Title:
METHOD AND APPARATUS OF SEPARATING RED CELLS FROM PLASMA IN WHOLE BLOOD
United States Patent 3762567


Abstract:
A method and apparatus for separating red cells from plasma in whole blood in which a diluent is added to whole blood to produce separation of the red cells. A double lumen cannula instrument may be used in withdrawing the blood from a patient and having an outer lumen for providing an anticoagulant diluent and an inner lumen for withdrawing blood plus diluent, the body portion of the instrument being approximately one inch long and made of a silastic material.



Inventors:
ALBISSER A
Application Number:
05/158676
Publication Date:
10/02/1973
Filing Date:
07/01/1971
Assignee:
HOSPITAL FOR SICK CHILDREN TORONTO,CA
Primary Class:
International Classes:
A61B5/15; A61M1/00; A61M25/14; (IPC1-7): A61B5/00
Field of Search:
210/73,83,84,65,532,534 128
View Patent Images:



Primary Examiner:
Spear Jr., Frank A.
Parent Case Data:


This is a division of U.S. Pat. application Ser. No. 708,668 filed Feb. 27, 1968, now U.S. Pat. No. 3,610,266.
Claims:
I claim

1. Apparatus for separating red cells from plasma in whole blood including:

Description:
This invention relates to a method of, and apparatus for, separating red cells from plasma in whole blood particularly of the type used in practice and in experimental research on animals and human beings.

In hospitals and research institutions, it is often necessary to withdraw blood from an animal or a human patient over long intervals of time and, in some tests, it may even be necessary to withdraw blood substantially continuously for between 2 to 5 hours. For example, in metabolic testing a sugar solution is injected into a patient at a regular predetermined rate or at regular predetermined intervals whilst at the same time a continuous sampling of the patient's blood is carried out. The blood is then subjected to certain testing procedures in order to ascertain its sugar content throughout the period of test. For this purpose, it is necessary to insert a cannula into the patient's vein or artery whereby the blood can be continually withdrawn. In order to prevent coagulation, it is, of course, necessary to provide an anti-coagulating diluent and therefore a double lumen cannula instrument is normally used whereby two concentric tubes are provided, the anti-coagulant diluent flowing towards the vein or artery along the outer tube whilst the mixture of blood and anti-coagulant diluent is withdrawn through the inner tube by means of a pumping action. Steps should, of course, be taken to ensure that the anti-coagulant diluent does not enter the bloodstream of the patient.

As will be clear from the above discussion, a double lumen cannula instrument must be provided to permit the anti-coagulant diluent to flow towards the vein or artery and to facilitate the withdrawal of the blood plus diluent away from the vein or artery. The tubes from the diluent-supplying means and the blood-pumping means must be connected each to the respective one of the two concentric tubes forming the double lumen cannula. In some instances, this has been achieved by obtaining a metal two-way stop cock, welding the handle of the stop cock so that it is in an open position whereby a cannula can be passed through the bore thereof for use in withdrawing blood whereby one end is inserted into the vein or artery whilst the other end is connected to the pump. A plastic sheath was fitted over that end of the cannula which was to project into the vein or artery and slid along the external surface of the cannula and over the respective part of the stop cock so as to be sealed thereto by means of a sealing compound. A sealing compound was also sometimes used to seal the cannula to the metal stop cock so as to provide a fluid-tight seal. However, in practice, it was found extremely difficult to maintain a fluid-tight seal and, furthermore, the metallic stop cock was found to be relatively heavy resulting in a discomfort to the patient when used over a long period of time. The metallic stop cock was found to be cumbersome in use and spaces within its body resulted in blood clots being formed in the withdrawn blood.

It is an object of the present invention to provide a method of separating red cells from plasma in whole blood wherein there is less tendency for blood clotting to occur.

Accordingly, there is provided a method of separating red cells from plasma in whole blood including the steps of causing a flow of diluent and a flow of whole blood to meet and continue along a single flow path whereby red cells separate from plasma; and providing separate flow paths for said red cells and said plasma.

From another aspect it is an object to provide apparatus for separating red cells from plasma in whole blood.

According to this aspect there is provided apparatus for separating red cells from plasma in whole blood including a first part having two passageways therein, a first passageway for whole blood flow and a second passageway for the flow of diluent; said first and second passageways meeting to provide a common flow passageway.

