CA2194870A1 - Rapid determination of blood sedimentation rate - Google Patents

Abstract

An apparatus and method for rapid determination of erythrocyte sedimentation rates for a blood specimen (29) which can be linearly transposed to Westergren sedimentation rates. The method includes the steps of inducing accelerated rouleaux formation in the specimen (29) in an amount sufficient to begin settling at substantially the decantation rate for the specimen. In one embodiment a structure (27) which produces a very thin cross-sectional region (37) of the specimen (29) inside the lumen (23) of a specimen container (21) is provided to accelerate rouleaux formation. In an alternative embodiment (120), accelerated rouleaux formation is accomplished using a centrifuge (122). A third embodiment employs a movable rod (223) mounted inside the specimen tube (221) to induce accelerated rouleaux formation. All embodiments of the process next employ gravity settling the specimen in a near horizontal oriented container (21, 121, 221). Thereafter, the amount of settling occurring is determined. A sealed specimen container (21, 121, 221) which permits thorough mixing of blood in a very small diameter container for use in performing the method also is provided.

Description

~ WO96101990 2 rq~io RAPID DETERMINATION OF BLOOD SEDIMENTATION RATE

~I'T~'t~TNTrAT. FTT'T.n The present invention relates, in general, to a method and apparatus for detDrmi nl ng the set~l1 ng rate ofsolids in a liquid-solid mixture, and more particularly, relates to methods and npparatus for ~QtQrmining the ~LyLI.~yLe or red blood cell sQ~ Lion rate in whole blood.

~AI ~ ~ IUNI~ ,AT~T
The rate at which erythrocytes or red blood cells settle through blood plasma in a whole blood spQ~i- has long been the subject of medical study. It has been found that the se~i- ation rate of blood can be significantly increased by a wide range of inflammatory conditions and ~icQ~cQc. Various attempts have been made to automate blood SD~i- Lation apparatus and to correlate settl ing or sQ~i- Lation rates and patterns to infl~ L~LY
conditions.

The original laboL~tuLy studies, however, are still regarded as the standards. More particularly, there is a Wintrobe se~i- LuLion method and a Westergren SQ~;- LuLion method. The Westergren method is most widely used and employs a 300 m;ll; ter long settling tube with the lower 200 m; 1 l ;- ' QrR being graduated.
The tube is filled to the 200 m111 i- ' ~~- mark with approximately 0.8 milliliter of blood and 0.2 milliliters Wo96/ol99o 2 bq ~8 7i~9 ~-2- r_l,v~

of anticoa~llAnt diluent which i8 allowed to gravity settle over a one or a two hour period. The amount of set~1;ng in a one hour period in a Westergren settling tube i8. generally regarded as the standard for blood s~'; tion rate.

In any blood 6~; L~tion study the Er~n; is first thù~uuyl.ly mixed so that the eLy~hLouyLes are evenly distributed thLuuyhuuL the sper;- . In the Westergren method, after mixing, the 300 millimeter tube is brought to a vertical orientation for gravity settl;ng and the settling clock started. After one hour the amount of set~1;ng which has ocuuLLed, as d~t~mm;n~d by the distance that the interface between the plasma and the eLyLhLu~yLes has traveled downward, is measured.

Various attempts have been made to automate the Westergren settling process. United States Patent No.
4,0il,502 to Williams, et al., for example, disclose6 an automated 5~A;- Lation measuring system which mea~uL~ Ls are taken every 15 seconds for one hour (or two hours) and a blood se~;- LaLion curve is pIuduced as a result of these mea~uL- Ls. United states Patent No. 4,848,900 to Ruo, et al. is a similar blood sP~ a~ion automated system in which a blood 6~i -a~ation curve is also generated over a one hour period.

While the amount of se~ tion varies for each ~peci- , as influenced by ;nfli tULy conditions, the general shape of blood s~;- L~tion curves is guite similar as a result of the settling rh~- - which are operative. Most 5~A; Lation curves, therefore, have three phases which can be clearly identified. First, there is a "lag phase" in which settl;nq is very slow and gradual. Next comes a "decantation phase" in which rapid, virtually linear, settling occurs. Finally, there ~ W096/01990 21 q4870 '~ ~4~'~ i ' Y Jl-is a "syneresis phase" in which the rate of set~l;nggreatly slows towards the end Or the one hour settl;nq period.

