Microdroplet test apparatus

A microdroplet test apparatus for use in HLA typing. The apparatus is an improvement upon the well known Terasaki tray which is in use for determining HLA antigens by measuring lymphocyte cytotoxicity. The apparatus includes a tray having a plurality of microtest wells wherein the sides of the microtest wells are designed to promote localization of lymphocytes or other cells being tested at the microtest well bottom. The apparatus further includes a cover having either rods or tubes which extend down into the test solution present in the microtest well to thereby control the amount of test solution which is present in the cylindrical optical view path through which the test solution is viewed by microscope for test result measurements. This allows control of the amount of test solution in the optical view path in order to reduce the amount of solution which must be looked through when cells are being counted or alternatively, increasing the amount of test solution present in the view path in order to maximize detection when a colored reaction product in the test solution is being measured.

BACKGROUND OF THE INVENTION 
The present invention relates generally to an improved microdroplet test 
apparatus used in determining human leukocyte antigens (HLA antigens) by 
measuring lymphocyte cytotoxicity. More specifically, the present 
invention relates to an improved microdroplet test tray design and 
improved tray cover structures which are designed to enhance the accuracy 
and usefulness of the microdroplet test system. 
The microdroplet lymphocyte cytotoxicity test was introduced in 1964 by 
Terasaki, P. I., McClelland, J. D.: Microdroplet Assay of Human Serum 
Cytotoxins. Nature 204: 998-1000, 1964. Since that time, the microdroplet 
lymphocyte cytotoxicity test has gained universal acceptance as the method 
of choice to test for HLA antigens. 
The basic microlymphocyte cytotoxicity test consists of reacting 0.001 
milliliters of lymphocytes with 0.001 milliliters of a specific antibody 
which is known to be reactive with a specific HLA antigen. If the 
lymphocytes contain the specific antigen being tested for, the antibodies 
will bind to the lymphocytes. Measurement of the degree of binding between 
the specific antibody and the lymphocytes is accomplished by adding 0.005 
milliliters of rabbit complement into the test well containing the 
lymphocytes and antibody. The rabbit complement promotes lysing of those 
lymphocytes which have reacted with the antibody. Measurement of the 
antibody-lymphocyte reaction is then measured by viewing the test solution 
in the test cell with a 10.times. microscope to determine the degree of 
lymphocyte lysis. 
In order to provide a suitable means for handling these minute quantities 
of reagents and additionally to provide an apparatus in which numerous 
tests can be carried out simultaneously, a plastic tray with multiple 
microtest wells was developed. These trays, which are commonly known as 
"Terasaki Trays" are widely known and used for microdroplet testing of HLA 
antigens. The microtest wells in the Terasaki tray are circular 
frusto-conical wells having an inwardly sloping funnel-shaped straight 
side wall of constant slope. The inwardly sloping side wall terminates at 
the microtest well bottom. The microtest well bottom has a circular cross 
section which is designed to be equal to the area which is visible in the 
single field of a 10.times. microscope objective when the test wells are 
viewed from directly above and adjacent to the tray. This allows the 
technician to view the entire test reaction (i.e. cell lysis) in one field 
of the microscope. 
Although the Terasaki tray has experienced wide popularity and is well 
suited for its intended purpose, some difficulty has been experienced with 
lymphocytes sticking or not flowing completely down the constant slope 
microtest well side wall. It is critical that the entire sample of 
lymphocytes to be tested be placed at the bottom of the microtest well. If 
the lymphocytes become stuck or otherwise adhere to the upper portions of 
the microtest cell side wall, a false positive test result is possible. It 
therefore would be desirable to provide an improved tray in which the test 
wells are shaped to prevent retention of lymphocytes on the microtest cell 
side walls to thereby promote localization of the lymphocytes at or near 
the microtest well bottom. 
During HLA testing, one or more reagents which are themselves colored or 
which produce a colored product may be utilized. Further, certain test 
solutions may be opaque. These colored test solutions make it difficult to 
count the number of lysed leukocytes remaining at the bottom of the 
microtest well after completion of the test. It would be desirable to 
provide an apparatus in which the amount of colored solution through which 
the technician must look to view the leukocytes can be reduced. 
