US 2959665 A
Description (OCR text may contain errors)
NOV. 8, 1960 c, RHEEs 2,959,665
ADAPTER FOR USE IN BLOOD PLASMA PROTHROMBIN TIME DETERMINATION Original Filed Feb. 6, 1956 2 Sheets-Sheet 1 M. C. RHEES Nov. 8, 1960 ADAPTER FOR USE IN BLOOD PLASMA PROTHROMBIN TIME DETERMINATION Original Filed Feb. 6. 1956 2 Sheets-Sheet 2 INVENT OR ATTORNEY United States Patent ADAPTER FOR USE IN BLOOD PLASMA PRO- THROMBIN TIME DETERMINATION Mark C. Rhees, 4411 W. Othello St., Seattle, Wash.
Original application Feb. 6, 1956, Ser. No. 563,519,
now Patent No. 2,878,715, dated Mar. 24, 1959. Divided and this application Jan. 23, 1959, Ser. No. 788,635
4 Claims. (Cl. 219-43) This application is a division of my copending United States patent application Serial No. 563,519, filed February 6, 1956, issued March 24, 1959, as Patent No. 2,878,715.
The present invention relates to blood plasma prothrombin time determinations and provides an instrumentality useful in practicing a method of making such determinations.
Important objects are to improve the accuracy of such determinations by providing novel apparatus for controlling temperature and eliminating errors inherent in the prior art apparatus and methods.
The invention involves apparatus for use in photoelectric detection of the instant of clotting by the use of a standard type of spectrophotometer and comprises a novel adapter provided by the invention for maintaining a sample of thromboplastin-plasma mixture at a constant, accurately controlled temperature and positioning the sample in the spectrophotometer.
In addition to the enhanced precision effected by the new device, an important advantage of the invention is the simplicity of the mechanical equipment and the ease with which the procedure for using it can be learned by relatively inexperienced personnel, with the result that the human equation error is substantially eliminated so that different workers arrive at uniform determination figures in separate tests of the same mate-rial.
In the accompanying drawings, which illustrate in certain preferred forms the apparatus comprising the invention,
Figure l is a perspective view of the complete apparatus;
Fig. 2 is a side elevational view of the new adapter or test tube holder;
Fig. 3 is an exploded perspective view of the adapter;
Fig. 4 is a top plan view of the adapter;
Fig. 5 is an axial sectional view of the adapter on a relatively enlarged scale;
Fig. 6 is a transverse sectional view taken on the line 66 of Fig. 5;
Fig. 7 is a transverse sectional view taken on the line 7-7 of Fig. 5; and
Fig. 8 is a bottom plan viev. of the adapter.
In Fig. l the reference numeral 1 designates a spectrophotometer of known type, such as a Coleman Junior Model 6 manufactured by Coleman Instruments, Inc, of Maywood, Illinois. This instrument has the usual well W, which in Fig. l is shown filled with the adapter 2 provided by the present invention, as well as an adjusting knob 3 and a galvanometer dial 4. Alternatively a colorimeter of standard type, such as a Leitz or Spectronic 20, could be substituted for the spectrophotometer shown.
The power supply 5 comprises simply a casing containing a volt A.C. stepdown transformer of the door bell type having an input cord 6 and an output cord 7 connected to the adapter and designed to deliver about 8 volts. The casing may conveniently be provided with a well W, similar in size to that of the well W of the spectrophotometer, for holding the adapter 2 when not in use.
The adapter 2 comprises an outer casing 10 of generally tubular form preferably made of machined aluminum, dyed black and anodized. The upper end of the casing is inwardly flanged, as shown at 11, so that the orifice, which in the illustrated embodiment is designed to accommodate rather snugly a 10 mm' culture tube or cuvet 12, is of slightly smaller diameter than the bore 13, which is cylindrical and extends uniformly through the open bottom end of the casing, which is surrounded by an axially directed peripheral flange 14. Just above its open bottom the casing is drilled diametrically to provide two aligned transverse sight openings 15.