More specifically there is provided apparatus also including a further part into which the common flow may flow; said further part including a first branch passageway for the passage of said red cells therealong and a second branch passageway for the passage of said plasma therealong.

Apparatus for separating red cells in blood according to the present invention may include a double lumen cannula instrument comprising a mounting of a non-metallic material, said mounting being formed with a first bore extending therethrough and of such a diameter as to be capable of receiving a cannula extending through the mounting, the mounting including a second bore extending from the exterior of said mounting into a cavity at the junction of said first and second bores, said first bore being of one diameter at one end and of a slightly greater diameter at the other end, whereby when said cannula is in position in said bore with its penetrating end protruding out of said other end, a fluid flow is possible from said second bore, into said cavity within said mounting and out through said other end to the exterior of the mounting.

An embodiment of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a diagrammatic representation of a double lumen cannula instrument used in the method according to the present invention;

FIG. 2 is a diagrammatic cross-sectional view of a mould for forming the instrument of FIG. 1;

FIG. 3 shows a rod for insertion in the mould of FIG. 2 to ensure correct formation of the double lumen cannula instrument;

FIG. 4 is a diagrammatic representation of part of a red cell blood separator unit for use with a double lumen cannula instrument as shown in FIG. 1 or independently thereof;

FIG. 5 diagrammatically illustrates a further part of a red cell blood separator unit; and

FIG. 6 is a diagrammatic representation of a separator system.

The double lumen cannula instrument as shown in FIG. 1 comprises a body portion 1 including an integral neck portion 2 formed as one unit, by a moulding operation, from a moulding compound, Silastic "A" RTV (Dow Corning). The overall length of the body portion, including the neck portion, is approximately 1 inch.

During the moulding of the cannula instrument, the body portion 1 is provided with a bore 3 of a first diameter extending for a first distance towards the neck portion 2 from the opposite end 4 of the body portion 1. The cannula instrument is also provided with a second bore 5 of a second greater diameter, the second bore extending from the region 6 of its junction with the first bore 3 and towards the neck portion 2. The bore 5 extends through the neck portion 2 so as to form an orifice 7 to the exterior of the neck portion.

The body portion 1 is also provided with a third bore 8 extending through the body portion from the exterior thereof and opening into the second bore 5 in the region 6. The third bore 8 is shown substantially at right angles to the second bore 5 although this is not, of course, essential to the invention.

In use, a 20 gauge lumen-forming cannula 9, i.e., a smaller diameter stainless steel tube, is inserted into the bore 3 with a substantial part of its length projecting forwardly of the orifice 7 in the neck portion 2 and a part projecting backwardly from the opposite end of the body portion 1 whereby a pumping device (not shown) can be attached by suitable tubing to that end of the lumen-forming cannula 9. It will be appreciated that, in use, the free end of the cannula 9 is inserted into the vein or artery of a patient whereby blood may be withdrawn for sampling. The blood vessel of a patient is diagrammatically illustrated in FIG. 1 and is identified by the numeral 10.

In FIG. 1, there is also shown a plastic outer lumen sheath 11 of a medical cannula which is of such a length that its end projects approximately 1 mm. beyond the end of the inner lumen 9 when the two are inserted into a blood vessel 10. The plastic lumen sheath 11 fits onto the neck portion 2 of the double lumen cannula instrument and because of the properties of the silastic material from which the body portion is moulded, a fluid-tight seal is achieved between the neck portion 2 and the plastic sheath 11. It will thus be seen that an inner lumen is formed by the lumen-forming cannula 9 whilst an outer lumen is formed between the external surface of the inner lumen-forming cannula 9 and the inner surface of the concentric plastic lumen-forming cannula sheath 11.

In use, an 18 gauge stainless steel tube 12 having a length of approximately three-fourth inch is inserted into the third bore 8 so as to form a fluid-tight seal therewith. Apparatus (not shown) is connected to the stainless steel tube 12 to supply an anti-coagulant diluent therealong and along the outer lumen formed between the tube 9 and the sheath 11 towards the blood vessel 10. Pumping means (not shown) is connected to the end of the tube 9 remote from the blood vessel 10 whereby blood may be withdrawn from the blood vessel for sampling purposes. Due to the pumping action, the anti-coagulant diluent is also drawn along the lumen 9 which therefore carries a mixture of blood and diluent so that substantially no diluent is passed into the blood vessel 10.

