patients scheduled to undergo elective, posterior segment surgery. Participants received 1% voriconazole solution, preserved with 0.01% benzalkonium chloride, hourly for four doses or four times a day for 3 days. Vitreous humour was obtained at the start of surgery and analysed using a validated high-performance liquid chromatography assay. RESULTS: The minimum and maximum voriconazole concentration after hourly dosing (n = 5) was 0.1 and 1.1 microg/mL, respectively, whereas the median was 0.3 microg/mL. Samples were taken between 0.8 and 5.4 h after the last dose, with the median at 1.2 h. Almost all voriconazole concentrations from the four times a day group (n = 5) were below the limit of quantification. CONCLUSIONS: One per cent voriconazole eye drops are able to penetrate into human vitreous humour. Adequate concentration for treatment of sensitive Candida species can be achieved upon hourly administration.

Stability of extemporaneously prepared voriconazole ophthalmic solution.

PURPOSE: The stability of extemporaneously prepared voriconazole ophthalmic solution was studied. METHODS: Voriconazole solutions (2% and 1%) were reconstituted from the i.v. formulation. After thorough mixing, 3-mL samples of each of the resulting 2% and 1% solutions were filtered into eyedroppers. Three samples for both solutions were analyzed in triplicate at each time point. The 2% voriconazole ophthalmic solutions were stored at 2-8 degrees C, 25 degrees C, and 40 degrees C. The 1% voriconazole eye drops were stored at 2-8 degrees C. The 2% voriconazole solution samples were analyzed at time 0 and at weeks 1, 2, 4, 8, 16, and 32. The 1% solution samples were analyzed at time 0 and at weeks 6 and 14. Stability was measured using high-performance liquid chromatographic analysis. RESULTS: The 2% voriconazole ophthalmic solution demonstrated excellent stability at 2-8 degrees C and 25 degrees C for up to 16 weeks. The voriconazole solution displayed no significant change in pH at all time intervals. No change in visual appearance or clarity was observed in the 2% voriconazole eye drops at any point of the study for all study temperatures. Voriconazole 1% solution was stable at 2-8 degrees C for up to 14 weeks. CONCLUSION: Voriconazole 2% (20 mg/mL) solution preserved with 0.01% benzalkonium chloride prepared as alternative antifungal eye drops was stable for 16 weeks when stored at 2-8 degrees C and 25 degrees C and for 8 weeks when stored at 40 degrees C, while voriconazole 1% solution was stable at 2-8 degrees C for up to 14 weeks.

Prospective open-label study of the administration of two-percent voriconazole eye drops.

Thirteen human subjects scheduled for elective anterior segment eye surgery received hourly 2% voriconazole eye drops 4 hours presurgery. No side effects were reported. Significantly, the voriconazole concentration in the aqueous humor of the eye was similar to that reported for the 1% voriconazole solution, suggestive of concentration-independent absorption.

Penetration of voriconazole, 1%, eyedrops into human aqueous humor: a prospective open-label study.

OBJECTIVE: To determine the therapeutic efficacy of adjuvant use of voriconazole, 1%, eyedrops in the treatment of refractory fungal keratitis. METHODS: A prospective open-label trial was conducted to determine voriconazole levels obtained in human aqueous humor after administration of a 1% solution, preserved with 0.01% benzalkonium chloride, every 6 hours for 3 days, or hourly for 4 doses. Ten participants were selected among patients scheduled to undergo elective anterior segment surgery, and samples were tested using validated high-performance liquid chromatography. RESULTS: The mean (SD) voriconazole concentrations after hourly dosing (n = 5) was 1.90 (1.12) microg/mL and after a single dosing every 6 hours (n = 5) was 0.94 (1.21) microg/mL, respectively. The mean (SD) sampling times after the last administration of eyedrops were 1.1 (0.5) hours after hourly dosing and 2.1 (0.6) hours after a single dosing every 6 hours. CONCLUSIONS: Voriconazole, 1%, eyedrops are well tolerated and penetrate into human aqueous humor when administered at hourly or 6-hourly intervals. They are effective in treating Candida and Aspergillus keratitis, are substantially more affordable than oral therapy, and have less potential to cause systemic adverse effects.

Cyclosporin monitoring in Australasia: 2002 update of consensus guidelines.

Therapeutic drug monitoring of cyclosporin (CsA) has been established as part of the routine clinical treatment of patients following organ transplantation for more than 20 years, and based on contemporary knowledge, many consensus guidelines have been published to assist clinics and laboratories attain optimal strategies for patient care. This article addresses the newer directions in CsA monitoring, with particular reference to the Australasian situation that has evolved since the 1993 Australasian guideline. These changes have included the introduction of alternative assay methodologies, changed CsA formulation from Sandimmun to Neoral throughout Australasia, and alternatives to trough concentration (C0) monitoring, especially 2-hour concentration (C2) monitoring and associated validated dilution protocols to accurately quantitate the higher whole blood CsA concentrations. The revision was prepared following a recent survey of all Australasian CsA-monitoring laboratories where discordant practices were evident.