A core member 16, made of the same material as the casing 10, is contained within the casing and has a cylindrical bore 17 in which the tube 12 makes a loose fit. For snugly seating the core member in the casing, the lower end portion 18 of the core member has an external diameter equal to the diameter of the bore of the casing, and the length of the core member is such that its upper end engages the flange 11 at the top of the casing when the closed bottom 19 of the core member is aligned in the same plane as the bottom of the casing (at the root of the flange 14). Most of the length of the core member is of reduced outside diameter, as shown at 20, and the lower portion 18 is slotted lengthwise, as shown at 21 in Fig. 3, to accommodate two leads of the heating wire 22, which will be hereinafter described. Just above its bottom the core member is diametrically bored to provide oppositely aligned sight openings 23, which register with the casing sight openings 15 when the core member is in place, as shown in Fig. 5.
A cap 25, made of the Same material as the casing and core member, has an axially projecting internal flange complemental to the flange 14 at the bottom of the casing and makes a tight friction fit over the casing bottom, providing a hollow chamber containing a small thermostat 26 of the kind made by Fenwal Corporation, Ashland, Mass.
An annular rubber gasket 27 may be interposed between the thermostat and the closed bottom of the cap 25 to cushion the thermostat and press it up against the bottom 19 of the core member 16. The cap is drilled to provide a small opening 28 for insertion of a tool, such as a small screwdriver, for effecting adjustment of the regulator screw 29 of the thermostat.
The terminals 30 of the thermostat are connected to the leads of the heating wire 22 which extend up through the slot 21 for connection with the power supply output cord 7 which enters the casing 19 through a hole in its upper side wall. The heating wire 22 in the preferred, illustrated embodiment of the invention consists of approximately 25 inches of cotton covered #34 Nichrome steel wire disposed in ten turns around the upper, smaller diameter portion of the core member, the remainder of the length of the wire being folded on itself and fitted into the slot 21. The arrangement of this wire is important, as will be explained hereinafter. For clarity of illustration the twisted condition of the wire and its position in the slot 21 are not shown in Fig. 3.
As will be seen in Fig. 3, the thermostat and heating wire turns are in series and are supplied by the cord 7 from the power supply 5. For the type of instrument shown in Fig. 1, in which the well is of plastic having good heat insulating properties, it is appropriate to supply about five watts at 8 volts. With instruments having metal wells somewhat more power should be used.
The thermostat is adjusted to maintain a temperature of 37 C. It is important that the device function-with precision to maintain this temperature, and it is for this purpose that the wiring arrangement be made in the following manner:
Of the 25 inches of wire, ten turns are made around the reduced portion 20 of the core member. The remainder is twisted and fitted into the slot 21. If all-of the wire were disposed in additional turns, the result would be too much lag at the thermostat and the ternperature could not be maintained accurately at-the setting. Moreover, if part or all of the wire were disposed nearer the thermostat, the control would be too sensitive and the tube 12 would not reach the desired temperature.
Power of five watts at eight volts is preferred for a spectrophotometer of the kind selected to illustrate this disclosure of the invention because such power results in the thermostat keeping the circuit closed about half of the time, thus maintaining the temperature within a cycle range of not more than 0.5 C. Excessive power would bring the tube to too high a temperature before the thermostat would open the circuit, so that a cycle range of as much as several degrees would result.
For use with the type of instrument shown in Fig. l the casing has a small pin 35 projecting from the upper portion of its side wall. This serves to align the registering openings 15 and 23 with the light beam when the adapter is inserted in the well W, as shown in Fig. 1.
In use the thermostat 26 is regulated to keep the contents of the test tube 12 at a constant temperature of 37 C., which of course is normal blood temperature, and the method of using the apparatus is as follows:
(1) A quantity of freshly drawn blood is mixed with sodium citrate and the plasma is separated by centrifuging, all in a well known manner. The proportion recommended is 0.5 ml. of 0.1 M sodium citrate to 4.5 ml. of blood.
(2) Into each of several 10- by 75-min. cuvets is meas- UI'ed a 0.10-ml. portion of 0.02 M calcium chloride solu tion and the cuvets are placed in a 37 C. constant temperature water bath located near the spectrophotometer.
(3) Equal parts of plasma and thromboplastin reagent are thoroughly mixed in a test tube. It is convenient to use 0.40 ml. of plasma with 0.40 ml. of Difco thromboplastin reagent produced by Difco Laboratories, Incorporated, Detroit, Michigan, and to use 1 ml. serologic pipets for measuring these quantities.
(4) The tube containing the thromoplastin-plasrna mixture and the l-ml. pipet used in measuring the two ingredients of the mixture are placed for five minutes in the 37 C. water bath.
