Dolan, Christopher

Abstract Objectives To determine the influence of pH on recovery of analytes in body fluids (BFs), investigate the mechanism of pH interference, measure the frequency of abnormal-pH BFs received, and compare pH measured by meter and paper. Methods We performed pH titration in residual BFs. A low-pH BF was spiked and neutralized to investigate pH interference. We measured analytes on a Roche cobas c501 analyzer (Roche Diagnostics) and calculated the percent recovery. Measurement of pH using a meter and paper was conducted on 122 BF samples received in the laboratory. Results Enzyme activity in BFs was unaffected when pH = 7.4-8.5 lactate dehydrogenase, pH = 7.3-10.2 amylase, pH = 6.0-9.9 lipase, and pH = 1.3-11.7 all other analytes. BFs had mean (range) pH of 8.0 (5.1-8.9), with a mean (range) difference (paper ‒ meter) of ‒0.4 (‒0.6 to 1.1). Conclusions Irreversible loss of enzyme activity occurs in BFs at low pH. Few clinical BFs have pH < 7.0, but laboratories should incorporate pH measurement in BF workflows.

Abstract Introduction Measurement of hemoglobin A1c (HbA1C) is used for the diagnosis and management of patients with diabetes. Methods for measuring HbA1C are classified on the basis of charge differences (cation exchange chromatography) or structural differences (boronate affinity chromatography). Some cation exchange high-performance liquid chromatography (HPLC) analyzers may be prone to interferences from hemoglobin variants. Historically, our lab used two methods to report HbA1C results: cation exchange HPLC (VariantII) with reflex to boronate affinity HPLC (Ultra2) methods. A new analyzer (BioRad D-100) with improved interference detection and thresholds for interference was evaluated. The objectives of this study were (1) assess the comparability of HbA1c results between D-100, VariantII, and Ultra2; (2) evaluate the need for maintaining a reflex method; and (3) calculate cancellation rates before and after implementing D-100. Methods HbA1c was measured by cation exchange methods using VariantII Turbolink, D-100 (BioRad), and boronate affinity using Ultra2Affinity (Trinity Biotech) according to the manufacturer’s recommendations with the following analytical measuring ranges (AMRs), 4.0% to 17.6%, 4.0% to 18.0%, and 4.0% to 17.6%, respectively. D-100 was compared to VariantII (n = 26) and Ultra2 (n = 31) using residual patient samples from provider-ordered HbA1c having no chromatography flags and with the following chromatography flags on VariantII (n = 113): variant window (n = 41), HbA1c <4.0% (n = 24), HbA1a + HbA1b >5% (n = 19), HbF >5% (n = 9), P3 >10% (n = 6), HbA1c >17.6% (n = 4), unknown peak (n = 3), labile 5% to 15% (n = 3), labile >15% (n = 3), and HbS >60% (n = 1). The cancelation rates were calculated at baseline (January 1-31, 2018) and postimplementation of D-100 (September 20, 2018, to January 22, 2019). Results HbA1c results from D-100 were within ±0.3 or 5% with the following frequencies: Ultra2 in 96.2% (25/26) with VariantII in 96.8% (30/31) using samples with no chromatography flags. A total of 113 samples with abnormal VariantII flags were tested using D-100 with 82% (n = 92) not exceeding interference limits for result reporting. These D-100 results were within ±0.3 or 5% of Ultra2 results with the following frequencies: 38.5% (10/26) of samples below (n = 23) or above (n = 3) the AMR, 85.3% (29/34) of results with Hb variant flags, 68.0% (17/25) with unknown/minor peaks, and 71.4% (5/7) with HbF <15%. The clinical concordance was also assessed according to the following decision limits: normal <5.6%, prediabetic = 5.7% to 6.4%, and diabetic >6.5%. Results were concordant in 88% with variant peaks (12/13 w/HbA1c <5.6%, 8/11 w/HbA1c = 5.7%-6.4%, 10/10 w/HbA1c >6.5%), 84% with minor peaks (5/6 w/HbA1c <5.6%, 0/2 w/HbA1c = 5.7%-6.4%, 16/17 w/HbA1c >6.5%), and 86% with HbF (3/3 w/HbA1c <5.6%, 2/3 w/HbA1c = 5.7%-6.4%, 1/1 w/HbA1c >6.5%). Results above/below AMR had 100% concordance (>16.0% or <4.4%). The frequency of VariantII rule violation was 2.1% in January 2018, with 53 of 117 having results confirmed as <4.0% or >16.0% by Ultra2, 2 of 117 reported by Ultra2, and 62 being cancelled due to HbF >15% (n = 23) or variant Hb >60% (n = 39). The frequency of rule violation using only D-100 since implementation was 2.2%. Conclusion Implementation of D-100 has removed the need for boronate affinity reflex testing and maintained similar cancellation rates.

