1. Jin Zhang, Valentin Quesada, Juan De Castro, Jennifer Zawislak, Jorge Acevedo, Ed Jachimowicz, and Liliana Tejidor Beckman Coulter, Inc., Cellular Analysis Business Group, Miami, FL Abstract Background. The measurement of CD4+ cells in HIV-infected patients has evolved into a routine and high volume test in flow cytometry. CD4+ cell enumeration is measured with differing combinations of monoclonal antibodies, balancing performance with cost. A good balance is the PLG CD4 assay, using two monoclonal antibodies: anti-CD45 for resolving leukocytes and anti-CD4 for enumerating CD4+ cells. The current PLG assay is semi-automated and allows for a maximum of 32 blood samples in tubes to be processed in a batch. Methods. We have modified the FlowCare™ PLG CD4 assay so that the addition of blood, antibodies, lysing, quenching, fixation reagents, and Flow-Count™ Fluorospheres is performed in 96-well plates by an automated sample and liquid handling system (BioMek ® NX P ). The plate is then transferred to a flow cytometer equipped with a multi-plate reader for enumeration of CD4+ cells. For identification purposes, each individual sample is tracked throughout by bar code. There is an approximately 30% reduction in time between the semi-automated and automated systems. Results. Performance testing of 24 non-clinical and 70 HIV samples demonstrated a significant correlation between the FlowCare™ PLG CD4 assay prepared in 96-well plates on the BioMek ® NX P and the semi- automated tube method (r2 = 0.99). Aging studies with 9 non-clinical and 15 HIV specimens (up to 5 days post blood draw) showed no significant change in the absolute CD4+ counts and percent of CD4+ cells (p = 0.41/0.99). This application is undergoing clinical evaluation. Conclusions. The results indicate that the PLG assay using automated sample preparation in a 96-well plate system gives comparable performance to the semi-automated tube method. The ability to process larger batches of clinical samples in a shorter period of time allows for reduced costs, higher throughput, and more efficient use of laboratory staff. Suspension Medium 1 Xanthine compound 2.0-7.0 g Adenosine monophosphate 0.2-0.8 g Inosine0.2-0.8 g pH adjusting agents sufficient to obtain pH 6.0-6.5 Osmolarity adjusters sufficient to obtain250-350 mOsm/kg H2O Preservative 2.0-6.0 g QS to 1 liter with reagent grade water Suspension Medium 2 Propyl paraben 0.3 to 1.0 g Methyl paraben 0.5 to 1.0 g Procaine hydrochloride0.1 to 0.5 g Deoxycholic acid0.1 to 0.9 g Lactose 0.0 to 50.0 g Actidione0.1 to 0.6 g Trisodium citrate dehydrate 3.0 to 8.0 g Citric acid monohydrate 0.3 to 0.9 g Sodium dihydrogenphosphate monohydrate 0.8 to 2.5 mg Phenergan hydrochloride0.1 to 1.0 g Colistimethate, sodium 0.2 to 0.9 g Penicillin G., sodium 1.75 x 106 units Kanamycin sulfate0.2 to 0.8 g Neomycin sulfate 0.2 to 1.0 g 5’-AMP 0.4 to 1.0 g Adenine 0.2 to 0.8 g Inosine 0.4 to 1.0 g Dihydrostreptomycin sulfate0.2 to 1.0 g Tetracycline hydrochloride0.2 to 1.0 g 30% Bovine albumin 100 to 350 ml QS to 1 liter with reagent grade water Preparation of immature granulocyte analog and integration into a Reference Control WASHING STEPS Use 50 ml of non human whole blood from selected species collected in citrate anticoagulant. Centrifuge whole blood and aspirate the top layer including white blood cells, platelets and plasma. Wash packed red blood cells three times with PBS. The cell washing steps were a series of centrifugations (1000 rpm/5 minutes), followed by removal of the supernatant and re- suspension of the packed cells with PBS. Re-suspend washed cells in the residual PBS. ANALOG PROCESSING STEPS Add 1 ml of packed washed red blood cells to 49 ml of the Analog Processing Medium 1 and mix by inversion to form a cell processing suspension. Roll mix at a slow speed overnight at room temperature. Wash processed cells three times with PBS, as described in Washing Step. Re-suspend processed cells in Suspension Medium 1 or 2. Store resulting Immature Granulocyte Analog at room temperature Phosphate Buffered Saline Solution (PBS) Sodium dihydrogenphosphate0.2g Disodium hydrogenphosphate 7H2O 2.0g Sodium azide0.1g Sodium chloride9.4g QS to 1 liter with reagent grade waterpH approximately 7.4 Osmolarity315 to 345 H2O Analog Processing Medium 1 Disodium hydrogenphosphate 7H2O2.0 g Sodium dihydrogenphosphate0.2 g Sodium chloride 24.5 g Glutaraldehyde (25%) 40 ml QS to 1 liter with reagent grade waterpH approximately 7.4 Osmolarity 900 mOsm/kg H2O Analog Processing Medium 2 Sodium chloride 31.7 g Glutaraldehyde (25%) 40 ml QS to 1 liter with reagent grade waterpH approximately 6.5 Osmolarity 1000 mOsm/kg H2O Analog Processing Medium 3 Disodium hydrogenphosphate 7H2O2.0 g Sodium dihydrogenphosphate0.2 g Sodium chloride 16.5 g Glutaraldehyde (25%) 40 ml Materials and Methods INTEGRATED CONTROL COMPOSITION Provide a predetermined volume of Suspension Medium 1 containing stabilized human Red Blood Cells and a Platelet component. Add predetermined amounts of lymphoid and myeloid analogs. Add a predetermined amount of immature granulocyte analog. The analogs are added in proportions