While the ~ yLI.,o~yLes are more den~e than the plasma, they are small and thus have such a high surface area relative to volume that they do not readily se~;-through plasma as single cells. In the initial or lagphase, the~erul~, the ~LyU~ ~yLes must come in contact with each other to group together in clumps or clusters known as "rouleaux." Once a s-~frici~nt number of ~LyLI-~yLes have grouped in rouleaux, the rouleaux will begin to s~;- through the blood plasma toward the bottom of the s~ L~Lion tube. Thus, the initial se~;- L~Lion or lag phase may take 5 to 15 minutes for s~ffi~i~nt rouleaux clusters to form to enter the more rapid decantation phase. The lag phase portion of a se~;- L~Lion V8. time curve, therefore, is relatively flat and typically shows little ~ tion or . L
of the ~ LyLI-,v~yLe separation interface.

As the ~LyLI.,o~yLes in rouleaux settle, they contact other eLyLI.L~yLes which adhere and decrease the surface to volume ratio and hence the drag on the se~; Ling rouleaux. Additionally, however, the plasma at the bottom of the sr~;r L~tion tube must rise or be ~;~plac~d upwardly by a volume egual to the s~ Ling ~yLl-L~yLes. The settl;nq process, therefore, involves both downward migration or s~;- L~tion of the more dense ~LyLI.L~yLes in rouleaux and upward migration of the lighter plasma through the downwardly migrating rouleaux. The ~e~; Lion rate increases significantly and is fairly linear in the decantation phase or the mid-range of the s~;- L-~Lion process.

Toward the end of the s~;- L~Lion process, however, the erythrocytes begin to pack more tightly at the bottom wog6/olsso 2 1 9 4 87 0 , J i~

oi the tube. This narrows the pdLhw~yD for plasma to upwardly migrate to the pl r _Ly ~~ y Le Beparation boundary or interface. The pla~ma, therefore, has a more difficult time escaping from between the red cells in rouleaux as the packing density or h to~Llt rises.
SP~; Lation, thelcf~L~, again slows in this last or syneresis phase of Sr~i- tion.

Various attempts have been made to devise methods and apparatus for accelerating the ~t~rm;n~tion of oLyLhL~yLe 8r~;- L~tion rates. In my United States Patent No. 3,824,841, for example, a ~r~;- ;rn method i8 ~;~rlose~ in which spec; are centrifuged in vertically oriented settling tubes, with the tubes perio~i r~l 1 y rotated about their longitudinal axis. The erythrocytes seesaw back and forth across the tube and downwardly under a combination of gravity and centrifugal forces. The se~i- Lion time using this process is reduced from one hour to about three minutes, and the sr~i Lation rates measured using this process and apparatus can be related to the Westergren method using non-linear regression algorithms. This apparatus and method, however, have drawbacks in the form of the complexity of the apparatus, as well as the need to use non-linear regression algorithms.

Sr~ii L~tion studies also have been undertaken in shorter tubes and particularly 100 milli- ter settling tubes. The problem with this terhn;~lr is that the final or syneresis phase, in which the hematocrit is rapidly rising, occurs earlier, again requiring non-linear algorithms for correlation to Westergren se~;- Lation results.

Additionally, accelerated blood ~6i;- Lation has been measured using tilted or i nrl; n~ci 5~ tion tubes instead of vertically oriented tubes. using an ;nrl ;n~

~ W096/ol990 2 1 9 4 8 7 0 tube ta tube 100-200 m; 11;- ' Dr5 long and 2.5 r;ll; L
in diameter in~l ;no~ at about 30 or 45 degrees from vertical) 8ettl;ng ratcs can be --- ~d after only 20 minutes of set~l ing. The 8ettl ing rAte which is ~Dt~rm;nPd using such apparatus, however, is not a true W L~LYL~I s~; Ld~ion rate because once again the relationship between the hematocrit rise and the ~ILL of plasma i8 altered resulting in a nnnl;nDAr relationship between the test method. Thus, the settl1n7 is non-linear and the- - ~d rates must be related to Westergren rates by non-linear correlation algorithms.
Tilting of the tube reduces the settling time by about two-thirds but because there i8 in this method no shortening of the lag phase, 20 minutes is still required and a non-linear correlation to Westergren rates i8 also nec~:Ary.