As will be clear from the above, the double lumen cannula instrument shown in FIG. 1 may conveniently be used in blood sampling tests on both human patients and animals. Because of the particular construction of the lumen cannula instrument and the advantageous medical properties of the silastic material used, it has been found that patients find that it is not as cumbersome as the previously used metal cannula instrument and that during a sampling period of from two to five hours the silastic cannula instrument is not as heavy and uncomfortable on the patient's arm as was the previously used metal instrument. Furthermore, it has been established during use that the silastic cannula instrument is not as liable to form clots in the withdrawn blood as was the previously used metal cannula instrument. As will be clear, the double lumen cannula is particularly useful for in vivo study of a patient and permits the continuous withdrawal of blood from the respective vein or artery by permitting the simultaneous infusion of the anti-coagulant, which mixes only with the withdrawn blood, whereby preventing the formation of clots in the sample tube 9. The mixing of the withdrawn blood and the anti-coagulant occurs only at the tip of the inner lumen 9 which is approximately 1 mm. inside the input end of the outer tubular sheath 11. Thus, the anti-coagulant drawn along the inner lumen 9 does not enter the bloodstream of the patient.

The rate of removal of blood from the patient depends on the difference the flow rates of the blood sample and the anti-coagulant diluent along the outer lumen. The solutions may be pumped to and from the cannula by a peristaltic pump-an auto-analyzer proportioning pump, which establishes a flow rate dependent on the diameter of the tubing in the pump. This ensures not only that the same volume of blood per unit time is withdrawn from the patient but also that both flows stop if the pump is stopped.

Due to the pressure produced by the pump, there is of course more tendency for liquid to leak out of the junctions between the plastic and metal of the tubes 9 and 12. To avoid this, the bores 3 and 8 may be moulded during manufacture to be slightly less than the external diameter of the respective tube. The bores 3 and 8 may conveniently be moulded to 0.025 inches, 23 gauge, whilst the bore 5 may be moulded to be 0.063 inch. The undersized bores in a plastic rubber thus ensure a good pressure fit when the stainless steel tubes are inserted and a good plastic-to-metal seal is effected which does not leak due to the suction of the pump.

As mentioned above, the double lumen cannula instrument according to the present invention may be included in a system for sampling the blood of a patient and performing a separation process thereon or alternatively, in instituting certain established test procedures. The sequence of operations for withdrawl of whole blood in a continuous monitoring experiment may be symbolized in block form where the double lumen cannula instrument feeds into a block representing withdrawl which also has an input from a further block labelled anti-coagulant and diluent capable of supplying the anti-coagulant and diluent at a rate RA. An output from the withdrawl block is fed to a calibration and adjustment unit at a rate R1. The calibration and adjustment unit supplies an output at the rate (R1 + R2) to the input of a separator unit. The rate of flow R2 represents the pumping rate of a further dilution of the blood sample.

The output of the separator unit will consist of two separate outputs, one being a PLASMA output and the other being a RED CELL output.

The sequential operations in the system may be considered as follows:

