Document 0837
 DOCN  M9480837
 TI    Immunomagnetic capture of CD4+ cells from whole blood for HIV detection
       by PCR.
 DT    9410
 AU    Piccoli SP; Ekeze TE; Gross S; Mehta RR; Kerschner JH; Immunicon Corp.,
       Huntingdon Valley, PA.
 SO    Abstr Gen Meet Am Soc Microbiol. 1994;94:555 (abstract no. C-366).
       Unique Identifier : AIDSLINE ASM94/94313108
 AB    Rapid diagnosis of HIV infection may be greatly facilitated by direct
       identification of the provirus, regardless of the time course of
       antibody response. Toward this end, we have developed a system to permit
       specific isolation of CD4+ T-cells for subsequent genetic analysis by
       PCR. Samples of whole blood (50-75% v/v in PBS) were incubated with an
       anti-CD4 monoclonal antibody. A ferrofluid (colloidal magnetite, Fe3O4),
       coated with goat anti-mouse Fc antibodies, was added to magnetically
       label the target CD4+ cells. Samples were then subjected to a brief high
       gradient magnetic separation (HGMS) to isolate the cells. To ensure
       complete removal of erythrocytes, the cells were resuspended and
       magnetically washed. Lysis was accomplished in a Proteinase K/Tween 20
       buffer at ambient temperature. Following heat inactivation of the
       enzyme, aliquots were subjected to PCR. All results were confirmed by
       probe hybridization to the product. In an initial study, twelve blinded
       samples from previously diagnosed patients or negative controls were
       examined for the presence of HIV using this procedure. All samples were
       found to be in agreement with serology and clinical diagnoses (10 HIV
       seropositive and 2 HIV seronegative) by appropriate detection of the
       presence or absence of the provirus. The sensitivity of this method was
       found to be equivalent to others such as Ficoll-Hypaque cell separation
       or erythrocyte removal by ammonium chloride lysis, followed by WBC lysis
       to release DNA and/or phenol/chloroform extraction. Similar results with
       mock-infected samples indicated a sensitivity limit of 10 HIV genome
       equivalents added to the final cell lysate. CD4+ cells were isolated in
       approximately 95% yield with approximately 95% purity as analyzed by
       flow cytometry. These data suggest that biologically active ferrofluids
       represent a rapid (< 30 minutes) and efficient method for preparation of
       peripheral blood cell subsets for diagnostic analysis by PCR.
 DE    Acquired Immunodeficiency Syndrome/*DIAGNOSIS  Cell Separation/*METHODS
       Flow Cytometry  Human  HIV/*ISOLATION & PURIF  *HIV Seronegativity  HIV
       Seropositivity/*DIAGNOSIS  Lymphocyte Subsets/CYTOLOGY/PATHOLOGY
       Magnetics  Polymerase Chain Reaction/*METHODS  Proviruses/ISOLATION &
       PURIF  T4 Lymphocytes/CYTOLOGY/*MICROBIOLOGY/PATHOLOGY  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).