An immunoassay for urinary extracellular vesicles.

Although nanosized urinary extracellular vesicles (uEVs) are increasingly used for biomarker discovery, their isolation currently relies on time-consuming techniques hindering high-throughput application. To navigate this problem, we designed an immunoassay to isolate, quantify, and normalize uEV proteins. The uEV immunoassay consists of a biotinylated CD9 antibody to isolate uEVs, an antibody against the protein of interest, and two conjugated antibodies to quantify the protein of interest and CD9. As a proof of principle, the immunoassay was developed to analyze the water channel aquaporin-2 (AQP2) and the sodium-chloride cotransporter (NCC). CD9 was used as a capture antibody because immunoprecipitation showed that anti-CD9 antibody, but not anti-CD63 antibody, isolated AQP2 and NCC. CD9 correlated strongly with urine creatinine, allowing CD9 to be used for normalization of spot urines. The uEV immunoassay detected AQP2 and NCC with high sensitivity, low coefficients of variance, and stability in dilution series. After water loading in healthy subjects, the uEV immunoassay detected decreases in AQP2 and NCC equally well as the traditional method using ultracentrifugation and immunoblot. The uEV immunoassay also reliably detected lower and higher AQP2 or NCC levels in uEVs from patients with pathological water or salt reabsorption, respectively. In summary, we report a novel approach to analyze uEVs that circumvents existing isolation and normalization issues, requires small volumes of urine, and detects anticipated changes in physiological responses and clinical disorders.

The hidden secrets of a neutral pH-blood gas analysis of postoperative patients according to the Stewart approach.

BACKGROUND: The superiority of either the traditional or Stewart based approach to acid-base balance has focused primarily on analyzing metabolic acidemia, with little attention given to patients with neutral pH. In this study, we evaluate metabolic disturbances in patients in the immediate postoperative period focusing on patients with neutral pH, while comparing the Stewart and traditional approach. METHODS: We conducted a single center retrospective observational cohort study. Over a 17-month period, data on arterial blood gas analysis, electrolytes, and albumin on the morning after surgery were retrieved from patients admitted to the postsurgical high dependency unit (HDU). Albumin-corrected anion gap (AG), apparent (SIDa) and effective strong ion difference (SIDe), and strong ion gap (SIG) were calculated. RESULTS: Out of 1207 HDU admissions, 400 cases had a complete set of laboratory-data including albumin of which 281 presented with neutral pH (7.35 ≤ pH ≤ 7.45), 64 with acidemia (pH < 7.35) and 55 with alkalemia (pH > 7.45). In pH neutral patients, the following acidifying disturbances were found: SIDa was lowered in 101 (36%), and SIG was raised in 60 (21%). Base excess (BE) was decreased in 16 (6%) and corrected AG raised in 107 (38%). The alkalizing effect of hypoalbuminemia was present in 137 (49%). Out of 134 cases with normal BE and corrected AG, SIDa was lowered in 58 (43%). Out of 136 cases with normal SIDa and SIG, none had lowered BE and 28 increased AG (21%). Length of stay was significantly longer in patients with hypoalbuminemia, lowered SIDa, and increased corrected AG, but not decreased BE (hypoalbuminemia: 16 days vs. 10 days, P < 0.001; low SIDa: 15 days vs. 12 days, P = 0.015; increased AG: 16 days vs. 11 days, P < 0.001; low BE: 14 days vs. 13 days, P = 0.736). CONCLUSIONS: Metabolic disturbances, characterized mainly by the presence of lowered SIDa, increased AG, and hypoalbuminemia, are frequent in our population with apparent neutral acid-base balance based on pH and base excess. These changes on the morning after surgery are associated with increased length of stay.