1. The continuous withdrawl of blood mixed in situ with an anti-coagulant and diluent.

2. The calibration and adjustment of the dilution factor ##SPC1##

3. The continuous separation of plasma from the diluted whole blood.

4. The chemical analyses performed on both the separated plasma and the red cell suspension.

Whole blood may be continuously withdrawn from a vein or artery of the organism by means of a double lumen cannula according to the invention consisting of two sterile disposable parts. The irst part may consist of the plastic outer lumen sheath of an Argyle Medicut cannula (Argyle Catalogue No. AR-3218) inserted into the blood vessel in the usual manner. The second part of the double lumen cannula instrument may consist of the above-described double lumen instrument properly inserted into the first part. In practice, the plastic outer lumen sheath 11 will be inserted into the respective blood vessel by means of the tissue-piercing syringe which is normally available for taking a blood sample and ensuring that one enters the respective blood vessel. The plastic sheath normally surrounds the needle of the syringe and is inserted into the blood vessel together with the syringe needle. After withdrawing the plunger of the syringe to examine the sampled blood, the syringe needle is withdrawn whilst the plastic sheath remains inserted in the blood vessel. The lumen-forming cannula 9 of FIG. 1 is then merely inserted down the plastic sheath until it enters the blood vessel in substitution for the aforesaid needle with the tip of the plastic sheath 11 projecting 1 mm. (approximately one thirty-second inch) beyond the tip of the inner lumen-forming cannula 9 within the blood vessel 10 (FIG. 1). If necessary, the two lumen-forming components may be pushed further into the blood vessel. The pump is now started and the whole blood sample is drawn into the cannula tip at the rate RHb at the same time being properly mixed with isotonic diluent and anti-coagulant. The diluent is pumped to the cannula tip along the outer lumen formed by the concentric sheath 11 at the rate RA. The diluted blood is withdrawn from the cannula tip via the inner lumen at the rate R1, which is simply the sum of the rates RA and RHb. This configuration prevents anti-coagulant and diluent from entering the bloodstream of the organism.

In one constructed system according to the present invention, the blood sample mixed with anti-coagulant and isotonic diluent was drawn from the double lumen cannula by means of a peristaltic pump (Technicon proportioning pump -- single speed) fitted with a manifold (Technicon manifold) containing one pump tube (Technicon pump tubes) whose diameter defined the flow rate R1. Tygon tubing was used to connect the cannula to the input of the pump and this should, of course, be kept as short as possible (between two and three feet) and its internal diameter should, of course, be small -- about 0.025 inches. These constraints minimize longitudinal diffusion in the tubing and also filter the flow irregularities of the peristaltic pumping. Further dilution of the blood sample can be carried out at the peristaltic pump by mounting on the same manifold a second tube, pumping at the rate R2, as mentioned above. The total dilution factor DF can be determined by measuring the flow rate R1 and the combined flow rate (R1 + R2). From these two rates, the rate RA can be determined for any desired dilution factor by applying the relationship

RA = R1 - DF(R1 + R2)

where RA is the rate of flow to the double lumen cannula tip.

Thus, the rate of whole blood removal is given by the formula

RHb = R1 - RA

During experiments, it has been found that RHb should preferably be greater than 0.1 ml/min. min. in order to minimize the peristaltically induced fluctuations in the flow R1.

Accurate measurement of the flow rates R1 and (R1 + R2) can be simply achieved by using two pipettes and a stop watch. The diluent at flow rate R2 passes along a path through the proportioning pump rollers to a three-way stop cock capable of passing the diluent either to waste or into the flow line of the flow at rate R1 (blood plus anti-coagulant diluent), after the respective proportioning pump roller in the direction of flow. When the first-mentioned stop cock is in the correct position, the combined flow (R1 + R2) is passed to a further three-way stop cock inserted in the fluid line whereby the fluid flow may be arranged to enter a 5 ml. pipette.

To measure R1, the flow R2 is diverted to waste by the firstmentioned stop cock and the flow R1 is directed into a 2 ml. pipette by means of the second-mentioned stop cock. The time to pump a known volume of liquid is measured and the flow can then be calculated. For the flow (R1 + R2), as mentioned, a 5 ml. pipette is used and by this means both flows can be measured.

In FIG. 2, there is shown a mould for manufacturing the silastic cannula instrument of FIG. 1. The mould may conveniently be a two-piece mould made of the plastic referred to as Lucite (registered trade mark). This mould is shown in cross section in FIG. 2 and it will be appreciated that prior to drilling and milling the mould, two pieces of Lucite are fitted together and held by means of three thirty-second inch studs approximately 1 inch long and having a half-round end together with a threaded opposite end. Conveniently, Lucite blocks may be used having a length several times the size of the required cannula instrument and, for example, a Lucite block may be 4-3/8 inches long so that six moulds may be drilled and milled into each block so as to produce the body portions for six double lumen cannula instruments according to the present invention. Thus, two pieces of Lucite material would be required having dimensions seven-sixteenths × 1-1/4 × 4-3/8 inches. The dimensions required for each mould are indicated in FIG. 2 for convenience, the overall thickness of the moulded body portion arranged to be approximately 1/4 inch.

Referring to FIG. 2, it will be seen that the mould 20 includes a main space 21 within which the main body portion 1 will be formed. The neck portion 2 (FIG. 1) will be formed within the neck space 22 which continues through into a space 23 corresponding to the second bore 5 of the double lumen cannula instrument of FIG. 1. The wall of the mould 20 is provided with a drilled, or otherwise formed, bore 24 having a diameter of 0.025 inch and emerging into the main space 21 of mould 20.

