Nandakumar, Vijayalakshmi

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 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.

Abstract Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of macrophage-inducible C-type lectin (Mincle) for host defense against Pneumocystis. Binding assays implementing soluble Mincle carbohydrate recognition domain fusion proteins demonstrated binding to intact Pneumocystis carinii as well as to organism homogenates, and they purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with PCP expressed increased Mincle mRNA levels. Mouse macrophages overexpressing Mincle displayed increased binding to P. carinii life forms and enhanced protein tyrosine phosphorylation. The binding of P. carinii to Mincle resulted in activation of FcRγ-mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after P. carinii challenge, critical in downstream inflammatory signaling. Mincle-deficient CD4-depleted (Mincle−/−) mice showed a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina pneumonia. Interestingly, Mincle−/− mice did not demonstrate worsened survival during PCP compared with wild-type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle−/− mice. Of note, the P. murina–infected Mincle−/− mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared with infected wild-type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection.

ABSTRACTWe explored differential polarization of macrophages during infection using a rat model ofPneumocystispneumonia. We observed enhanced pulmonary M1 macrophage polarization in immunosuppressed (IS) hosts, but an M2 predominant response in immunocompetent (IC) hosts followingPneumocystis cariniichallenge. Increased inflammation and inducible nitric oxide synthase (iNOS) levels characterized the M1 response. However, macrophage ability to produce nitric oxide was defective. In contrast, the lungs of IC animals revealed a prominent M2 gene signature, and these macrophages effectively elicited an oxidative burst associated with clearance ofPneumocystis. In addition, duringP. cariniiinfection the expression of Dectin-1, a critical receptor for recognition and clearance ofP. carinii, was upregulated in macrophages of IC animals but suppressed in IS animals. In the absence of an appropriate cytokine milieu for M2 differentiation,Pneumocystisinduced an M1 response bothin vitroandin vivo. The M1 response induced byP. cariniiwas plastic in nature and reversible with appropriate cytokine stimuli. Finally, we tested whether macrophage polarization can be modulatedin vivoand used to help manage the pathogenesis ofPneumocystispneumonia by adoptive transfer. Treatment with both M1 and M2 cells significantly improved survival ofP. carinii-infected IS hosts. However, M2 treatment provided the best outcomes with efficient clearance ofP. cariniiand reduced inflammation.