(5) When the mixture has come to temperature, one of the cuvets containing the 37 C. calcium chloride solution is quickly wiped dry and placed in the warm adapter in the spectrophotometer well. The wavelength is set at near 500 m. and the galvanometer indicator is adjusted so that it is just visible in the extreme right side of the scale opening.
(6) Two-tenths ml. of the test mixture is drawn into the tip end of the warm l-ml. pipet and forcibly blown into the cuvet in the adapter, and at the instant of blowing a stopwatch is started. Immediately then the pipet is palmed in such a way that the cuvet can be removed from the adapter and mixing is completed as by striking a few sharp blows on the hand. The cuvet is then returned to the adapter, and the pipet is wiped and returned to the tube containing the thromboplastin-plasma mixture. This operation should be completedwithin an elapsed time of five seconds. When the cuvet is finally placed in the adapter, the galvanometer indicator will return to about the center of the scale and come to rest. The end-point of the determination is the instant at which the indicator makes a positive move toward the left. At this instant the stopwatch is stopped, and the elapsed time noted is the prothrombin time.
It is recommended that the test be repeated at least two more times and the average value taken.
It may be pointed out that if oxalated plasma is usedthe slow precipitation of calcium oxalate may not allow the indicator to come to rest when the cuvet containing the mixture is placed in the spectrophotometer well.
It will be appreciated by those skilled in the art that the controlled constant temperature made possible by the new apparatus, employed as hereinabove explained, is productive of a high degree of uniformity in results so that errors attributable to variations in temperature, not only as between different determinations of the same plasma, but also during the making of a single determination, are effectively eliminated.
While. the invention has been explained in terms of a single preferred construction, it is, to be understood that variations from the disclosed embodiment may be prac-, ticed without departing from the essential principles of the invention as defined by the broader of the appended claims. Thus, for example, while aluminum is the preferred material for the casing and core member because of the high coefficient of heat-conductivity of aluminum, other materials could be substituted. So also with other features of the disclosed structure, as will be understood.
1. An adapter for use in subjecting a prothrombinplasma mixture to a photoelectric beam comprising an outer casing having an axial bore and a hole in the upper portion of its side wall, a tubular core having a bore of uniform diameter throughout its length and having an upper outer surface of relatively small diameter and a lower outer surface of relatively large diameter concentrically disposed in said bore with the surface of relatively large diameter snugly seated in the lower portion of the bore of the outer casing and with the surfaceof relatively small diameter spaced radially inwardly from the adjacent surface of the casing bore, a winding of electric resistance wire surrounding the core and disposed within said space for heating the core, axially spaced bottoms for the core and easing, a thermostatic element contained within the space between the bottoms, the bore and easing having transversely aligned openings just above the core bottom for passing a light beam through a test tube supported on said core bottom, and a circuit including the winding and thermostatic element in series and terminating in leads extending from the casing through said hole in the upper portion thereof.
2. An adapter for use in subjecting a prothrombinplasma mixture to a photoelectric beam in the well of a spectrophotometer, said adapter comprising an outer casing having a smooth cylindrical outer surface adapted to fit snugly into said well and an inner core having a closed bottom and a smooth cylindrical bore adapted snugly to receive a culture tube resting on said bottom, said casing having a radially inwardly flanged upper end defining an orifice for said tube and said core having a lower portion of large diameter snugly fitting the inner surface of the casing and an upper portion of smaller diameter engaging and terminating at the flanged upper end of the easing and providing an annular, axially elongated space between the core and the casing, an electric heating wire Wound about the upper portion of the core within said space and having lead-in terminals extending through a hole in the upper part of the casing, and the lower portions of the core and casing being providedwith diametrically aligned sight openings for passinga b eamoflight through a tube resting on the bottom of the core when the adapter is in place in the well of the spectrophotometer.
3. An adapter as claimed in claim 2, in which the outer casing is provided with a chamber beneath the bottom of the core, with a thermostat contained within the chamber and in series with the heating wire.
4. An adapter as claimed in claim 2, in which the outer casing is provided with a chamber beneath the bottom of the core, a thermostat is contained within the chamber, the lower portion of the core is slotted lengthwise, and Wires connecting the thermostat with the heating wire are disposed in said slot.
References Cited in the file of this patent UNITED STATES PATENTS FOREIGN PATENTS France Oct. 7, 1953