In FIG. 3, there is shown a rod 25 for insertion within the mould of FIG. 2 to ensure that the bores 3, 5 and 8 (FIG. 1) are properly formed. The rod 25 is of one-sixteenth inch diameter and 1 inch total length. At one end it is provided with an axially located bore 26 and a transverse bore 27 as shown in FIG. 3.

During a moulding operation, the end 28 of the rod 25 is inserted in the space 23 (FIG. 2) with the major portion of the rod 25 projecting into the space 21. To ensure that the bores 3 and 8 are properly formed, two pieces of wire, such as piano wire, are used. The first piece of wire having a diameter of 0.024 inch and a length of approximately 1/2 inch is inserted in the bore 26 of rod 25 so as to project upwardly through the top of the mould 20. The second piece of wire having a diameter of 0.024 inch and a length of approximately 3/4 inch is inserted in the bore 27 so as to project horizontally through the bore 24 in the mould 20 of FIG. 2. Thus, when the silastic "A" RTV (Dow Corning) moulding compound is injected into the mould, the bores 3, 5 and 8 will be properly formed.

The steps in the moulding operation may be summarized as below.

1. Ensure that the components of the mould are clean and dry.

2. Assemble the Lucite pieces of the mould together using, for example, three thirty-seconds inch diameter threaded studs approximately 1-1/4 inch long.

3. Insert the rod or wire into the axially located bore 26 of rod 25.

4. Clamp the assembled combination of rod 25 and the abovementioned rod or wire with a pair of hemostats and insert the end 28 of rod 25 into the 1/8 inch diameter hole 23 in the LUcite mould.

5. Align the bore 27 in rod 25 with the 0.025 inch diameter bore 24 in the mould 20 of FIG. 2.

6. Push the end of the respective rod or wire through the 0.025 inch bore 24 in mould 20 and into the bore 27 of rod 25 (FIG. 3).

7. Prepare the moulding compound, silastic "A" RTV (Dow Corning) moulding compound, and inject it into the mould, starting at at the bottom and filling the mould to the top so as to ensure that no air bubbles are trapped by the silastic material.

After the silastic material has properly set and catalyzed, the mould may be taken apart as follows.

1. Remove the rod or wire from the bore 27 by merely pulling it straight out.

2. Remove the threaded bolts or studs holding the two parts of the Lucite mould together.

3. Carefully separate the two parts of the Lucite mould.

4. Remove the silastic double lumen attachment from the Lucite mould.

5. Carefully pull out the remaining rods or wires, i.e., the rod 25 and the wire previously inserted into the bore 26 thereof.

6. Insert the two pieces of stainless steel tubing 9 and 12 of FIG. 1 into the moulded silastic body portion.

The stainless steel tubing constituting the lumen-forming cannula 9 and the stainless steel tube 12 of FIG. 1 must be carefully checked to ensure that no burrs, fragments of steel, or dirt are introduced into the final instrument. For the instrument of FIG. 1, the stainless steel tubing 9 should be of 20 gauge and approximately 4 inches long and, in step 6, is passed axially through the moulded body portion. The tube 12 should be of 18 gauge stainless steel tubing and approximately 3/4 inch long and, in step 6, would be inserted perpendicularly into the moulded body portion. In this way, a double lumen cannula instrument as shown in FIG. 1 would be constructed.

Sterilization of the double lumen cannula instrument may be effected by any of the usual methods.

The double lumen cannula instrument described above may conveniently be used for in vivo monitoring of blood parameters in response to drugs and/or other substances in humans or animals. General cannulations involving a double lumen catheter of any length can employ the double lumen cannula instrument to provide the anti-coagulant to the site of blood removal. Provided that the blood vessel is found and entered quickly, the procedure in using the described double lumen cannula instrument is simple and straight forward, taking only a minute or so, and once the pump is started the blood sample may be withdrawn continuously for as long as necessary and advisable. The double lumen cannula instrument has proved, in use, to be both rigid and strong and it has been discovered by experimentation that practical cannula instruments constructed of the above-mentioned silastic rubber moulding compound are particularly useful for medical purposes. Furthermore, the described double lumen cannula instrument may be regarded as a disposable item, for use in one operation only.

