Buffy coat

Speck-, fatty, lardaceous. amyloid, speck-ahnlich, -artlg, a. fatty, lardaceous. Speck-glanz, m. greasy luster, -haut, /. (Physiol.) buffy coat. 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

The limit of detection of the assay was estimated to be 200 parasites/ml of blood. The detection limit is well within the range of sensitivity needed to diagnosis trypanosomiasis, as the parasitemia may vary from 5000 to 1,500,000 parasites/ml (Vickerman, 1974). The bDNA assay was compared with buffy coat microscopy for detection of T brucei in 56 blood samples (36 buffy coat positive and 20 buffy coat negative by microscopy). There was complete concordance between the results of the two tests in terms of identifying specimens as positive of negative. However, the numbers of parasites observed by microscopy were lower overall than those calculated with the bDNA assay. The authors suggested that the excess of leukocytes in the buffy coat could interfere with the microscopic detection of typanosomes, resulting in lower apparent parasitemia than the true value. 

Control slides should be used to monitor the staining. Specimens containing protozoa are best for controls however, feces containing inflammatory cells or added buffy-coat leukocytes also are satisfactory. 

Remove about 0.5 mL of the top most layer in the tube (the buffy coat of the plasma), and add it to a tissue-culture flask containing 5 mL of Ham s F-10 medium supplemented with 10% fetal bovine serum and 400 pL of phytohemagglutinin. Incubate for 72 h at 37°C in a carbon dioxide incubator (see Note 4). 

PBMCs were isolated from buffy coats of healthy blood donors by density centrifugation according to the standard procedures and cultured in complete cell medium. 

The antiviral and antiproliferative effects of individual IFN-a subspecies differed from buffy-coat IFN-a [30,31]. Unlike recombinant purified IFN-a2, which is species specific, buffy-coat huIFN-a has broad species effects, producing responses in humans, bovines, and felines. The clinically observed side effects of purified recombinant IFN-a2 and naturally produced buffy-coat IFN, however, were noted to be similar [32,33]. 

Subsequently Cantell at the Finnish Blood Bank used this strategy to develop a large-scale process of preparing leukocyte interferon from virus-stimulated buffy-coat lymphocytes pooled from blood donors [3]. This allowed production of comparatively large amounts of crude interferon, but effi-... 

Key Words Buffy coat lymphocyte carcinoma enrichment cell culture polycarbonate membrane. 

The following section describes the steps necessary to isolate carcinoma cells (cell line, MCF-7) from a buffy coat. A filtration step is added at the end of the density gradient centrifugation procedure to concentrate cells onto a polycarbonate membrane, which is a convenient vehicle for analysis by immunocytochemistry. 

Buffy coat (leukocyte fraction collected from peripheral blood available from a Red Cross or a Blood Bank). 

Buffy coat (white blood cell fraction) obtained from Red Cross must be kept in cold storage until use. Take an aliquot of the buffy coat and dilute with HBSS to determine cell concentration. Either a hemacytometer or an automated particle counter can be used for cell count (see Note 3). 

Based on the total number of cells present in the buffy coat, seed viable carcinoma MCF-7 cells to a final percentage of 0.1% (see Notes 4 and 5). 

Dilute the cell suspension (buffy coat spiked with the cancer cells) with HBSS at different ratios prior to centrifugation. Suggested ratios range from 1 1 to 1 5, cell suspension HBSS. 

Determining cell concentration in a buffy coat sample using a particle counter can be difficult. If the laboratory has access to hematological material, it is recommended to use the Unoppette system (Becton Dickinson), after diluting the sample. Results from particle counter and manual counting should not differ by more than 5%. 

This protocol mentions a 0.1% of rare cells seeded into the buffy coat. It is possible to increase or reduce the percentage of cells. However, less than 0.1 % of cells might make it difficult to obtain reliable recovery data, and a larger percentage would not be a good representation of rare cells circulating in blood. 

We developed a PEEK-WC hollow-fiber (HF) membrane bioreactor for the maintenance of human peripheral lymphocytes as a model system for the in-vitro investigation of disease pathogenesis, chemical effects and individual drug sensitivity. Peripheral lymphocytes isolated from the donor s human buffy coat were cultured in the shell compartment of the PEEK-WC-H F bioreactor. Lymphocytes in the PEEK-WC-HF membrane bioreactor produced IL-2 and IL-10 throughout the culture period of 14 days (Figure 19.5). 

The obtained results demonstrated that a PEEK-WC-H F membrane bioreactor is able to support the proliferation and functions of human peripheral lymphocytes isolated from the buffy coat of healthy individuals. Therefore, the lymphocyte HF membrane bioreactor can be used as a valuable tool to maintain viable and functional lymphocytes and as an in-vitro model for pharmacological and adoptive immunotherapy. 