to resemble white blood cell subpopulations and immature granulocytes in Normal and Abnormal human whole blood. The following integrated reference control compositions were formulated: Composition A 30% Lymphoid analog made of human RBC 70% Myeloid analog made of processed goose RBC Composition B 30% Lymphoid analog made of Human RBC 49% Myeloid analog made of processed Goose RBC 21% Immature granulocyte made of processed Emu RBC Composition C 25% Lymphoid analog made of Human RBC 58% Myeloid analog made of processed Emu RBC 21% Immature granulocyte made of processed Alligator RBC USE OF THE HEMATOLOGY REFERENCE CONTROL ON A HEMATOLOGY ANALYZER FOR MEASUREMENT OF IMMATURE GRANULOCYTES USING A DC IMPEDANCE MEASUREMENT The reference control compositions were analyzed on an experimental hematology analyzer. In the analysis, an aliquot of 28 µl of the reference control or a blood sample was diluted with 6 ml of Coulter® LH Series Diluent (Beckman Coulter, Inc., Miami, Florida) in a WBC bath, mixed with 1 ml of a lytic reagent to lyse red blood cells. The lytic reagent contained 25.0 g/L of Tetradecyltrimethylammonium bromide (from Dishman), 15.0 g/L of Igepal SS-837 (an ethoxylated phenol from RhÔne-Poulenc), 4.0 g/L of Plurofac A38 prill surfactant (from BASF Corp.), and had a pH of 6.2. The experimental hematology analyzer was a modified LH750 (product of Beckman Coulter, Inc., Miami, Florida), equipped with non-focused apertures of a length of 100 µm and a width of 80 µm for measuring the prepared sample mixtures as described above. The sample mixture was drawn through a set of three apertures by a constant vacuum. The white blood cells were enumerated by DC impedance measurement, and a WBC histogram (size vs cell count) produced after pulse editing. Results Figure 1 illustrates a DC histogram of a normal whole blood sample analyzed on the experimental hematology analyzer. The white blood cells in the normal sample exhibit a bi-modal distribution, with the lymphoid subpopulation on the left and the myeloid subpopulation on the right. No cell population located on the right side of the myeloid subpopulation. Figure 2 illustrates a DC histogram of a clinical sample containing immature granulocytes. The manual reference reported about 12% of immature granulocytes (IG), including metamyelocytes, myelocytes and promyelocytes, which showed on the right side of the myeloid subpopulation. This sample had only 7% lymphocytes, and the majority of the white blood cells were myeloid population. Figure 2A illustrates DC histogram of another clinical sample containing about 6% of immature granulocytes including metamyelocytes and myelocytes, which are indicated by the large cells extending into the right-most region of the histogram. The manual reference also reported 5 NRBC per 100 WBC in this sample. As shown, NRBCs are located on the left side of the lymphoid population, which was differentiated from the white blood cells. Figure 3 and 4 illustrates DC histograms of reference control compositions A and B. As shown, the histogram of the reference control composition A resembles the cell distribution of the normal blood sample, and the histogram of the reference control composition B resembles the cell distribution of the clinical sample containing immature granulocytes. Figure 5 and 6 illustrates DC histograms of a normal whole blood sample and a clinical sample containing immature granulocytes analyzed on the same experimental hematology analyzer with a larger dynamic range of the measurement. With the larger dynamic range, normal white blood cells distributed in approximately only half of the histogram. Some of the immature granulocytes in this example were very large, and extended to the extreme right of the histogram. Figure 7 illustrates the DC histogram of reference control composition C analyzed on the experimental hematology analyzer with the same dynamic range shown in Fig. 5, which resembles the cell distribution of the clinical sample containing immature granulocytes. Conclusions The control composition offers an automated cost effective way of monitoring immature granulocytes enumeration on low cost analyzers. The flexibility of the process allows the manufacture of a wide range of analogs that encompass multiple detection systems. The analog can be measured by radio frequency, impedance and optical methodologies that reflect the size of cells. Analogs can be combined to simulate the broad range distribution of immature granulocytes seen in clinical samples. The immature granulocyte analog can be used as a stand alone product or integrated into a reference control containing stabilized red blood cells, mature white blood cells, platelets, reticulocytes and NRBC components enabling quality control of hematology systems with a single product Acknowledgements We are grateful to Jing Li, Staff Scientist, Beckman Coulter Inc, Miami, Florida for her collaboration in the evaluation of the Immature Granulocyte Reference Control. An Automated Cell Prepration Method For FlowCARE™ Pan-Leukogated (PLG) CD4 Assay in a 96-Well Plate Offers Increased Throughput and Ease of Use