Turning now to another aspect, I have designed apparatus for separating blood by a new method.

In medical practice and research on animals and humans, it is sometimes necessary to separate whole blood into its constituent components. Centrifugation methods have previously been used in separators to separate the respective components from diluted whole blood. As is known, some chemical analysis must be made on plasma rather than on whole blood and therefore separated flows, one of diluted plasma and the other of diluted plasma plus the cells, are obtained by separation techniques so that the appropriate chemical analysis may be made.

By pumping a whole bloodstream into a diluent stream so dilution occurs continuously, hence dynamically (dynamic dilution), whole blood may be separated into two laminar streams. The upper stream will consist mainly of diluent and plasma whilst the lower stream will consist of concentrated red cells and some plasma. In other words, the settling time of red cells can be greatly enhanced when a small flow of blood is pumped into another flow of diluent. Settling then occurs in about one second and the liquid proceeds in two layers along the tubing of the apparatus used.

In FIG. 4, there is shown a part of a red cell blood separator unit.

The unit shown in FIG. 4 comprises a first part 30 having a cross-sectional shape as shown so as to form two passageways, a first passageway 31 for the flow of diluent and a second passageway 32 for the flow of whole blood. The passageway 32 joins the passageway 31 at right angles thereto and the output flow of the first part 30 continues in a single flow path along an outlet passageway 33 in-line with the passageway 31. A passageway 34 of a nipple part 35 is provided in-line with the passageway 31, the part 35 being held in abutting relationship with the part 30 by means of a Tygon (trade mark name) sleeve 36. A length of Tygon tubing 37 is provided on the end of the nipple part 35 whereby the flow thereto may be fed to subsequent apparatus.

The first part 30 may conveniently be a glass T fitting type D0 or D1. (Technicon)

It is important to note that air segmentation of the streams is not used because the air bubble tends to remix the blood cells and the plasma. Separators are effective to remove the lower layer of red cells. In FIGS. 5, there is shown a further part of a red cell blood separator unit whereby the concentrated red cells and some plasma may be separated from the partially cell free diluted plasma. The incoming laminar flow of diluted plasma and settled red blood cells travels along a length of diluted plasma and settled red blood cells travels along a length of tubing 38, through a nipple part 39 and through an in-line passageway 40 of a further part 41. The nipple part 39 and the further part 41 are held in abutting relationship by means of a sleeve 42.

The combined flow of diluted plasma and settled blood cells travels along the passageway 40 into a junction region 43 where the concentrated red cells travel along a lower branch passageway 44 whilst the partially cell free diluted plasma travels along an upper branch passageway 45. The concentrated red cells travelling along the lower passageway 44 will, in fact, include some plasma therein but by using three cascaded separator units I have been able to separate in excess of 99 percent of the red cells. This figure is, of course, subject to rechecking.

The partially cell free plasma flowing along passageway 45 may, of course, be passed through a second and then a third separator to further accomplish separation. I believe that the first separator will remove along passageway 40 a volume of fluid corresponding to about twice expected red cell volume. Subsequent separators do approximately the same, but the net flow is less because the red cell concentration has already been reduced by the first separator unit.

For proper settling of the whole blood, the tubing connecting each separator unit should be preferably maintained horizontal and the locus of its path should be kept smooth with only gradual changes. Smooth transition along the passageways formed by the respective tubing's internal diameters and the glass fittings must be maintained to avoid turbulence and non-laminar flow which results in mixing of the two streams.

A slight downward slope of the whole apparatus should enable the red cells to slide and flow at the same velocity as the supernatant plasma and diluent.

Obviously, completeness of separation and volume of cell free plasma bear to each other a reciprocal relationship.

At the present moment, I do not have a concrete theroretical explanation of the settling phenomenon. It does, however, occur in solutions at room temperatures as well as in ice-water and with diluents as different as 0.15 molar NaCl and 0.15 M LiNO3. In experiments, isotonic lithium nitrate has been used as a diluent because the lithium ion acts as an internal reference in the flame photometry of sodium and potassium.