Following venous blood collection into a plastic syringe, add 25-35 mL of blood to the tube prepared in step 1.) Recap the tube and mix by inverting 4-5 times. Loosen the cap to prevent small volumes of blood collecting at the top of the tube. Allow to stand at room temperature for approx 30 min until there is a clear separation between the upper leukocyte rich buffy coat layer and the red cell layer. 

Carefully collect the upper buffy coat layer with a plastic pipet without disturbing the red cell layer. 

It is preferable but not essential to screen PBMCs from different donors to identify a donor whose cells replicate HIV efficiently and do not carry the A 32 C025 mutation. Alternatively, leukocyte enriched buffy coats may be purchased from blood transfusion centers. Mixing PBMCs from two donors results in a mixed lymphocyte reaction which can increase cellular activation and hence HIV replication. 

Freshly collected venous blood is anticoagulated with K-EDTA (1 mg/ml blood) or heparin (5 IU/ml heparin sodium) and centrifuged at 3000 rpm for 7 min. The supernatant (plasma) and the buffy coat are removed and discarded. The packed erythrocytes are resuspended in autologous plasma containing 0.25 % human albumin and the haematocrit value is fixed at 10 %. The red blood cells are altered by one or several of the stress factors mentioned above. 

Centrifuge at 1000 X g for 10 min. Using a Pasteur pipette, carefully remove the plasma and buffy coat from the pelleted red blood cells. 

Whole blood is centrifuged at 600 x g for 10 min at 4°C in graduated centrifuge tubes using a swing out rotor. The plasma and buffy coat (a layer of white blood cells over the erythrocytes) are removed by aspiration. Packed erythrocytes are washed twice in 10 vol. of isotonic 5 mM phosphate buffer pH 7.4, or other buffer as required, by centrifugation at 600 x g for 10 min at 4°C. 

Woik perfi)rmed at the Theodor Kocher Institute was sipported by a giant 31 -42336.94 from die Swiss National Science Foundation. We thank the Central Laboratoiy of the Swiss Red Cross Blood Transfusion Service, Berne, for the supply of buffy coats. 

Lieden G, Hilden JO. Febrile transfusion reactions reduced by use of buffy-coat-poor erythrocyte concentrates. Vox Sang 1982 43(5) 263-5. 

Collect the white huffy coat by removing 10 mL from the interface of the red blood cells and the plasma. The buffy coat may not be visible, or it may look like a gray paste covering the top of the red layer. Regardless of the appearance, collect 10 mL from the interface as just described. 

Transfer the buffy coat to a sterile, 50-mL screw-cap centrifuge tube containing 10 mL of filtered PBS P/S and mix by swirling gently. 

Aspirate 10 mL of LSM into a 10-mL pipet and insert the tip into the bottom of the tube containing the diluted buffy coat. Slowly pipet the LSM under the diluted cell layer. 

Buffy coat from human blood donation. 

The use of PBMCs is usually reserved for those few compounds that show good activity in the primary screen. PBMCs are more difficult to infect than lymphoblastoid cells and more virus is used 100 TCID50. The release of sufficient p24 for measurement usually takes at least 5 d. If only a few compounds are to be evaluated at one time it is possible to increase the number of replicates at each concentration (5-10) since more variation is observed between wells. PBMCs are prepared from human blood donations either from a transfusion center or from a donor panel of healthy volunteers. Transfusion centers can provide a "buffy coat," which is a leukocyte concentrate of one unit of blood containing red blood cells (RBCs) and platelets. It is usually necessary to obtain ethical permission to work with PBMCs from donors because their blood will be tested for p24. The method describes the separation of PBMCs from a buffy coat or a heparinized whole-blood donation from a healthy volunteer. 

PBMCs should be prepared in a Class 2 cabinet and latex gloves should be worn. Cut the top left hand tubing on the buffy coat bag. Use round-ended scissors wiped with 70%. Carefully pour 25-mL aliquots of the contents of the bag into 50-mL centrifuge tubes. 

Add 25 mL RPMI wash medium to each tube, thus diluting the buffy coat 1 2. Depending on the volume of the buffy coat 4-6 tubes will be needed. Pipet up and down several times to mix. 

Invert the Ficoll-Paque and check that it has reached room temperature and add 15 mL to four sterile centrifuge tubes. If the buffy coat has a large volume (>80 mL), then steps 5-13 can be repeated if the maximum number of PBMCs present are required. 

Using a 10 mL plastic pipet, carefully layer 25 mL of the buffy coat-RPMI mixture on top of the Ficoll-Paque. This is achieved by allowing the buffy coat to run slowly down the inside of the centrifuge tube. The centrifuge tube should contain no more than 40 mL at this stage. 

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