Creatinine clearance in selection of living kidney donor among the Malaysian population: is it safe?

BACKGROUND: Assessment of donor renal function is made by the measurement of Glomerular Filtration Rate (GFR). Exogenous markers are preferred over creatinine clearance and are widely used for measuring GFR. However, they are difficult to obtain, costly and laborious. This is a study to look into the safety and accuracy of creatinine clearance for renal assessment among the living kidney donors in the Malaysian population. METHODS: This is a retrospective, single-centre study comprising 105 living kidney donor candidates from the year 2007 to 2020. By comparing against 51-Chromium ethylenediamine-tetraacetic acid (51Cr-EDTA), we analysed creatinine clearance for correlation, bias, precision and accuracy. RESULTS: The study group had a mean age of 45.68 ± 10.97 years with a mean serum creatinine of 64.43 ± 17.68 µmol/L and a urine volume of 2.06 ± 0.83 L. Mean measured GFR from 51Cr-EDTA was 124.37 ± 26.83 ml/min/1.73m2 whereas mean creatinine clearance was 132.35 ± 38.18 ml/min/1.73m2. Creatinine clearance overestimated 51Cr-EDTA significantly with a correlation coefficient of 0.48 (p < 0.001) and an accuracy of 78.10% and 64.0% within 30% and 20% respectively of 51Cr-EDTA. CONCLUSION: Creatinine clearance is an acceptable and affordable alternative for donor renal assessment in the absence of exogenous markers with an emphasis on adequate urine collection followed by using measured GFR in selected cases.

Effect of indexed glomerular filtration rate according to age.

BACKGROUND: To find out up to what age there is dispersion in glomerular filtration rate (GFR) values when normalised by body surface area (BSA) and extracellular fluid volume (ECFV), based on the hypothesis that these values differ in childhood age. METHODS: A retrospective study was performed on patients aged 0-85 years, with renal pathology, who were administered i.v. 51Cr-EDTA. GFR was obtained using the Ham and Piepsz (children) or Christensen and Groth (adults) formula. Results were normalised by BSA and ECFV. RESULTS: The cut-off point is defined as the age that discriminates between values ± 10 points apart. Using a ROC curve analysis, this age was set at 11.96 years with a sensitivity of 0.83 and a specificity of 0.85. The area obtained was 0.902 (95%CI 0.880-0.923). The results were corroborated by linear regression stratifying by age. For children under 12 years of age, the Pearson correlation was 0.883 (95% CI 0.860-0.902). For those aged 12 years or older, this coefficient was 0.963 (95%CI 0.957-0.968). According to our results, when normalising GFR by BSA and by ECFV there are different behaviours according to age. CONCLUSION: For children older than 12 years both normalisation methods can be used, but for children younger than 12 years there are differences. We believe that in children under 12 years of age, GFR should be normalised by ECFV.

NK Cell Isolation and Cytotoxicity by Radioactive Chromium Release Assay and DELFIA-EuTDA Cytotoxicity Assay.

Cytotoxicity assays are important in vitro tools to measure the lysis of desired target cells via an effector immune cell of choice. Specific lysis of the target cells can be determined by labeling the target cells with a radioactive isotope or fluorescent molecule, co-incubating it with an effector cell, then measuring the release of the labeled molecule in the supernatant. Here, we describe and compare different cell cytotoxicity assays using a chromium-51 (51Cr) release and DELFIA EuTDA fluorescent assay using K562 as the target cells and peripheral blood mononuclear cell (PBMC) derived natural killer (NK) cells as the effector cells.

Age-specific normal reference ranges for 99mTc-DTPA glomerular filtration rate to use with two-sample slope-intercept method and Jodal Brochner-Mortensen correction.

Glomerular filtration rate (GFR) varies with age, the calculation method, and the correction factor for slope-intercept overestimation. Hence, any normal reference range accompanying the results should be suitably adapted to the method used. For Nuclear Medicine Departments using a two-sample slope-intercept method, the lack of appropriate age-specific normal reference range has been a hindrance to adopting the recently updated Jodal Brochner-Mortensen (JBM) correction over other older and more widely used methods. A retrospective analysis of the routine GFR calculation and clinical reports generated locally from 2006 to 2020 was carried out. GFR was calculated with 99mTc-DTPA plasma clearance using a two-sample slope-intercept method with JBM correction. Age-specific normal range equations were developed from normal healthy subjects. Published normal reference ranges were modified with appropriate correction reversal and compared with the locally developed reference ranges. Age-specific normal GFR reference ranges for 99mTc-DTPA with slope-intercept method and JBM correction were developed and validated with current literature. Normal reference range (Mean ± 2SD) for Normalised GFR (ml min-1 (1.73m2)-1) within 95% confidence limits suitable for use with JBM correction is 100.6 ± 35.2 for children above 2 years and 102.9 - 0.00629 × (Age)2 ± 19.4 for adults. Availability of age-specific normal GFR reference ranges applicable to the target population and appropriately tailored to the calculation method and correction factor enables Nuclear Medicine Departments to update their calculation methods in line with the current literature and also facilitates accurate reporting and evaluation of the calculated GFR results.

Reduced erythrocyte lifespan measured by chromium-51 in patients with type 2 diabetes undergoing long-term hemodialysis.

