Lakshmi V. Ramanathan

Treatment of multiple myeloma with monoclonal antibodies and the dilemma of false positive M-spikes in peripheral blood

OBJECTIVES To characterize the effect of three humanized IgG κ monoclonal antibodies (daratumumab, isatuximab, and elotuzumab) on the interpretation of results generated by protein electrophoresis, immunofixation, free light chain, and heavy/light chain assays performed on human serum. METHODS Healthy volunteer serum and serum from multiple myeloma patients were supplemented with clinically relevant concentrations of each of the three monoclonal antibodies. These specimens then underwent analysis via serum protein electrophoresis, immunofixation, serum free light chain quantification, heavy/light chain quantification, total IgG, and total protein. In addition, serum specimens from patients who had undergone treatment with elotuzumab for multiple myeloma underwent similar analysis. RESULTS Addition of the study drugs to serum from both the healthy donor as well as multiple myeloma patients resulted in a visible and quantifiable M-protein on SPEP and a visible IgGκ band by IFE. Increases were also noted in total IgG, IgGκ, and IgGκ/IgGλ-ratios. Analysis of serum from multiple myeloma patients receiving study drug showed similar findings with an additional IgGκ band and quantifiable M-protein with similar migration patterns in specimens drawn after administration. CONCLUSION The treatment of multiple myeloma patients with monoclonal antibodies results in a visible and quantifiable M-protein that has the potential to falsely indicate poor response to therapy.

Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum

A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.

Sensitive simultaneous quantitation of testosterone and estradiol in serum by LC–MS/MS without derivatization and comparison with the CDC HoSt program

BACKGROUND Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. METHODS Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes. RESULTS The lower limits of quantitation (LLOQs) of E2 and Te were 5pg/mL and 1ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5-600pg/mL and 1-1,170ng/dL for Te. Assay accuracy was determined both by comparison with a LC-MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N=10) and between-day (N=20) CVs at concentrations spanning the AMR were less than 7% for both analytes. CONCLUSION We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC-MS/MS without chemical derivatization and using commercially available calibrators.

Analytical performance of the Abaxis Piccolo Xpress® point of care analyzer in whole blood, serum, and plasma.

OBJECTIVES To examine the analytical performance of 14 comprehensive metabolic panel analytes on the Abaxis Piccolo Xpress® Point of Care analyzer in serum, plasma, and whole blood. DESIGN AND METHODS Precision was evaluated by running two levels of control material over multiple days. Linearity was evaluated using material provided by the manufacturer and the College of American Pathologists (CAP) linearity surveys. Accuracy was evaluated by comparing the results from 60 patient specimens on the Piccolo Xpress® with the Ortho Vitros® 5600 analyzer. The method comparison was performed on all three specimen types intended for use on the Piccolo Xpress®: serum, heparinized plasma, and whole blood. Manufacturer suggested reference ranges for all 14 analytes were tested in serum and plasma specimens from 23 healthy volunteers. RESULTS High precision (CV ≤ 10%) was noted for all the analytes. Linearity was found to span the clinically useful range for all analytes. The method comparison demonstrated minimal proportional bias and good correlation for most of the analytes in all three matrices tested. The only exceptions were for sodium and total CO2, for which either significant proportional bias and/or poor correlation was noted in all three matrices. Significant bias was noted for AST in serum as well as for total bilirubin in plasma and whole blood. CONCLUSION The Piccolo Xpress® allows for the delivery of CMP results in a footprint small enough to be stored in a biological safety cabinet, while providing satisfactory performance for the majority of analytes.

D-lactic acidosis mediated neuronal encephalopathy in acute lymphoblastic leukemia patient: An under diagnosis

BACKGROUND D-lactic acidosis, also referred as D-lactate encephalopathy, has been reported in patients with short bowl syndrome (SBS). CASE REPORT The neurologic symptoms include altered mental status, slurred speech, and ataxia. Onset of neurological symptoms is accompanied by metabolic acidosis and high anion gap. We present here a case of D-lactic acidosis in a patient with acute lymphoblastic leukemia (ALL) who developed severe neurological symptoms and metabolic acidosis due to vancomycin-resistant enterococci (VRE) infection, and elevated D-lactic acid.

Diagnostic Utility of Measuring Free Light Chains in the Cerebrospinal Fluid of Patients With Multiple Myeloma

Multiple myeloma involvement of the central nervous system is rare, portends a poor prognosis, and requires rapid clinical intervention. The gold standard for diagnosis is cytology, which is highly insensitive. Abnormal free light chain detection in cerebrospinal fluid was more reliably concordant with clinical diagnosis of central nervous system multiple myeloma than cytology. Free light chain levels in cerebrospinal fluid dynamically correlated with response to therapy. Assessment of free light chains in cerebrospinal fluid can be included in the diagnostic algorithm to diagnose central nervous system multiple myeloma and monitor response to therapy.

