Arrow Point Partners, Inc. 484 East Carmel DriveSuite 282Carmel, IN 46032US
Technical Library
Cell Biology Research - Additional Information
Gradient Separation of lymphocytes and nylon wool separation of B and T cells Product Line Overview
SCIENTIFIC BACKGROUND:
Among the most important recent advances in diagnostic immunology have been the use of fluorescence-activated flow cytometry instrumentation and the development of monoclonal antibodies against immune cells. Flow cytometry with monoclonal antibodies has allowed identification and quantification of subsets of the major lymphocyte populations and even further subdivisions that differ in biologic function, maturation stage, and activation.
ISOLATION OF LYMPHOCYTES FROM PERIPHERAL BLOOD
No matter what the reason for deciding to conduct an assessment of the lymphocyte population of a patient, the requisite first step is to isolate a representative sample of white cells from the patient's peripheral blood: The most commonly used procedure is for separation of the mononuclear cells by density gradient centrifugation of whole blood. This procedure is performed by carefully layering diluted whole blood over a polysucrose - sodium metrizoate medium (Ficoll-Paque, Lymphoprep, Histopaque, etc). The diluted blood is added to the gradient by gently pipetting with the tubes held at an angle or by pouring the blood onto the separation medium. This latter method requires considerable practice and is not recommended for beginners. To obtain good separations, it is critical that a clear separation be kept between the dense polysucrose - metrizoate and the blood layer before centrifugation. Due to the extreme care that must be exercised when pipetting blood on to the separation medium, alternative methods have been devised. In one such procedure a sample of the diluted blood is placed in a centrifuge tube and Ficoll-Paque (or equivalent separation medium with a density of 1.077) is added by being underlayered under the blood. This method creates cleaner interfaces than those obtained when blood is layered over the Ficoll, since there is less disturbance of the surface of the Ficoll and less mixing.
READY-FOR-USE CELL SEPARATION TUBES
It is not surprising that various manufacturers have sought to introduce time saving innovations to solve some of the problems inherent in cell separation techniques. One of these is the LEUCOPrep® method of Becton Dickinson, which contains a semi-rigid gel, and Ficoll in cell separator tubes (LEUCOPrep®). Sigma Chemical Co. sells a pre-filled tube, the Accuspin®, which contains Histopaque and a semi-permeable plastic "frit". After centrifugation, the lymphocyte layer is separated by the frit from possible contamination by the precipitated red cells. A new, innovative ready-to-use product is ARROW POINT'S patented ARROW-SEP™ tube. The heart of the ARROW-SEP™ is a plastic insert that allows the blood sample to be poured directly into the tube alleviating the need for slow and careful addition of the blood. Secondly, a one-way feature of the insert allows passage of materials during the centrifugation step but prevents the flow of the separation medium during shipping. After centrifugation, if desired, the upper lymphocyte-containing fraction may be poured off without risk of contamination from the erythrocytes, which are trapped under the insert. The insert is made from an inexpensive plastic that adds very little to the cost of the procedure. The reasons why Becton-Dickinson and Sigma methods are not more popular are primarily economic; the cost per tube is substantially more than for a centrifuge tube filled by the technician from a bulk supply of Ficoll-Paque.
THE MARKET FOR ARROW-SEP™
Customers for ready-to-use blood separation tubes are found in the clinical immunology and oncology laboratories of all large hospitals and medical centers. Research laboratories in many diverse fields - from immunology and cancer to virology and parasitology - form another important market. The main application of these tubes is for separation of white blood cells from whole peripheral blood. The white cells are then tested by various procedures including the latest FACS-monoclonal antibody methods. Results have important implications in cancer diagnosis, in tissue typing, and in the identification of inherent or acquired immune system deficiencies. Due primarily to the high cost of the ready-to-use separation tubes on the market now, most users of this technology purchase centrifuge tubes and Ficoll separately and prepare blood separation tubes as part of their routine laboratory practice. While this may be more economical from the standpoint of raw materials purchase, preparation of separation tubes is time consuming and their application is technically difficult. The ARROW-SEP™ and ARROW-SEPmaxi™ products described in this letter provide, for the first time, a competitively priced ready-for-use alternative to the "home made" blood separation tube. ARROW-SEP™ tubes are manufactured with either 2 or 3 ml separation medium in 15 ml centrifuge tubes or with either 10 or 15 ml separation medium in 50 ml centrifuge tubes. For laboratories that have stocks of Ficoll or Lymphoprep, the tubes can be ordered empty for charging with separation medium immediately before use.
THE NEXT STEP - SEPARATION OF B AND T LYMPHOCYTES
Organ transplantation, from tissue typing of potential donors and recipients to understanding rejection phenomena, is heavily dependent upon studies of HLA antigens. Today's technology requires separation of mixed lymphocyte populations into B and T lymphocyte fractions (the T cells may then by further differentiated into "helper", "killer", or "suppressor" subtypes). After isolation of the white blood cells in a ARROW-SEP™ tube the next step is to separate T and B cells with ARROW POINT'S new ARROW-SORB™ ready-to-use nylon wool columns. Nylon wool columns - exploiting the property of B cells, but not T cells, to adhere to nylon wool - are the technique of choice in many laboratories because of the simplicity of the technique and the short time necessary to recover T-enriched as well as B enriched populations. This method, for which literature references have been accumulating for nearly 30 years, yields excellent B and T preparations (>90% purity). Arrow Point's ARROW-SORB™ columns are sterile, ready-to-use polypropylene tubes packed with specially treated nylon wool. The top of the column is closed with a silicon rubber plug which may be penetrated by syringe needle. Outflow is controlled by a unique twist valve. ARROW-SORB™ columns are manufactured to rigorous specifications ensuring consistent performance and reproducible results. Additional details on each product can be found later in this document on the noted page.