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Cell Biology Research & Vaginal pH Self-Testing - Additional Information
Gradient Separation of lymphocytes and Vaginal pH Self-Testing Product Line Overview
Gradient Separation
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.
Vaginal pH Self-Testing
VH-SENSE for self-testing for bacterial vaginitis. The test indicates abnormal vaginal discharge acidity by identifying changes in the pH level. The test cutoff is set at pH 4.7 (+0.3/-0.2 pH units; there may be variations due to sensitivity to buffer capacity). When the vaginal secretion has a pH above this
cutoff, the test tip will be stained (or partially stained) Blue or Green.
The sensitivity of a regular pH test is about 77-78%. Therefore, a regular pH test will miss at least 22% of cases (22% false negative indications) in which BV is accompanied by normal pH.
VH SENSE supports diagnosis of BV by detecting two disorder parameters in a vaginal secretion: pH level above normal and watery secretion - with low buffer capacity.
VH SENSE is a testing swab coated with unique chemical composition. The swab tip reacts as a pH indicator due to the incorporation of Nitrazine yellow in the polymer matrix. In addition, the has hydrophobic characters in contrast to the hydrophilic character of commercial pH papers like Nitrazine paper®. This makes the sensitive to buffer capacity of a solution as well as to pH. The lower the buffer capacity, the greater the sensitivity of the to pH. The tip changes color in lower pH than comparable solutions that have a higher buffer capacity.
The color of the swab tip will change from yellow to Blue or Green when the swab comes into contact with vaginal secretions whose pH and /or buffer capacity is abnormal. The color changes when the secretion pH is above 5.2 units. In addition, the color changes will occur in a dynamic range between pH levels of 4.3 to 5.2, depending on the levels of the secretion buffer capacity.
SCIENTIFIC BACKGROUND -
BACTERIAL VAGINOSIS (BV), TRICHOMONAS VAGINALIS (TV), YEAST INFECTION - CANDIDIASIS
Vaginitis - Vulvovaginitis, is an inflammation or infection of the vagina. It can also affect the vulva, which is the external part of a woman's genitals. It is symptomatically observed by a watery discharge with burning and itching of the vulva.
Vaginitis is common, especially in women in their reproductive years. It is one of the most common reasons for gynecological consultation. It usually happens when there is a change in the balance of bacteria or yeast that are normally found in the vagina. There are different types of vaginitis, and they have different causes, symptoms, and treatments.
Normal levels of vaginal pH fluctuate between 3.8 and 4.5. A normal, healthy vagina contains both Lactobacillus sp. ("good bacteria") and harmful bacteria. Normally, the Lactobacillus flora are predominant and produce lactic acid (and H2O2) to keep the vaginal pH level between 3.8 and 4.5.
Various factors may cause a loss of lactobacillus bacteria in the vagina, including use of: antibiotics, vaginal medications, systemic hormones, contraceptive preparations, and douches; as well as sexual intercourse and STDs. Some of these situations may cause the harmful bacteria to overgrow. When this occurs the harmful bacteria “over control”, which can lead to bacterial vaginosis (BV). With BV and parasitic infections the vaginal pH usually increases to 5.0 or 6.0 and above, which are above the normal range.
Bacterial Vaginosis (BV)
BV is the most common cause of abnormal vaginal discharge in women of childbearing age, but may also be encountered in menopausal women, and is rather rare in children. The prevalence in the United States is estimated to be 21.2 million (29.2%) among women ages 14–49, based on a nationally representative sample of women who participated in NHANES 2001–2004. The following are other findings from this study:
• In Caucasian women the prevalence is 5-23%
• In African and American blacks 45-55%
• Mexican Americans 32%
• In Asian women the prevalence is less well studied, but in general around 20-30%.
BV is caused by high concentration of gram-negative rods of anaerobic bacteria like: Gardenerella vaginalis, Mycoplasmas hominis Ureaplasma, Prevutella spp., Mobiluncus spp., Peptostreptococcus spp.
This flora replaces the normal flora: Lactic acid and H2O2 to produce Lactobacillus sp.
BV doesn’t usually cause any vaginal soreness or itching, but often causes unusual vaginal
discharge.
BV is not serious for the vast majority of women, although it may be a concern if symptoms of BV develop in pregnancy and there is a history of pregnancy-related complications.
Around half of women with BV have no symptoms.
Trichomonas Vaginalis (TV)
TV is a globally occurring anaerobic Uorgenital Protozoa, which colonizes the epithelium of the human urogenital tract. Although often asymptomatic, TV infections can cause inflammation in the cervix, the vagina, and the urethra. Based on estimates of the World Health Organization (WHO) from 2008, trichomoniasis constitutes the most prevalent non-viral sexually transmitted disease (STD) worldwide, affecting more than 3.7 million people every year.
Health disparities persist in the epidemiology of TV infection in the United States: Of the women tested, 13% of black women were affected compared with 1.8% of non-Hispanic white women. TV infection affects >11% of women aged ≥40 years, and a particularly high prevalence has been detected among STD clinic patients. In the Republic of Korea, 10.4% of women complain of vaginal symptoms and signs, or were reported to be infected with TV.
Trichomonas infections are asymptomatic in as many as 50% of male and female patients.
Women and men are infected with comparable frequency, but in men symptoms are normally mild and infections are cleared by the host’s immune system within weeks.
In women, however, TV infections can persist for many years, and symptoms, mainly pruritus caused by inflammation and odorous vaginal discharge, can attain a severity which is debilitating.
Yeast infection - Candidiasis
Candidiasis is an infection caused by a yeast called Candida. Vulvovaginal candidiasis (VVC) is an infection caused by a Candida species that affects millions of women every year. Although candida albicans is the main cause of VVC; the identification of non-candida albicans candida (NCAC) species, especially candida glabrata, as the cause of this infection appears to be increasing. Candida normally lives inside the body (in places such as the mouth, throat, gut, and vagina) and on skin without causing any problems. Sometimes Candida can multiply and cause an infection if the environment inside the vagina changes in a way that encourages its growth. Candidiasis in the vagina is commonly called a “vaginal yeast infection.” Other names for this infection are “vaginal candidiasis”, “vulvovaginal candidiasis”, “candidal vaginitis”, or "trush infection".
Although most vaginal candidiasis is mild, some women can develop severe infections involving redness, swelling, and cracks in the wall of the vagina.
Approximately 75% of all women will experience at least one episode of VVC during their lifetime.
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