Multiple simultaneous testing media

• US 6,165,739 A
• Filed: 08/20/1999
• Issued: 12/26/2000
• Est. Priority Date: 03/13/1996
• Status: Expired due to Term

• Current Assignee: Thorlabs Incorporated
• Original Assignee: Compucyte Corporation (Thorlabs Incorporated)
• Inventors: Clatch, Richard J.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/372,115
• Time in Patent Office: 494 Days
• Field of Search: 435/29, 435/283.1, 435/286.5, 435/287.1, 422/50, 372/43
• US Class Current: 435/29
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: G01N 1/312 for samples mounted on planar substrates
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

A stationary testing media device made of a glass or plastic plate and conveniently a microscope slide, wherein multiple simultaneous tests are conducted on a single multifurcated testing sample. The plate or slide is partitioned into two or more testing zones with each of the zones having been preferably pre-loaded with a stable test reactant. The testing zones are physically separated from one another but are connected, with a fluid flow connection, to a single sample reservoir which simultaneously feeds, such as by capillary action, each of the testing zones with a test portion of the testing sample. The testing zones retain the individual portions for a time sufficient to effect the individual zone reactions with pre-loaded reagents. An optional outflow reservoir is utilized to capture excess sample flow and flushing fluids. Cell samples undergoing laser scanning cytometry are able to be simultaneously reacted, such as with varying fluorescent dyes, to provide a series of test profiles with a single sample and testing procedure.

Claims

1. A method of conducting at least two simultaneous tests on a single test sample with a laser scanning cytometer, wherein the single test sample is located on a single glass or plastic plate, said method comprising the steps of:

a. providing the plate with a test sample reservoir and at least two separated sample portion troughs in fluid connection with the reservoir;

b. providing each of the sample troughs with a stable reactant for a test sample, with said stable reactant being different in each sample trough in order to perform different tests on the test sample at the same time, and with said different stable reactants being provided in the sample troughs either before or after the placing of a fluid test sample in the respective sample troughs;

c. placing a fluid test sample containing cells in said reservoir wherein fluid connection means causes portions of the test sample to flow into each of the at least two separated sample portion troughs;

d. maintaining sufficient cells in each of the portion troughs for a time sufficient for the cells to react with the stable reactant within the individual portion troughs; and

e. removing excess reactant cells and scanning each of the portion troughs, containing cells reacted with the stable reactant, with a laser scanning cytometer, whereby test results can be meaningfully obtained by the laser scanning cytometer with minimized background noise.

2. The method of claim 1, wherein depth of each of the sample portion troughs is of predetermined dimensions and wherein the method further comprises the steps of:

i) precisely diluting the fluid test sample prior to placement in said reservoir;

ii) performing an assay by scanning the fluid test samples in each sample portion trough with the laser scanning cytometer over a fixed known area by fixing an extent scanned by the laser scanning cytometer that is digitized and keeping track of the number of known step-size steps that are moved in a perpendicular direction to complete the assay;

iii) calculating a volume of the assay; and

iv) thereby determining an absolute count and an undiluted absolute count of scanned cells in each sample portion trough.

3. The method of claim 1, wherein the method further comprises the steps of:

i) precisely diluting the fluid test sample prior to placement in said reservoir;

ii) performing an assay by determining the depth of each of the sample portion troughs;

iii) scanning the fluid test samples in each sample portion trough with the laser scanning cytometer over a fixed known area by fixing an extent scanned by the laser scanning cytometer that is digitized and keeping track of the number of known step-size steps that are moved in a perpendicular direction to complete the assay;

iv) calculating a volume of the assay; and

v) thereby determining an absolute count and an undiluted absolute count of scanned cells in each sample portion trough.

4. The method of claim 3, wherein the depth of each trough is determined by separately focussing a laser of the laser scanning cytometer on indicia printed on two inner surfaces of each trough which define the depth of the trough and measuring the distance between focus points so obtained.