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ABSTRACT: We have previously reported a flow rate calibration method for the determination of absolute CD4(+) T-lymphocyte counts that removes the need for the addition of latex beads to each sample. However, a limitation with this approach is that a calibration factor (CF) needs to be applied to adjust for differences in viscosity between latex bead suspensions and biological specimens. We have also demonstrated the value of using stabilized whole blood samples in external quality assessment (EQA) studies; such samples have a stable absolute lymphocyte count for over 1 year, at 4 degrees C. It was successfully demonstrated that this material can be used as a flow rate biocalibration (FRB) material for use as a flow cytometric control to provide a sample with a known CD4(+) T-lymphocyte count. Such material has advantages over latex bead technology as it can act as a full process control as well as having the same matrix and viscosity characteristics as the test material, thus removing the need for a CF.
In this study, we have analyzed 268 consecutive normal, abnormal, and HIV(+) samples using FRB, incorporating the PanLeucoGating approach and compared this to the MultiSet method, defined as the predicate.
Percentage similarity statistics revealed the following: 0-3,000 CD4(+) cells/mul mean percentage difference (MPD; bias) 1.2%, 95% CI of 5.6-8%; 0-200 CD4(+) cells/microl MPD of 1.25%, 95% CI of 11.63-14.13%; 201-500 CD4(+) cells/microl MPD of 1%, 95% CI of 4.6-6.6%.
This study demonstrates that stabilized whole blood can be used for FRB. It has the advantage of being a full process control, in addition to costing less than latex beads with highly comparable results. As bench top flow cytometers are extremely stable, this is a low cost and robust alternative to bead based methods for generating absolute CD4 counts.
Cytometry Part B Clinical Cytometry 06/2006; 70(3):154-62. · 2.53 Impact Factor
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ABSTRACT: The derivation of reliable CD4(+) T lymphocyte counts is vital for the monitoring of disease progression and therapeutic effectiveness in HIV(+) individuals. Flow cytometry has emerged as the method of choice for CD4(+) T lymphocyte enumeration, with single-platform technology, coupled with reference counting beads, fast becoming the "gold standard." However, although single-platform, bead-based, sample acquisition requires the ratio of beads to cells to remain unchanged, there is no available method, until recently, to monitor this.
Perfect Count beads have been developed to address this issue and to incorporate two bead populations, with different densities, to allow the detection of inadequate mixing. Comparison of the relative proportions of both beads with the manufacture's defined limits enables an internal QC check during sample acquisition. In this study, we have compared CD4(+) T lymphocyte counts, obtained from 104 HIV(+) patients, using TruCount beads with MultiSet software (defined as the predicated method) and the new Perfect Count beads, incorporating an in house sequential gating strategy.
We have demonstrated an excellent degree of correlation between the predicate method and the Perfect Count system (r(2) = 0.9955; Bland Altman bias +27 CD4(+) T lymphocytes/microl).
The Perfect Count system is a robust method for performing single platform absolute counts and has the added advantage of having internal QC checks. Such an approach enables the operator to identify potential problems during sample preparation, acquisition and analysis.
Cytometry Part B Clinical Cytometry 02/2004; 57(1):47-52. · 2.53 Impact Factor
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ABSTRACT: Uncontrolled apoptosis contributes to tubular cell deletion in renal scarring. Caspase-3 has a central role in the execution of apoptosis and may provide a target for regulating cell death. Here we evaluate three caspase inhibitors: B-D-FMK (pan caspase inhibitor), Z-DEVDFMK (predominantly Caspase-3 inhibitor) and Z-VAD-FMK (predominantly Caspase-1 and -3 inhibitor) to ameliorate apoptosis induced by cisplatin in rat proximal tubular (RPT) cells.
Caspase-3 activity (substrate cleavage assay) and protein (immunocytochemistry and Western blotting), apoptosis (Annexin V flow cytometry, in situend labelling of fragmented DNA, light/electron microscopy and DNA laddering) and cell survival (trypan blue exclusion and propidium iodide flow cytometry) were determined in RPT cells exposed to cisplatin with and without caspase inhibitors.
Cisplatin induced a dose-dependent increase in Caspase-3 activity and 8-fold of increase in apoptosis (p < 0.01) when applied for 24 h at 100 microM. B-D-FMK (40 microM), Z-DEVD-FMK (15 microM) and Z-VAD-FMK (22 microM) almost completely inhibited the 25-fold increase in Caspase-3 activity and decreased apoptosis from 15.9 +/- 4.4 to 2.0 +/- 0.6% (p < 0.01), 15.0 +/- 2.2 and 15.0 +/- 2.2% respectively. DNA ladders were visible in cisplatin-treated cells, which disappeared following addition of B-D-FMK and decreased with Z-VAD-FMK and Z-DEVD-FMK. Cisplatin reduced cell survival to 61% by trypan blue exclusion. B-D-FMK and Z-VAD-FMK increased this to 87 and 75%, but Z-DEVD-FMK had no significant effect.