In general, the use of non-linear algorithms becomes less reliable in relating results to Westergren s~ L~tion rates as the ED~i- Lation rate in~L-aGes. High se~i- L~Lion rates usually indicate the ~L~s~ e of ;nfli toLy conditions. Thus, the non-linear effects induced by rapid se~ir L~tion tend to decrease the correlation accuracy to Westergren rates for spec;-which are most affected by disease and other conditions25 sought to be discovered or analyzed by the sD~ir Lation process.

Accordingly, it is an object of the present invention to provide an a~L~Lu_ and method for eLyLI.l~yLe blood se~i- L~Lion which can be rapidly accomplished and yet is capable of high correlation by linear transposition to Westergren s~ - Lation rates.
.

Another object of the present invention is to provide an erythrocyte s~ Lion method and apparatus which wo96lolsso 2 1 9 4 8 7 0 can be used repeatedly on the same 8peci to rapidly determine and verify the blood s~A1 -L~tion rate.

Another object of the present invention i8 to provide a blood s~Ai~ L~tion apparatus and method in which the whole blood spo~i , from the moment of veni~u,-~Lu~e, remains in a sealed c~nt~i n~r.

Still a ~urther object of the present invention i5 to provide a blood s~A1 LaLion apparatus and method in which smaller blood sreci~ are required and mixing of the blood ~pe~i- can be readily accomplished in very atmall volumes.

Another object of the present invention is to provide an i ~v~d ~peri- container for use with processes reguiring mixing of small liquid volumes.

Still another object of the blood ~Ai Lion a~ LuD
and method of the present invention is to provide a sedimentation process having increased àccuracy, relatively low cost, and suitability for semi-automated use by relatively unskilled paL -'i~Al per~llllel.

The blood ~eAi~ L~tion method and apparatus of the present invention have other objects and features of advantage which will become apparent from, and are set forth in more detail in, the ~r ying drawing and following Best Node Of Carrying Out The Invention.

DISCLOSUR~ OF INVENTION
The method of the present invention provides a process for accelerated determination of erythrocyte s~Ai- Lation in whole blood which can be correlated with a very high confidence level to Westergren settling rates using linear data tL~ o~ition. The present method greatly accelerates the lag phase by using one of several ~ W096/01990 2 ~ 9 4 ~ 70 ~ r-te~hn1~lpc~ Al liChPC the gravity decantation phase rapidly by orienting the sreci container in a manner reducing the time required for settling, and essentially eliminates the syneresis phase, again by orienting the specimen c~nt~inp~ to avoid plasma trapping as Luv~it rises.

The method for accelerated ~P~PrminAtion of eLyLhLv~lLe 5P~; ~aLion of the present invention comprises, briefly, in~ ing rouleaux formation in a time period su_stantially less than the Westergren lag phase time period for the same spec;- and in an amount sllfficiPnt for the speri- to enter the decantation phase. In the preferred form of lag phase acceleration, a portion of the spPri is formed into a very thin ~LV~ se_Lion in the cpe~; container through which a fluid current i5 induced to flow 80 that contact between individual erythrocytes is enhAnred and rouleaux formation in such portion occurs rapidly. This thin cros5-5Prti~nAl portion of the container is advAnt~geollcly provided by a rod positioned inside the lumen of a tubular container and extending beyond the top surface of the ~pP~i- to cause a veil of spPci- material to form by capillary attraction between the rod and container. Il~r eOvèl ~ the very thin ~LV~ ~ectional portion of the spPci- is oriented preferably at about 20 to about 30 degrees from horizontal for gravitational - v~ L of the rapidly formed rouleaux down through the crPci to seed the #rPri- and ~ the decantation phase.