The microtest tray can also be used for various testing procedures in which 
a colored product is produced. In these situations, it is many times 
desirable to increase the depth of test solution in the microtest well 
without increasing the total amount of reagents and cells utilized in 
order to increase the detection limits of the procedure. It would be 
desirable to provide some type of apparatus which could be combined with 
the Terasaki Tray to provide this desired increase in optical path through 
the test solution in order to maximize color intensity for measurement. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a microdroplet test apparatus is 
disclosed for use in determining HLA antigens by measuring lymphocyte 
cytotoxicity. The present apparatus basically includes a microdroplet 
testing tray having a plurality of microtest wells at spaced locations. 
The microtest wells are designed to receive test solutions which typically 
will include lymphocytes and selective cytotoxic reagents such as 
antibodies and complement. The microtest wells include an upper rim 
located on the tray top which defines an opening in the tray which is of a 
sufficient surface area to allow convenient introduction of small 
quantities of lymphocytes and reagents into the microtest wells. The 
microtest wells further include a bottom having a surface area less than 
the surface area of the well opening and a well side wall extending 
between the upper rim and the well bottom. The microtest well includes a 
central cylindrical zone extending vertically upward from the well bottom 
and defining a vertical view path wherein lymphocyte cytotoxicity or any 
other test result is visually measured by viewing vertically down through 
the test solution in the vertical view path. 
As a particular feature of the present invention, the side wall of the 
microtest wells has a vertical cross section which is arcuate. The slope 
of the arcuate side wall is less than vertical adjacent the microtest well 
rim and increases to a substantially vertical slope adjacent the well 
bottom. The arcuate shape and continuously increasing vertical slope of 
the microtest well wall promotes localization of the lymphocytes toward 
the well bottom to insure complete reaction of lymphocytes and antibodies 
in the test well. 
As an additional feature of the microdroplet test apparatus, a tray cover 
is provided which has a top surface, bottom surface and a perimeter. Means 
are associated with the perimeter of the tray cover for providing a seal 
between the tray cover and the microtest tray to thereby prevent 
evaporation of the test solution. Further, the cover tray is provided with 
view path control means located on the cover which extend downward from 
the cover bottom and are insertable within the microtest well for 
controlling the amount of test solution present in the vertical view path. 
This thereby provides selective control of the depth of solution in the 
vertical view path through which the technician must look when taking 
visual measurements. 
As one particular feature of the present invention, the view path control 
means is provided by a solid rod of optically clear plastic or glass which 
is integral with and extends downward from the cover plate in the view 
path and into the test solution. The solid rod displaces test solution 
from the optical view path thereby reducing the depth of solution which 
must be viewed through. By viewing down through the optically clear rod, 
the technician can now clearly view cells or other desired items present 
on the microtest well bottom even though the solution may be colored or 
turbid. 
As another feature of the present invention, the view path control means 
may be provided by a tube which extends through the tray cover and down 
into the test solution. The tube is positioned so that the internal 
conduit of the tube is coaxial with the vertical view path. The internal 
conduit and tube are appropriately sized so that a portion of the test 
solution is drawn up into the conduit by capillary action to thereby 
increase the depth of test solution present in the view path. This feature 
is useful when the reaction product or test result involves the 
development of a color which must be measured either visually or by 
suitable photo detectors since it increases the color intensity in the 
view path and thereby increases the accuracy of the measurement and allows 
detection of smaller quantities. 
The above-discussed and many other features and attendant advantages of the 
present invention will become apparent as the invention becomes better 
understood by reference to the following detailed description when 
considered in conjunction with the accompanying drawing.

DETAILED DESCRIPTION OF THE INVENTION 
The improved microdroplet test apparatus in accordance with the present 
invention is shown generally at 10 in FIGS. 1 and 2. The test apparatus 10 
includes a tray 12 and a cover or coverplate 14. The tray 12 is preferably 
made from plastic or other relatively inert material and includes a 
plurality of microtest wells 16 which may be molded, machined or otherwise 
formed in the tray 12. The tray 12 also includes a top surface 18, a 
bottom surface 20 and a perimeter 22. 
As best shown in FIGS. 3-6, the microtest wells 16 include an upper rim 24 
on the tray top surface 18 which defines an opening 26 which is of a 
sufficient surface area to allow introduction of lymphocytes or other 
types of cells and reagents into the microtest well 16. The microtest well 
16 further includes a bottom 28 and a well side wall 30 extending between 
the upper rim 24 and the well bottom 28. The microtest well 16 includes a 
central cylindrical zone shown in phantom at 32 which defines a vertical 
view path wherein lymphocyte cytotoxicity or other measurable 
characteristics are measured by viewing vertically down through the test 
solution present in the vertical view path 32. 