INTRODUCTION: A reduced erythrocyte lifespan potentially explains the low hemoglobin A1c values found in hemodialysis patients. However, data supporting this notion in patients with type 2 diabetes is unclear. We evaluated the erythrocyte lifespan in patients with type 2 diabetes undergoing long-term hemodialysis and investigated potential predictors of erythrocyte lifespan. METHODS: Long-term hemodialysis patients with type 2 diabetes and type 2 diabetes patients without nephropathy (estimated glomerular filtration rate > 60 mL/min/1.73 m2 ) were included. The erythrocyte lifespan was measured using chromium-51 (51 Cr)-labeled erythrocytes. Blood radiotracer activity was measured six to nine times over a period of 3-5 weeks to determine the erythrocyte lifespan of each patient. Biochemical markers were obtained five times over 16 weeks and associated with the erythrocyte lifespan. FINDINGS: Type 2 diabetes patients undergoing hemodialysis (N = 13) had a significantly shorter median erythrocyte lifespan of 49.7 (interquartile range [IQR] = 44.1-58.6) days compared with 64.2 (IQR = 62.6-83.5) days in the control group (N = 10) (P ˂ 0.001) with a difference between medians of 14.5 (95% confidence interval = 8.1-38.8) days. In the hemodialysis group, no association could be detected between the erythrocyte lifespan and markers of hemolysis, level of inflammation, or urea. DISCUSSION: A reduced erythrocyte lifespan was detected in type 2 diabetes patients undergoing long-term hemodialysis. This may contribute to the reduced hemoglobin A1c values observed in the type 2 diabetic hemodialysis population. An association could not be detected between the erythrocyte lifespan and biochemical markers of hemolysis or inflammation.

Glomerular filtration rate: comparison of simultaneous plasma clearance of 99mTc-DTPA and 51Cr-EDTA revisited.

The issue of whether 99mTc-DTPA can replace 51Cr-EDTA for measurement of plasma clearance as a surrogate for glomerular filtration rate (GFR) is of great relevance to daily clinical practice. Prompted by the shortage of 51Cr-EDTA we conducted a head-to-head comparison in patients attending our department for GFR determination. The two tracers (3.7 MBq of 51Cr-EDTA and 8 MBq of 99mTc-DTPA) were administered intravenously immediately after each other, and the standard number of blood samples were drawn. Fifty-four patients were enrolled. In 51 of these, single-sample measurement was performed with the following results: GFREDTA was 84.6 ± 23.3 mL/min, GFRDTPA was 84.2 ± 24.7 mL/min. The mean difference was 0.4 ± 2.8 mL/min, p = 0.32, and results based on the two tracers were highly correlated (r = 0.995). GFRDTPA exceeded GFREDTA at high GFR values (difference < 0 at GFREDTA >91.4 mL/min) and vice versa (difference > 0 at GFREDTA < 91.4 mL/min). However, differences fell within few GFR units that most often will have no clinical consequence. We therefore conclude that 99mTc-DTPA can replace 51Cr-EDTA for single-sample determination of GFR in a clinical setting.

51Cr-release to monitor NK cell cytotoxicity.

Natural killer (NK) cells are cytotoxic lymphocytes that belong to the innate lymphoid cells. They have the ability to attack tumor cells that lack ligands of inhibitory NK receptors and/or express ligands of activating NK receptors. The most important ligands of inhibitory NK receptors are major histocompatibility complex (MHC) class I molecules, which are down regulated on many tumor cells. Several ligands of activating NK receptors, in contrast, are up regulated on tumor cells due to cellular and genotoxic stress. Due to these features, NK cells constitute an important element of the tumor immunosurveillance and are explored as a cellular tool for tumor immunotherapy. Therefore, it is important to be able to monitor the cytotoxic activity of NK cells reliably in preclinical animal models as well as in patients. It is also of interest to determine the susceptibility of tumor cells toward NK cell-mediated cellular cytotoxicity. The 51Cr-release assay monitors the lysis of target cells by NK cells and is suitable to address both questions. We describe here the experimental set-up of this classic cytotoxicity assay in detail.

A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry.

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.

Ikaros deficiency in host hematopoietic cells separates GVL from GVHD after experimental allogeneic hematopoietic cell transplantation.

The graft-versus-leukemia (GVL) effect following allogeneic hematopoietic stem cell transplantation (allo-HCT) is critical for its curative potential. Hwever, GVL is tightly linked to graft-versus-host disease (GVHD). Among hematological malignancies, acute lymphoblastic leukemia (ALL) is the most resistant to GVL, although the reasons for this remain poorly understood. Clinical studies have identified alterations in Ikaros (Ik) transcription factor as the major marker associated with poor outcomes in ALL. We have shown that the absence of Ik in professional host-derived hematopoietic antigen-presenting cells (APCs) exacerbates GVHD. However, whether Ik expression plays a role in resistance to GVL is not known. In this study we used multiple clinically relevant murine models of allo-HCT to explore whether Ik expression in hematopoietic APCs and/or leukemic cells is critical for increasing resistance to GVL and thus inducing relapse. We found that Ik deficiency in host APCs failed to enhance GVL despite increased GVHD severity. Mechanistic studies with bone marrow (BM) chimeras and tetramer analyses demonstrated reduced tumor-specific immunodominant (gag+) antigen responses in the [B6Ik-/-→B6] group. Loss of GVL was observed when both the leukemia cells and the host APCs were deficient in Ik. We found that calreticulin (CRT) expression in host antigen-presenting dendritic cells (DCs) of Ik-/- animals was significantly lower than in wild-type animals. Rescuing CRT expression in Ik-/- DCs improved leukemic-specific cytotoxic T cell function. Together, our data demonstrate that the absence of Ikaros in host hematopoietic cells promotes resistance to GVL despite increasing GVHD and thus provides a potential mechanism for the poor outcome of Ik-/- ALL patients.