In one alternative form of the present process, lag phase acceleration is A: , lichP~ by centrifugating the spP~i , rather than creating a capillary veil, to form rouleaux rapidly. In still a further alternative process, a c~nf;nPd cross-sP~tionAl portion of the ~pPC;- container is used and combined with a change of the ~reri- c~ntAinP~ configuration to accelerate wos6/olsso 21 9~870 ~ 3~lr- ~

the lag phase. This form of lag phase acceleration is _1;8~P~ by holding a rod, for example ~~gnPtic~ y, against an upper side of a horizontally oriented container lumen and then releasiDg the rod to gravitate down through the Sre~i~ to a lower side of the lumen.

The present process further inrlll~Pr~ the step of gravity settling the eLyU rvuyLes, after ro~ a--y formation, with the ~rPci container oriented to 8U'oStAnti A 11y reduce the time required for the de~nt~Lion phase below that which would be required in the Westergren process for the same speri . Such gravity settling is A~ 1 jChpd, for example, by maintaining the speci container oriented at 30 dcgrees or less to the horizon.
This orientation, together with the PL~S~nCe of the rod on the upper side of the tube lumen, provides two protected nhAnnPlc through which plasma can escape thus sir3nifl~Antly shortening the syneresis phase of se~i LaLion. The method of the present invention inn~ c as a final step dPtermining the amount of 2 o seS i- Lation which has occurred in the 9pPC i - , preferably by reorienting the Epr~Ci~ n container from its near horizontal orientation for gravity settling to a near vertical oriemtation for cp~;- L~Lion mea~uL L.

The apparatus for accelerated determination of erythrocyte sedimentation rate is comprised, briefly, of an elongated fipe~i tube having a lumen formed to contain a blood EpPci~ therein and preferably in~ ing a very thin cross-cPctinn~l portion. The thin cross-sectinnAl portion of the lumen advantageously can beprovided by mounting a rod, adhesively or m-gnpticAlly~
in the Cper-i tube next to a wall of the tube and in a position to extend above a top surface of the spPrir 80 that a veil of blood will form between the rod and tube wall.

~ W096/01990 21 ~4870 ~ /L ~
g . .~
The present apparatus also includes spec;- tube orienting assembly which can hold the ore~i tube in a manner orienting it for accelerated rouleaux f inn~
for example at about 30 degrees from a horizontal plane.
The orienting assembly also orients the speci tube for accelerated gravity settling during the decantation phase, again at about 30 degrees or less to a horizontal plane. Preferably the orienting assembly is further formed to enable m-nirl~lation of the speci tube to enable ErDci mixing and reoriDntAt;nn from a gravity settl ing orientation to a SD~;- Lation ~etDrmin~tion orientation.

In a first alternative : 'i L of the apparatus the orienting assembly is also formed for centrifugation of lS the Cpeci- tube, and in a second alternative: ~i L
the orientation assembly is formed to selectively hold and release a rod mounted in the creri- tube.

In another aspect of the present invention, a specin container for mixing small volumes of liquid, such as blood spe~i- -, is provided. The crDri- container has an elongated bore with an elongated member mounted therein to define a resulting elongated lumen with a channel portion of the cross sectional area of the lumen being formed to be sufficiently thin in LL~nDv~L~e cross section that a liquid-mixing gas bubble cannot enterthis channel portion of the lumen and the liquid can flow along the channel.

n~rRTpTIoN OF T~ DRAWING
FIGURE 1 is a top peL~e~Live view of an eLyLllLuuyLe 30 B~i- Lation a~aLaLus constructed in accordance with the present invention.

WO96/01990 " ~ "~' " Y~1/L

FIGURE 2 is a top pelD~euLive view UULLI ~l..."~;ng to FIGURE 1 with the blood EpDCi--- tubes oriented for accelerated rouleaux formation and gravity settl ing.

FIGURE 3 is an enlarged, top plan view in cross section of the apparatus taken D~.~La--Lially along line 3-3 in FIGURE 1.

FIGURE 4 is a greatly enlarged, side elevation view, in cross section, of a ~pDci- cnnt~inPr tube ~ul~DLL~uLed in ~cuuLdance with the present invention.