Abstract Background Body fluid samples (BFs) with low pH have demonstrated underrecovery of enzyme activity. The aims of this study were (1) measure the frequency of abnormal pH in BFs received for clinical chemistry testing, (2) calculate recovery of analytes in BFs undergoing pH titration, and (3) investigate the mechanism of pH interference. Methods Residual BFs submitted for physician-ordered testing to the Central Clinical Laboratory (Mayo Clinic) were utilized for this study. BFs were centrifuged 10 minutes (3,200 rpm) prior to pH measurement (pH meter, Accumet X15; Fisher Scientific) and analysis. A Roche Cobas c501 (Roche Diagnostics) analyzer was used to measure amylase, lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine, glucose, albumin, total protein, bilirubin (all Roche Diagnostics reagents), and lipase (Sekisui Diagnostics). pH was measured in 122 BFs received between 10/23/2018 and 10/26/2018 within 24 hours of receipt. pH titration was performed using pooled BFs of similar source/type. Each pool was titrated over a range of pH (≤2 to ≥10) in 1-unit increments by addition of a fixed volume of acid (6M HCL) or base (6M NaOH). Analytes were measured before and after acid/base addition. Average percent recovery was calculated (measured/expected × 100%) from n = 3 to 9 pH measurements. A low pH BF pool was spiked (<5% by volume) with a high-concentration BF to investigate mechanism of enzyme underrecovery. Additionally, low pH BF pool was neutralized by addition of base and % recovery calculated. For all titration/spiking experiments, a control sample having the same volume of diluent (7% bovine serum albumin or saline) was used to account for the effects of dilution. Results BFs received (n = 122) had mean (SD) pH = 8.0 (0.6) with 6.6% (n = 8) having pH <7.0 and 2.5% (n = 3) having pH <6.0. All recovery (%) and pH data are expressed as mean (range) values. Amylase recovery (initial pH = 8.4-8.9) was 1.3 (0.9-2.2)% at pH = 1.6 (1.0-2.2) (n = 6), 55.6 (37.4-67.0)% at pH = 4.4 (3.5-5.4) (n = 6), 81.4 (74.0-85.2)% at pH = 6.2 (6.1-6.5) (n = 3), 90.1 (84.4-95.4)% at pH = 7.5 (7.3-7.7) (n = 3), 98.8 (96.2-101.7)% at pH = 10.0 (9.8-10.2) (n = 3), and 14.5 (1.4-38.9)% at pH = 11.9 (11.5-12.1) (n = 3). LDH recovery (initial pH = 8.2-8.5) was 1.4 (0.1-3.7)% at pH = 1.7 (1.0-2.4) (n = 6), 18.3 (0.5-39.7)% at pH = 4.5 (3.5-5.4) (n = 6), 63.6 (54.9-68.4)% at pH = 6.2 (6.1-6.5) (n = 3), 85.9 (80.2-90.7)% at pH = 7.2 (6.8-7.4) (n = 3), 46.1 (36.4-61.0)% at pH = 10.0 (9.8-10.2) (n = 3), and 9.8 (0.0-28.9)% at pH = 11.3 (10.4-12.1) (n = 3). Lipase recovery (initial pH = 8.2-8.9) was 10.4 (<1-18)% at pH = 1.8 (1.0-2.4) (n = 6), 73.9 (62.6-85.5)% at pH = 4.2 (3.4-5.2) (n = 3), 92.4 (90.8-93.5)% at pH = 6.2 (6.0-6.5) (n = 3), 96.3 (95.6-96.8)% at pH = 7.3 (6.8-7.7) (n = 3), and 92.9 (89.3-96.8)% at pH = 9.9 (9.8-10.1) (n = 3) and 80.9 (0.0-78.6)% at pH = 11.3 (10.4-12.1) (n = 3). Creatinine, BUN, albumin, glucose, total protein, and bilirubin recovery were 97.9 (92.2-102.9)%, 100 (96.6-103.1)%, 99.9 (97.6-102.4)%, 100.2 (97.8-103)%, 100.2 (98.7-101.9)%, and 100.1 (92.-105.9)%, respectively, between pH 1.3-11.7 (n = 9). Recovery of amylase, LDH, or lipase was <1% after spiking enzymes into BF pool with pH <3. pH adjustment to normal (pH = 8.6-9.0) also resulted in recovery of <1%. Conclusion Enzyme activity in BFs was not affected (90%-110% recovery) when pH = 7.4-8.5 for LDH, pH = 7.3-10.2 for amylase, pH = 6.0-9.9 for lipase, and pH = 1.3-11.7 for all other analytes. An irreversible loss of enzyme activity occurs at low pH. Few clinical BFs have pH <7.0, but laboratories should have awareness.