The well side wall 30 has an arcuate cross section, as best shown in FIGS. 
3-6, wherein the slope of the side wall 30 gradually increases from less 
than vertical near the well rim 24 to substantially vertical at the well 
bottom. This is an improvement over the prior Terasaki trays where the 
side wall has a constant slope, since the increasing slope of side wall 30 
promotes localization of the lymphocytes at the well bottom 28. 
The cover 14 includes a top surface 34, a bottom 36 and a perimeter 38. 
Tongues or ridges 40 are provided (as best shown in FIG. 2) which extend 
around the cover perimeter 38 for making engagement with grooves 42 in the 
tray 12. The tongue and groove arrangement around the perimeters of the 
cover 14 and tray 12 provides means for sealing the cover 14 to tray 12. 
Other means for sealing the perimeters of the cover 14 to tray 12 may be 
utilized including various sealant materials, such as wax, polymer 
sealants or any other sealing system in which an air tight seal between 
the cover 14 and tray 12 is provided. 
The tongues 40 and grooves 42 provide a sealing arrangement which also 
provides for precise positioning of the cover 14 in relation to tray 12. 
In addition to the tongues 40 and grooves 42, pegs may be included on 
cover 14 (not shown) for insertion into holes 44 present in the tray 12 to 
provide additional accurate positioning of the cover 14 on the tray 12. 
As best shown in FIG. 1, it is preferred that both the tray 12 and cover 14 
be provided with alpha and/or numeric indicia shown generally at 46 and 
48. The indicia is provided to allow specific identification of each 
microtest well 16. 
Referring now to FIGS. 3 and 4, the tray 12 is shown containing a test 
solution 50. The test tray 12 is designed specifically for testing HLA 
antigens and therefore the test solution will generally include leukocytes 
(usually lymphocytes) and various specific antibodies and complement. Many 
times, the test solution 50 will be colored or may be turbid or opaque. In 
these situations, it is preferable to reduce the amount of test solution 
which is present in the verical view path 32. In accordance with the 
present invention, cover 52 is provided with a plurality of view path 
control means, such as solid rods 54 which are made from an optically 
clear material such as clear plastic or glass. The rod 54 includes a base 
56 which is attached to the cover bottom 58 by any convenient means. 
Preferably, the cover 52 and rod 54 are molded simultaneously together to 
form an integral cover. As shown in FIG. 4, when the cover 52 is 
positioned onto tray 12, the tip 60 of rod 54 extends into the test 
solution 50 so as to displace test solution from the view path 32 to 
thereby reduce the depth of test solution in the view path as shown at 62. 
The cross-sectional area and length of rod 54 may be varied depending upon 
the amount of test solution 50 which is desired to be diplaced from the 
vertical view path 32. For particularly turbid colored test solutions, 
longer rods may be preferred in order to remove as much test solution from 
the vertical view path 32 as possible while still leaving the lymphocytes 
or other cells at the microtest well bottom 28 for viewing. Preferably the 
rod 54 is tapered as shown so that the base 56 has a greater 
cross-sectional area than the tip 60. 
In other microtest procedures, test measurements or detections are based on 
the detection or measurement of the amount of color or other visual 
indicator present in the test solution. In these situations, it is 
desirable to maximize the amount of test solution present in the optical 
view path 32 as much as possible. As shown in FIG. 5, a test solution 64 
is present within microtest well 16. In accordance with the present 
invention, a cover 66 is provided which includes a view path control 
means, such as tube 68 which is attached to the cover and positioned to 
extend vertically downward into the microtest well 16 when the cover is 
placed in position on the tray 12 as shown in FIG. 6. The tube 68 includes 
an internal conduit 70 which has an open bottom end 72 and open top end 
74. As shown in FIG. 6, the test solution 64 is drawn up into the internal 
conduit 70 by capillary action to thereby effectively increase the depth 
of test solution 64 present in the view path 32 as shown at 76. For most 
cases, the amount of test solution pulled up into conduit 70 by capillary 
action will be sufficient to increase the amount of test solution in the 
view path 32 to desired levels. When it is desired to increase the amount 
of test solution in the optical view path 32 further, various apparatus 
such as vacuum systems or other conventional apparatus for creating 
negative pressure may be connected to conduit 70 in order to increase the 
amount of solution drawn into conduit 70. As can be seen from FIGS. 3-6, 
the present invention provides a particularly useful and simple means for 
controlling the amount of test solution which is present in the microtest 
well veiw path 32. 