Multiple Myeloma and Its Precursor Disease Among Firefighters Exposed to the World Trade Center Disaster

Importance The World Trade Center (WTC) attacks on September 11, 2001, created an unprecedented environmental exposure to known and suspected carcinogens suggested to increase the risk of multiple myeloma. Multiple myeloma is consistently preceded by the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and light-chain MGUS, detectable in peripheral blood. Objective To characterize WTC-exposed firefighters with a diagnosis of multiple myeloma and to conduct a screening study for MGUS and light-chain MGUS. Design, Setting, and Participants Case series of multiple myeloma in firefighters diagnosed between September 11, 2001, and July 1, 2017, together with a seroprevalence study of MGUS in serum samples collected from Fire Department of the City of New York (FDNY) firefighters between December 2013 and October 2015. Participants included all WTC-exposed FDNY white, male firefighters with a confirmed physician diagnosis of multiple myeloma (n = 16) and WTC-exposed FDNY white male firefighters older than 50 years with available serum samples (n = 781). Exposures WTC exposure defined as rescue and/or recovery work at the WTC site between September 11, 2001, and July 25, 2002. Main Outcomes and Measures Multiple myeloma case information, and age-adjusted and age-specific prevalence rates for overall MGUS (ie, MGUS and light-chain MGUS), MGUS, and light-chain MGUS. Results Sixteen WTC-exposed white male firefighters received a diagnosis of multiple myeloma after September 11, 2001; median age at diagnosis was 57 years (interquartile range, 50-68 years). Serum/urine monoclonal protein isotype/free light-chain data were available for 14 cases; 7 (50%) had light-chain multiple myeloma. In a subset of 7 patients, myeloma cells were assessed for CD20 expression; 5 (71%) were CD20 positive. In the screening study, we assayed peripheral blood from 781 WTC-exposed firefighters. The age-standardized prevalence rate of MGUS and light-chain MGUS combined was 7.63 per 100 persons (95% CI, 5.45-9.81), 1.8-fold higher than rates from the Olmsted County, Minnesota, white male reference population (relative rate, 1.76; 95% CI, 1.34-2.29). The age-standardized prevalence rate of light-chain MGUS was more than 3-fold higher than in the same reference population (relative rate, 3.13; 95% CI, 1.99-4.93). Conclusions and Relevance Environmental exposure to the WTC disaster site is associated with myeloma precursor disease (MGUS and light-chain MGUS) and may be a risk factor for the development of multiple myeloma at an earlier age, particularly the light-chain subtype.

Development of an Assay for Methotrexate and Its Metabolites 7-Hydroxy Methotrexate and DAMPA in Serum by LC-MS/MS

Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 μL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to 1.0 × 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.

A Simple and Sensitive Method for Quantitative Measurement of Methylmalonic Acid by Turbulent Flow Chromatography and Tandem Mass Spectrometry

A simple and sensitive method for the detection of methylmalonic acid in serum without derivatization has been developed. This method implements protein precipitation using methanol followed by additional sample clean up by turbulent flow liquid chromatography (TFLC). The sample was directly injected into the turbulent flow liquid chromatography tandem mass spectrometry system (TFLC-MS/MS) for online extraction followed by HPLC separation. The eluent was transferred to the mass spectrometer and ionized by heated electrospray negative ionization (HESI) and the analyte was quantified using a six-point calibration curve. The validated analytical measurement range (AMR) is 30–1,000 nMol/L. Dilutions of 10 and 200-fold were validated giving a clinical reportable range (CRR) of 30–200,000 nMol/L. The between-day and within-day imprecision values at concentrations spanning the AMR were less than 15%. This method was compared to an established LC-MS/MS method at a CLIA certified national reference laboratory and shows an excellent correlation with our TFLC-MS/MS method.

Pseudohypocalcemia in Cancer Patients: A Recommendation for the Postanalytical Correction of Serum Calcium in Patients with Hypoalbuminemia

To the Editor: Critical value reporting is a regulatory responsibility of all clinical laboratories. Efforts to reduce the number of calls that are not truly critical could have a significant impact on the clinical laboratory, medical team work flow, and patient care. At Memorial Sloan Kettering Cancer Center (MSKCC), 1 the clinical laboratory makes approximately 30 000 critical value calls a year with only 9 analytes accounting for nearly 75% of these calls. Calcium is one frequently called analyte, responsible for approximately 1000 critical calls a year. We evaluated the findings of critical hypocalcemia [critical value: <6.5 mg/dL (<1.62 mmol/L); reference interval (RI): 8.5–10.5 mg/dL (2.12–2.62 mmol/L)] in cancer patients with albumin concentrations <4.0 g/dL (RI: 4.0–5.2 g/dL). At MSKCC between 2013 and 2015, 38% of albumin results from a comprehensive metabolic panel (CMP) were below 4.0 g/dL. There are several published formulae to correct serum calcium in patients with hypoalbuminemia. The Payne formula {corrected total calcium (mg/dL) = total calcium (mg/dL) + 0.8 × [4.0 − albumin (g/dL)]} is the most widely used and based on the observation that each 1-g/dL reduction in albumin <4.0 g/dL …