FIGURE 5 is an end view, in cross section, taken substantially along the p]Lane of line 5-5 in FIGURE 4.

FIGURE 6 is a graph of se~i Lation rate data using the apparatus of FIGURES 1-5, as compared to Westergren sP~i Lation data for modified blood sp~ from the same patients.

FIGURE 7 is a top p~ P~Iive view of an alternative ~ L of an ~LyU~rOuy Le 8P~; r Lation apparatus constructed in accordance with the present invention.

FIGURE 8 is an enlarged, side elevation view, in cross section, of a ~pPr1- container designed for use with the apparatus of FIGURE 7.

FIGURE 9 is a further enlarged, cross sectional view taken substantially along the plane of line 9-9 in FIGURE
8.

FIGURE ~A is a cross sectional view COLL~ 7;ng to FIGURE 9 of an alternative : ' 'i L of the ~pPCir -collection tube of FIGURE 8.

~ wo96lolsso 2 ~ q 4 8 70 ~ Pcl/~

FIGURE 9B is a cross s~ct~n~l view CULL~ 1ng toFIGURE 9 of another alternative : ' 'i- t of the ~rerir-- collection tube of FIGURE 8.

FIGURE 10 is a schematic l~pl.s_.,LaLion of the speri-tube of FIGURE 8 after drawing of a Ereci-FIGURE 11 is a schematic representation of the speci-tube of FIGURE 8 during mixing.

FIGURE 12 is a schematic ,~pl~s~..Lation of the Rreci tube of FIGURE 8 during centrifuging.

FIGURE 13 is a schematic ~L~s~lLation of the ~pe~i-tube of FIGURE 2 at the start of gravity settling.

FIGURE 13A is an enlarged, cross ~cct~n~l view of the 5p~cir tube at the start of settling.

FIGURE 13B is an enlarged, cross sectional view of the spDr;- tube after significant settling has OC~ur ~d.

FIGURE 14 is a schematic L~pL~se..Lation of the speri-tube of FIGURE 8 during the step of ~t~rmining the amount of s~i- Lution.

FIGURE 15 is a graph of s~ir-ntation rate data using 20 the apparatus of FIGURES 7 and 8, as compared to Westergren se~i- tution data for unmodified blood spaci from the same patients.

FIGURE 16 is a graph of sedimentation rate data using the apparatus of FIGURES 7 and 8, as compared to u 25 Westergren s~i- LuLion data for modified blood ~ r~ci- ~ from the same patients.

WO96101990 2 1 ~ 4 8 7 0 ~ C

FIGURE 17 i6 a side elevation view, in cross section of nn alternative ~ L of a cpec; c~ntnlnDr constructed in a-~uLdan~-e with the present invention.

~T MODE OF ~YING OUT TM~ lN V~
The method and ~ L~LU~ of the present invention achieve rapid ~LyLh~o~yLe s~i~ Lation results which can be linearly related to Westergren 5~Ai- Lation results.
Essentially two key principles are employed to greatly accelerate the time required for se~;- tion. First, the lag phase of the conventional W LeLyL_..
se~i LaLion method is accelerated by in~11ring rouleaux formation in the spe~i rapidly, until there i5 sufficient rouleaux to cause erythrocyte settling at substantially the decantation rate for the crec~ .
Next, the cpe~i is gravity settled in a container formed and oriented so that the e yLhlu1yLes can settle through upwardly migrating plasma in a manner which is substantially nni ~ by the increasing hematocrit.

The method and apparatus of the present invention allow a blood specimen se~i L~tion to be accelerated across the lag phase, gravity settled through the decantation phase in a much shorter period of time, and settl ing in the syneresis phase to essentially be eliminated. The result is that 8~5i- L~tion data that can be linearly tr~ncroced to Westergren data can be obtained in five minutes or less.