It is expected that the flow pattern of the red cells in the small bore tubing and the sudden encounter with a new environment, namely that of the diluent, causes a change whereby the cells drop out of suspension. To contrast this effect, a well-agitated prediluted sample of blood settles slowly, very slowly compared to the settling of the cells when diluted dynamically, i.e., with both solutions in motion. Further experiments should be carried out to appreciate the hydrodynamic properties of these diluted suspensions of red blood cells.

A chemical analysis of the plasma obtained from the separator units described above may be made in a modified auto-analyzer system. Since the sample is prediluted both in the double lumen cannula instrument and at the first proportioning pump, it is only necessary to modify the auto-analyzer so that further dilution does not take place. The simultaneous electrolytes channel of one auto-analyzer determines Na+, K+, Cl-, CO2 in the separated plasma; while another auto-analyzer channel measures glucose in the separated red cell suspension.

The advantages of the continuous separation technique described above are believed to be as follows:

1. The cells are separated anaerobically, rapidly, and continuously.

2. The separation occurs `one line` as it were.

3. The gravitational stresses applied to the cells are insignificant.

4. The cells are not packed tightly together so that there is less chance of their contents exchanging with their environment.

5. The separated plasma is removed from the cells almost immediately.

The system also introduces several advantages:

6. The dilution factor can be easily controlled by adjusting RA.

7. The flow rate RHb of blood withdrawn can be made very small, 0.01 ml/min. so that frequent measurements may be made.

8. The system is particularly useful in continuous monitoring work because it eliminates the need for discrete batch centrifugation and thereby permits complete automation and integration of the withdrawal-analysis system.

There are also some possible disadvantages:

1. The measured plasma concentrations depend on the sample hematocrit because the whole blood is diluted first and then separated; in contrast to first separating the plasma from the cells and then diluting the plasma for analysis. The latter method is believed to introduce no hematocrit dependence.

The described separator method and apparatus may be used in conjunction with the double lumen cannula instrument illustrated in FIG. 1 or, alternatively, it may be used separately therefrom or with other suitable apparatus.

I have constructed a multiple separation unit for continuous separation using pieces of glass and tubing, etc. manufactured by the Technicon Company.

The arrangement is shown diagrammatically in FIG. 6 and it will be seen that heparinized, diluted whole blood flow from a double lumen cannula instrument, such as shown in FIG. 1, is fed along one input line whilst an isotonic diluent NaCl or LiNO3 is fed along another input line. The flow rates at various parts of the arrangement are indicated in ml/min. and a Technicon proportioning pump is utilized to ensure continuous blood withdrawal. The pump tube shoulder colours are indicated on the respective lines.

Identification of the respective parts in FIG. 6 is indicated in the following list.

A--tygon tubing 0.065" ID.; Length, 4 ft. minimum

B--tygon tubing 0.065" ID.; Length, 8 in. minimum

C--tygon tubing 0.065" ID.; Length, 8 in minimum

D--tygon tubing 0.025" ID.; Length, any

E--tygon tubing varies I.D.

F--tygon tubing varies I.D.

G--tygon tubing varies I.D.

Lengths, adjusted to properly phase flows from separators

R--roller of Auto Analyzer Proportioning Pump (Technicon)

W--glass fitting D2; (Technicon)

X--glass fitting C3; Debubbler (Technicon)

Y--glass fitting C2; Debubbler (Technicon)

Z--glass fitting G0; Cactus (Technicon)

The interconnecting tubes A, B and C serve the additional purposes of maintaining laminar flow as well as allowing settling of the red cells. It is necessary that they be placed to avoid any sharp bends or twists which would cause mixing of the two ribbons of fluid in the tubes.

All connections should be made properly using the appropriate nipples so that laminar flow is maintained throughout.

The red cell collecting tubes should be adjusted in length and internal diameter so that the three flows are in phase at the summation point Z. In this way, an analysis, such as glucose, performed on the red cell suspension will not suffer from excessive "mIxing" or loss of `response` to a step change in concentration.

To maintain the `response` of the separators it is also advantageous to place the separators in a descending cascade allowing the more viscous red cells to slide down a ramp thereby maintaining equal the velocity of both separated streams.

For different conditions, different pump tubes may be used. Perhaps only two instead of three separators need be used. The configuration shown is not necessarily the best; it removes better than 99 percent of the red cells, but proper adjustment of the orientation and settling lengths can increase this figure.