Six milliliters of peripheral venous blood were withdrawn from each participant under strict sterile conditions following standard biosecurity and international safety procedures and divided into two aliquots. Blood was withdrawn at the time patients were first diagnosed clinically with HCC and before any medical therapy or surgical intervention. The first 3 mL of blood was placed into EDTA anticoagulant vacutainers for complete blood count using Sysmex KX-21 (the automated haematology analyser KX-21 manufactured by Sysmex Corporation, Kobe, Japan) and FC assay. The rest of the blood was transferred into a polymer serum gel separator tube with a clot activator (Kremsmünster, Upper Austria, Greiner Bio-One GmbH, Australia) left for 15 min at room temperature (24 °C) to clot, followed by 10-min centrifugation at 10,000× g at 4 °C. Sera obtained were aliquoted into Eppendorf tubes and stored at −80 °C until biochemical assessment.
Kx 21
Manufactured by Sysmex
Sourced in Japan, Germany, United States, India
The KX-21 is a compact, automated hematology analyzer designed for routine blood cell analysis. It provides a rapid and accurate assessment of common blood parameters, including red blood cell count, white blood cell count, and platelet count.
Automatically generated - may contain errors
Lab products found in correlation
Cobas c501, by Roche (7 mentions)
Cobas c311, by Roche (3 mentions)
Cobas integra 400 plus, by Roche (2 mentions)
Vitros dt60, by Ortho Clinical Diagnostics (2 mentions)
Cellquest software, by BD (1 mentions)
Xp 300, by Sysmex (1 mentions)
Synchron cx9 alx, by Randox (1 mentions)
Multitest reagents, by BD (1 mentions)
Facscalibur instrument, by BD (1 mentions)
Selectra junior, by Vital Scientific (1 mentions)
Trucount tube, by BD (1 mentions)
Triglyceride assay kit, by Cayman Chemical (1 mentions)
Cobas c111 analyser, by Roche (1 mentions)
Hanks balanced salt solution (hbss), by BD (1 mentions)
Lymphoprep, by BD (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Crf 1, by Oriental Yeast (1 mentions)
Male sprague dawley rat, by Charles River Laboratories (1 mentions)
Trisodium citrate, by Sarstedt (1 mentions)
Titrisol iron standard, by Merck Group (1 mentions)
154 protocols using kx 21
1
Peripheral Blood Sampling for HCC
2
Comprehensive Hematological Assessment Using Sysmex KX-21
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A hematology analyzer (Sysmex KX-21) was used for the analysis of CBC, employing 2 mL of whole blood (hemoglobin, total leukocytes count, hematocrit, and platelet count). For the analysis of the complete blood count, EDTA-containing vials were used. Before the process, all the samples were placed on the roller for 5 min at room temperature. As the samples mixed, CBC vials were introduced to the chamber of the Sysmex KX-21 for analysis of the complete blood counts.
The normal reference range of hemoglobin was 13–18 g/dL in males and 12–15 g/dL in females. The total leukocyte count normal reference range was 2.6–8.3 × 10³ mm³. Some control patients had borderline leukocyte counts due to infections other than HIV infection, i.e., seasonal fever. The normal reference range for platelet count was 140–440 × 10³ mm³. The Biochemical tests like LFTs and RFTs were performed on Biosystems BA200, a fully automated clinical chemistry analyzer.
The normal reference range of hemoglobin was 13–18 g/dL in males and 12–15 g/dL in females. The total leukocyte count normal reference range was 2.6–8.3 × 10³ mm³. Some control patients had borderline leukocyte counts due to infections other than HIV infection, i.e., seasonal fever. The normal reference range for platelet count was 140–440 × 10³ mm³. The Biochemical tests like LFTs and RFTs were performed on Biosystems BA200, a fully automated clinical chemistry analyzer.
3
Blood Cellular Analysis Workflow
4
Refined Retro-orbital Blood Collection
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The refined retro-orbital bleeding method, utilizing the lateral approach described by Sharma et al. (2014) , was employed to obtain blood samples of adequate volume and quality. This technique involves gently removing the eyelid and stretching the upper eyelid to expose the eye, allowing for careful retrieval of blood using a Pasteur pipette with minimal vein irritation. After collection, the blood samples are divided into two portions. One half is deposited into an EDTA aliquot tube for hematological assays, while the other undergoes centrifugation. The centrifuged samples are further processed to isolate the serum intended for future biochemical analysis.
The Sysmex KX-21 (Sysmex Corp., Japan), an automated blood analyzer, was utilized to determine a comprehensive panel of hematological parameters, including total red blood cell (RBC) count, hemoglobin (HGB) level, mean RBC hemoglobin concentration (MCHC), hematocrit (HCT), white blood cell (WBC) count, platelet (PLT) count, and WBC differential.
For the biochemical analyses, serum samples were collected and analyzed for liver enzymes such as aspartate aminotransferases (AST) and alanine aminotransferases (ALT). Additionally, serum kidney parameters including urea and creatinine were quantified using an automated serum biochemistry analyzer from Beckman Coulter (Japan).