The overall size of tray 12 and cover 14 are typically around 2 inches by 3 
inches. The microtest wells are arranged in an array which preferably 
includes about 60 microtest wells 16. The overall area covered by the 
microtest well array is preferably around 11/2 inch by 21/2 inches. Each 
microtest well is preferably approximately 3/16 inch in diameter at the 
upper rim 24 with the microtest well bottom 28 having a circular surface 
area which is equivalent to the field of view seen through a 10 power 
microscope when the microscrope lens is placed adjacent the cover for 
viewing down through the vertical view path 32. 
Although the microdroplet testing tray 12 and cover 14 may be used in a 
wide variety of microtest procedures, it is particularly well suited for 
use in the microdroplet testing for HLA antigens. The following is 
exemplary of a preferred use for the microdroplet test apparatus in 
accordance with the present invention. 
0.001-ml of the desired specific antisera is put into the tray wells 16 
with a serum dotting machine. This is followed by the addition of 0.005-ml 
of mineral oil. This sequence permits the dispensing of antisera into the 
center of the well. Without a machine it is necessary to reverse the 
sequence, that is, oil is added to the trays first and the antisera 
second, because the sera would evaporate before oil could be added. Manual 
adding of sera to the trays containing oil must be done carefully since 
the droplets tend to float on oil and stick to the sides of the test 
wells. To check proper placement, the sera are stained with phenol red to 
aid in visual observation. All these procedures are carried out on a table 
with built-in fluorescent lights beneath the table surface. It is 
important to examine the trays with this type of lighting to insure that 
all dots are in place. When a serum is missing it should be either added 
by hand or the well cancelled out by marking the bottom of the trays with 
a marking pen, since the absence of the serum in a given well will not be 
known once the tray is stained and fixed. 
After preparation, the trays are stored at -70.degree. C. Storage for more 
than several months often results in the formation of bubbles. Trays 
should not be stored in a CO.sub.2 atmosphere on dry ice since the acidity 
tends to destroy the activity of sera. When shipped in dry ice, trays are 
enclosed in either a tightly sealed glass container or an aluminum sealed 
container. 
It is important that all sera be centrifuged at high speeds to eliminate 
debris and lipids that come to the top of the tube. Sera are clarified 
either by ultracentrifugation at 35,000 rpm for 40 min. or by 
centrifugation of Beckman tubes at 15,000 rpm for 15 min. with the Beckman 
microfuge placed inside a refrigerator. The advantage of the Beckman tubes 
is that the lipids that rise to the top can be cut off with a razor blade. 
With ultracentrifugation, the serum is withdrawn from below by piercing 
the cellulose tube with a syringe and needle. This step is important to 
assure that the sera within the wells are as clean as possible. The 
presence of debris tends to make reading difficult and increases reading 
errors. As previously described, the use of the tray cover shown in FIGS. 
3 and 4 reduces this problem. 
Lymphocytes are added to the typing trays in 0.001-ml volume with the use 
of a 0.05-ml syringe attached to a repeating dispenser. The dotting of the 
trays requires careful addition of the lymphocytes and mixing of the 
microdroplets within wells. The arcuate sides of the wells 16 makes this 
step easier by promoting localization of the cells near the well bottom. 
An electrostatic mixer or a wire is used to insure that the wells are 
mixed. Trays are incubated in a controlled temperature incubator at 
25.degree. C. 
Following 1/2 hour incubation, 0.005-ml of complement is added. After a 
further 1-hr incubation at 25.degree. C., 0.005-ml of eosin dye is added, 
followed 2 min. later by 0.005 ml of formaldehyde. The trays are then 
covered with cover 52. Prevention of evaporation of the reagents as well 
as containment of formaldehyde is accomplished by the tongue and groove 
seal 40 and 42. Formation of bubbles in the tray can be reduced by storing 
trays in the refrigerator. Trays can be read with a 10 power microscope 
over a 1 to 2 week period after sealing by counting the number of unlysed 
cells present on the microtest well bottom. 
Having thus described exemplary embodiments of the present invention, it 
should be noted by those skilled in the art that the disclosures within 
are exemplary only and that various other alternatives adaptations and 
modifications may be made within the scope of the present invention. 
Accordingly, the present invention is not limited to the specific 
embodiment as illustrated herein and is only limited in accordance with 
the following claims.