Cisplatin causes an increase in RPT apoptosis that is associated with increased Caspase-3 activity. All caspase inhibitors were equally effective at reducing Caspase-3 activity, however the pan caspase inhibitor B-D-FMK was more effective at preventing apoptosis and increasing cell survival. Therefore, pan caspase inhibition offers the greatest potential for the prevention of renal tubular cell deletion by uncontrolled apoptosis.
Nephron Experimental Nephrology 01/2004; 96(2):e39-51. · 1.86 Impact Factor
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ABSTRACT: CD4(+) T-lymphocyte enumeration is vital for monitoring disease progression in individuals positive for the human immunodeficiency virus (HIV), and as a result, there is a need to develop cost-effective protocols that provide accuracy, precision, and affordability. Recently, PanLeucoGating has been shown to fulfill these requirements; however, although comparable to state-of-the-art single-platform protocols (SP), there is still a requirement for an accurate total white cell count. To overcome this limitation, we recently developed a flow-rate based calibration method that enables the PanLeucoGating protocol to be used as a SP approach, and in this study show that this approach can be used for CD4(+) T-lymphocyte enumeration.
A total of 113 HIV samples were analyzed using three protocols: (a) state-of-the art SP bead-based method (MultiSet; predicate protocol), (b) PanLeucoGating protocol used as a dual-platform (DP) approach, and (c) the newly developed flow rate-based SP approach. We demonstrate that flow rate calibration can be achieved easily and that the method is highly comparable to the state-of-the-art SP method.
A high correlation was observed between the predicate protocol and the SP PanLeucoGating approach over the whole range of CD4 counts tested (r(2) = 0.9928; bias 8 cells/microl), including the clinically relevant range (e.g., 0-200 CD4 cells/microl; bias 0 cells/microl). For batched samples, the cost of providing a CD4(+) T-lymphocyte count was reduced to approximately US $1.
The SP PanLeucoGating is a cost-effective approach to CD4(+) T-lymphocyte enumeration that maintains accuracy and precision.
Cytometry Part B Clinical Cytometry 10/2003; 55(1):8-13. · 2.53 Impact Factor
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ABSTRACT: Reports suggest that flow rate (FR) is constant on bench top flow cytometers. Therefore, if FR is constant, the volume acquired in a fixed time period will also be constant, enabling absolute leucocyte counting using flow rate calibration (FRC).
FR stability was ascertained on a standard FACSCalibur by counting TruCount beads suspended in phosphate buffered saline over 120 s. Studies using two lysing solutions (FACS lysing solution and PharM Lyse) and corresponding sample lysates established a lysing solution calibration factor (CF). Absolute CD4(+) T-lymphocyte counts on 10 peripheral blood samples determined using FRC were compared with the predicate method TruCount/MultiTEST, incorporating MultiSET software. Linearity studies were also performed at three different flow rates.
A high degree of linearity over a wide range of counts (50 to >1,600 CD4(+) T lymphocytes/microl) at all three pressures was observed. Importantly, there was no significant difference from the predicate method when appropriate lysing solution CF was used.
Using a simple calibration procedure and incorporation of an appropriate lysing solution CF, we show that FRC can easily be performed. The technical details that underpin this novel approach for absolute leucocyte enumeration are provided.
Cytometry Part B Clinical Cytometry 09/2003; 55(1):1-7. · 2.53 Impact Factor
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ABSTRACT: Major causes of interlaboratory variation in low-level WBC counting are the gating strategies and staining methods employed. To overcome these limitations, a stable low-level WBC control preparation (termed daily run control [DRC]) was developed that when coupled with a new gating strategy will enable international standardization.
Both a whole blood preparation (stability of more than 12 months; target WBC count of 20 cells/microL with defined fluorescence values) and a new gating strategy were developed and used with a staining kit (LeucoCOUNT [Becton Dickinson BioSciences] providing the basis for standardization). These were then combined and used to crosscalibrate seven different flow cytometers. After standardization with the DRC, comparative studies were undertaken with fresh samples with a WBC range of <1 to 60 cells per microL.
The developed gating strategy enabled the DRC WBCs to be positioned to within four channels of the expected target fluorescence 1 value (2.3% variation) and within three channels of the target fluorescence 2 value (0.7% variation) on all evaluated instruments. Subsequent analysis of any sample meant that the WBCs always occupied the same "sample space," irrespective of flow cytometer platform and without the need for repositioning of the analysis region and/or gate.
The cross calibration and standardization of flow cytometers used for low-level WBC counting (irrespective of platform) are attainable with this United Kingdom National External Quality Assessment Scheme strategy. Its adoption should reduce interlaboratory CVs and provide a practical approach for the rapid identification of operator and machine problems.
Transfusion 06/2002; 42(6):738-46. · 3.22 Impact Factor
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ABSTRACT: The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4(+) T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4(+) T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration.
Cytometry 05/2002; 50(2):102-10.
