Photodensitometer for minimizing the refractive effects of a fluid sample

A vertical photometer for measuring an optical characteristic of a fluid sample having a meniscus and for minimizing the optical effect of the meniscus, comprising:

• a light source;
• means for collecting light from the light source to form a light beam defining an optical axis;
• means for positioning a central portion of a meniscus on a fluid sample substantially perpendicular to the optical axis;
• and
• means for focusing the light beam to define a cone of light having a reduced diameter portion at the central portion of the meniscus so that refraction of the beam by the meniscus is minimized.

A photometric method for substantially locating a vertical light beam within a central portion of a fluid well bottom wherein the fluid well has a coordinate position on a plate having a known manufacturing variance, comprising the following steps:

• selecting a beam diameter at the well bottom smaller than the well bottom diameter;
• grossly positioning the plate so that the center of the well is positioned transverse to a radial path through the axis of the light beam within a predetermined tolerance which is smaller than the well bottom radius less the beam radius;
• using a plate having a manufacturing variance for the coordinate position of the well which is less than the predetermined tolerance;
• and
• moving the beam and plate relative to one another along a parallel path to the radial path and within the predetermined tolerance so that all of the beam passes through the well bottom at some point on the parallel path.

A photometric method for measuring the optical density of a fluid sample, comprising the following steps:

• containing the fluid sample in a fluid well having an open top and a substantially transparent bottom;
• forming a light beam having a central optical axis;
• focusing the light beam to provide a substantially narrowed portion thereof;
• grossly positioning the center of the well transverse to a radial path through the optical axis within a predetermined tolerance;
• axially positioning the well in a radial plane such that a central portion of a fluid meniscus on the fluid sample can be illuminated by the substantially narrowed portion of the focused beam to minimize refraction of the beam by the meniscus;
• and
• moving the beam and well relative to one another along a parallel path to the radial path in discrete steps so that all of the beam passes through the well bottom at some point on the parallel path.

A method for determining a central location of a light beam within a well having upwardly projecting sidewalls and a substantially flat, transparent portion, comprising the following steps:

• moving the light beam and the well relative to one another along a path such that all of the beam passes through the transparent portion at some point on the path;
• measuring the intensity of the light transmitted through the substantially transparent portion;
• detecting a maximum intensity measurement;
• and associating the detected maximum with a central beam location wherein all of the beam has passed through the substantially transparent portion.

A method for determining the HLA type of cells, comrprising the following steps:

• introducing a human leukocyte suspension into a plurality of wells having open tops and substantially transparent bottom portions wherein the wells are coated to enhance the binding of the leukocytes to the wells;
• centrifuging the cell-containing wells thereby forming supernatant;
• removing the supernatant and washing the cell-containing wells;
• adding monoclonal antibody specific for an alloantigen into at least one well, wherein differing monoclonal antibodies are put in different wells resulting in the formation of monoclonal antibody/cell complexes;
• washing the wells to remove nonspecifically bound monoclonal antibody;
• adding antibody conjugated with an enzyme or a receptor conjugated to an enzyme which binds to the monoclonal antibody/cell complexes;
• adding a colorimetric substrate to the wells to provide a fluid sample having an absorption maxima at a specific wavelength, wherein the optical density of the fluid sample at the wavelength is proprotional to the degree of formation of antigen-antibody complexes;
• generating a light from a light source substantially at the wavelength;
• collecting the generated light and forming a light beam havng a central optical axis;
• focusing the light beam to form a vertical cone of light;
• axially positioning the meniscus of the fluid sample substantially at the narrowest portion of the light cone so that a central portion of the meniscus illuminated by the light cone has a minimal refractive effect on the light beam;
• moving the light beam and the transparent portion relative to one another along a path such that all of the beam passes through the transparent portion at some point on the path;
• measuring the intensity of the light transmitted through the substantially transparent portion;
• and
• detecting the light transmitted through the fluid sample and therefrom determining the HLA types of the cells.