Leukocyte counts and differentials were performed on all individuals using the Act-5 Diff hematology analyzer (Beckman Coulter).
Act 5 diff
Manufactured by Beckman Coulter
Sourced in United States
The Act-5 Diff is a laboratory instrument designed for automated hematology analysis. It provides a comprehensive analysis of various blood cell parameters, including red blood cells, white blood cells, and platelets. The Act-5 Diff is capable of performing a five-part differential count, which categorizes white blood cells into different subpopulations.
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Lab products found in correlation
Abx micros 60, by Horiba (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Polymorphprep, by Axis-Shield (1 mentions)
Hrp conjugated anti human vwf, by Agilent Technologies (1 mentions)
Anti human vwf antibody, by Agilent Technologies (1 mentions)
Coulter epics xl mcl flow cytometer, by Beckman Coulter (1 mentions)
9 protocols using act 5 diff
1
Leukocyte Counts and Differentials
2
Validation of Hemoglobin Measurement Methods
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Samples of pooled capillary blood and venous blood were sent to the central laboratory the same day of sample collection. For both types of samples, Hb concentration was determined using the cyanmethemoglobin method [16 (link)], which we considered the gold standard, in the equipment Beckman Coulter Ac•T 5diff® (Beckman Coulter Inc., Danaher Corporation, CA, USA) [17 ]. Hb concentration from venous samples was additionally quantified by a conventional hematology analyzer using a non-polluting generic lysing reagent (Diagon-Dya Lyse, code number: 20212) in the equipment ABX Micros 60® (Horiba, Kyoto, Japan) according to the manufacturer’s manual [18 ]. Only one experienced technician handled the venous and pooled capillary blood samples and used the two hemocounters.
3
Quantification of Viral RNA in SIV Infection
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The Virology Core at the WaNPRC, led by Dr. Shiu-Lok Hu and Dr. Patricia Firpo, quantified viral RNA in the plasma of SIVΔB670-infected animals using a real-time quantitative PCR (RT-q-PCR) assay. The Virology Core also determined complete blood counts, using a Beckman Coulter® AC*T™ 5diff hematology analyzer (Beckman Coulter) as described previously [73 (link)].
4
Leukocyte Isolation from Maternal and Placental Blood
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The following day, immediately after delivery (approximately 12–16 hours after anthropometric measurements were obtained), we collected maternal peripheral blood by venipuncture of the forearm, placental intervillous blood by manually draining the cotyledons and the fetal membranes cut from the placenta. Leukocytes from maternal peripheral blood (MAT) and placental intervillous blood (PLA) were isolated by density gradient using Polymorphprep (Axis‐Shield, Norway) and following the manufacturer's protocol. Choriodecidual leukocytes (CHD) were isolated from the chorionic layer of the fetal membranes by enzymatic digestion following a previously described method (MacDonald‐Ramos et al., 2018 ).
Total cell count was obtained by a cell counter (AcT5diff, Beckman Coulter) and 10 × 106 leukocytes were stored in 1 ml of RNAlater (Ambion, U.S.A.) or 1 ml of TRIzol Reagent (Invitrogen Life Technologies, U.S.A.) at −70°C until further processing.
Total cell count was obtained by a cell counter (AcT5diff, Beckman Coulter) and 10 × 106 leukocytes were stored in 1 ml of RNAlater (Ambion, U.S.A.) or 1 ml of TRIzol Reagent (Invitrogen Life Technologies, U.S.A.) at −70°C until further processing.
5
Haematological Analysis via Act-5 Diff
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Haematology was performed on all individuals using the Act-5 Diff haematology analyzer (Beckman Coulter). Demographic details and other clinical parameters were shown in Table 1 .
6
Evaluation of von Willebrand Factor in Plasma
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Baseline complete blood counts, including platelet counts, were obtained at a regional reference laboratory using a Beckman Coulter AcT 5 Diff hematology analyzer (Beckman Coulter, Inc, Fullerton, CA, USA). For the evaluation of change in VWF antigen over time, VWF antigen was measured in stored plasma samples from all study participants at all available time-points (i.e., through day 14 or death). Plates were coated with anti-human VWF antibody (DAKO North America, Inc, Carpinteria, CA, USA, 1:600 dilution), incubated with samples (1:1000 dilution) and standards (serial dilutions of recombinant VWF, American Diagnostica, Stamford, CT, USA), then incubated with HRP-conjugated anti-human VWF (DAKO North America, Inc, Carpinteria, CA, USA, 1:8000 dilution). Bound antibodies were detected with the HRP substrate tetramethylbenzidine, the reaction was stopped with H2SO4, then the colour signal was read at 450 nm. Background signal was determined from blank wells included on each plate (assay buffer added instead of sample), and background optical density was subtracted from all samples and standards prior to analysis. Samples with optical densities below the lowest detectable standard were assigned the value of that standard.
7
Blood-based Diagnostic Protocols for HIV
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At recruitment, venous blood samples were collected. CD4+ lymphocyte count was analysed using a Coulter® Epics XL-MCL™ Flow Cytometer (Beckman Coulter, Brea, CA). Haemoglobin level and white blood cell counts were analysed using haematological Coulter AcT 5 diff, (Beckman Coulter, Brea, CA). HIV status was determined on two rapid tests done in parallel (SD Bioline HIV-1/2 3.0, Standard Diagnostics Inc., Kyonggi-do, South Korea; Determine HIV-1/HIV-2, Inverness Medical Innovations Inc., Delaware, USA). Discordant HIV test results were resolved using HIV UNIFORM II ELISA (Organon Teknia Ltd, Boxtel, Netherlands),
8
Hemogram Analysis via Beckman Coulter
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Hemograms were performed on all individuals using the Act-5 Diff hematology analyzer (Beckman Coulter, Brea, CA, USA).
9
Hematology Analysis Using Act-5 Diff Analyzer
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