The Sysmex KX-21 (Sysmex Corp., Japan), an automated blood analyzer, was utilized to determine a comprehensive panel of hematological parameters, including total red blood cell (RBC) count, hemoglobin (HGB) level, mean RBC hemoglobin concentration (MCHC), hematocrit (HCT), white blood cell (WBC) count, platelet (PLT) count, and WBC differential.
For the biochemical analyses, serum samples were collected and analyzed for liver enzymes such as aspartate aminotransferases (AST) and alanine aminotransferases (ALT). Additionally, serum kidney parameters including urea and creatinine were quantified using an automated serum biochemistry analyzer from Beckman Coulter (Japan).
5
Hematological Biomarker Analysis
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Blood samples were collected into EDTA spray-coated tubes, WBC, RBC, PLT and HGB were analyzed with an automated hematology analyzer (Sysmex KX-21, Sysmex Co., Japan).
6
Immunophenotyping of Leukocyte Subsets
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Automated white blood cell counts were performed on the Sysmex KX21 or XP300 (Sysmex, Norderstedt, Germany). Flow cytometric analysis of leukocytes from fresh whole blood was performed as described (5) . The following antibodies were used to identify the analyzed cell populations: CD3 (SK7), CD4 (SK3), CD8 (SK1), CD25 (2A3), CD194/ CCR4 (1G1), CD56 (NCAM16.2), and CD19 (SJ25C1) from Becton Dickinson (Heidelberg, Germany) and CD127 (HCD127) and CD161 (HP-3G10) from BioLegend (San Diego, CA). Samples were measured on a dual laser FACSCalibur cytometer (Becton Dickinson). Acquisition of samples was performed using Cellquest software (Becton Dickinson). Samples were analyzed with FlowJo software (version 10). Gating strategies for the different cell populations are shown in Supplementary Fig. 2.
7
Hematological and Biochemical Analysis
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On the tenth day of the experiment, blood samples (about 5 mL) from the jugular vein were collected from all groups in EDTA tubes for whole blood collection, and gel tubes for serum isolation. Strict aseptic conditions were observed during blood sampling. The automatic digital machine (Sysmex Kx-21, Sysmex Corporation, Kobe, Japan) was used to determine hematological parameters from whole blood. The ultraviolet-visible spectrometer (UV-VS) (T60, PG instrument, Leicestershire, UK) was used to measure biochemical parameters in serum.
8
Automated Hematological Analysis Protocol
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The hematological parameters, hemoglobin (Hb), Packed Cell Volume (PCV), Red Blood Cells (RBC), White Blood Cells (WBC), and platelets count (PLT), were determined using the automated hematologic analyzer SYSMEX KX21, a product of SYSMEX Corporation, Japan, as described by Dacie and Lewis, [18 ].
9
Comprehensive Blood Biomarker Profiling
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Blood samples were collected from all participants for routine laboratory investigations (complete blood count, fasting and 2-hour blood sugar, liver and kidney function tests, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and lipid profile). Blood chemistry was done using a fully automated autoanalyzer Synchron CX9 ALX (Randox, California, USA), and hematology parameters were obtained using an automated Sysmex KX-21 (Sysmex Corporation, Kobe, Japan).
For lipid profile measurement, after an overnight fasting, total serum cholesterol, triglyceride, and high-density lipoprotein cholesterol (HDL-c) were obtained using Synchron CX9 ALX (Randox), while low-density lipoprotein cholesterol (LDL-c) was estimated using Friedewald’s formula.
IL-6, NT-proBNP and resistin were measured in all participants using ELISA (Chongqing Biospes, China). This kit was based on a sandwich ELISA technology.
For lipid profile measurement, after an overnight fasting, total serum cholesterol, triglyceride, and high-density lipoprotein cholesterol (HDL-c) were obtained using Synchron CX9 ALX (Randox), while low-density lipoprotein cholesterol (LDL-c) was estimated using Friedewald’s formula.
IL-6, NT-proBNP and resistin were measured in all participants using ELISA (Chongqing Biospes, China). This kit was based on a sandwich ELISA technology.
10
Comprehensive Blood Biomarker Analysis
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An 8 ml blood sample was obtained by venipuncture to measure CD4+T cell count, HIV-1 viral load, hematology, glycaemia and blood lipids. The blood samples were collected and analyzed following biosafety procedures. The CD4+T cell count was performed on whole blood using a FACS-CaliburTM instrument with MultiTest reagents, TruCOUNT tubes and MultiTEST software (all from Becton Dickinson, USA). HIV-1 viral load was determined using a COBAS®TaqMan system 48 Analyser with COBASTaqMan HIV-1 test reagents (Roche Diagnostics, Germany) for automated amplification and detection of nucleic acid with limit of detection above 20 copies/ml. Haematological parameters were measured within six hours after blood sample collection using an automated hematology analyser, the Sysmex KX21 (Sysmex Corporation, Japan), and glycaemia and lipid profiles were analyzed by using the Selectra Junior (Vital Scientific NV, The Netherlands). All testing were performed at the laboratory of the Instituto Nacional de Saúde, Ministry of Health, Mozambique.
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