T.A~ phAC~ ~ccelera~ion The first step of the present process is to accelerate se~i- nt 1tion by greatly reducing the time which would normally be required for the specimen to pass through the lag phase and begin the linear decantation phase.
Several t~chni ~1eC for acceleration of blood se~ir- L~tion through the lag phase have been disuu~l d and will be described herein, but the preferred technique ~ wos6~1sso 2 ~ 9 4 8 7 0 can be described by reference to a speci tube which is particularly well suited for use in the method of the present and i6 shown in FIGURES 4 and 5. ~cc~n~ 1~1 1 y the same tube also is shown in FIGURES 8 and 9.

As will be seen from FIGURES 4 and 5, elongated Sp~ri tube or cnnt~in~r 21 is formed with a tube wall 22 which defines a tube lumen 23. Lumen 23 terminates in an open end 24 having a rubber stopper or other end closure member 26 mounted therein. As will be de6cribed in lo greater detail hereinafter, speci tube 21 advantag~ol~cly can be a partially evacuated, blood 5p~Ci- tube having a length of about 110 milli- Lers and a lumen diameter of about 6 mi 11 i tcrs. An anti-coagulant i6 placed in the tube before the ~p~ri- i8 drawn. ~he 6peci is taken by a needle 159 (shown in broken lines in FIGURE 8) which extends through stopper 26 in a conventional manner, and such blood speci-tubes, as thus far described and blood speri drawingte~hni~c, are well known in the medical profession.

While the present process is described by reference to preferred creci tube 21, it will be apparent from the following description that other forms of blood cpe~i containers can be used in the present process.

In order to provide severali L~..L advantages, aswill be set forth below and, particularly in order to provide a very thin ~L~ se_Lional portion of the sp~cir in lumen 23, tube 21 preferably has an elongated member or rod 27 mounted in the lumen. Rod 27 is preferably about 4 mi 11 ir ' ~rc in diameter and is secured or held by adhesive, fused glass or other means, against one side of lumen 23, in this case the upwardly oriented side 28.

In the preferred form, tube 21 has a substantially cylindrical lumen 23 and elongated member 27 is a WO96101990 2 1 9 4 8 7 ~ -14- ' Y~

cylindrical rod, but other lumen-rod configurations are suitable ~or use in the present invention.

As set forth in the ba~Luu-.d, the Westergren lag phase can reguire 5 to 15 minu1:es to complete. During this time rouleaux must be formed from individual ~LyLh~yLes in sufficient number to seed the speri- and start the linear or decantation phase of se~ii Lion.

In the present invention rouleaux formation is induced at an accelerated rate and sufficient rouleaux created to begin the decantation phase in a matter of seconds, for example, 30 seconds or less. The preferred manner of accelerating rouleaux formation is to provide a spPr; rnnt~inPr which will have a very thin cross-sectirn~l area in one portion of lumen 23. As will be seen in FIGURE 5, the combination of cylindrical rod 27 and cylindrical lumen 23 is that two areas of small ~LVpS section, namely, horn-like areas 31 and 32, exist proximate upper side 28 of lumen 23. Thus, blood specimen 29 in these areas is at least somewhat c~nfin~d.
While it may appear that horn-shaped cross -~ 9 ~ irr~l areas 31 and 32 are "small" or "thin" in cross section, in the absence of lengthwise current induced by the exaggerated settling of rouleaux in the veils, they do not by themselves produce the accelerated rouleaux formation of the method of the present invention.

As shown in FIGURE 4, upper end 33 of rod 27 ~LuL~dLa above top surface 34 or the- ;~rll~ of sp~ci -- 29 for a tube oriented as shown, in this case, oriented at an angle of 30 degrees to a hori~ontal plane 36. As will be seen, the blood ~p~ri~ forms an arcuate veil-like volume 37 in each of horn areas 31 and 32 proximate and up to the upper end 33 of rod 27. While impractical to show in the drawing, very thin spec; veil 37 will ~ WO96/0199~ 2 1 ~48 70 h ~ r~

extend up rod 27 by ~r; 11 Ary action to the last point of contact 38 of the rod with tube upper side wall 22.

As arcuate ~reci veil 37 extends upwardly toward point 38, its cross section thins and in fnct tapers down to what is believed to be a relatively amall multiple of the red ceil ~i~ or~ for example, n veil cross section of 10 cell ~; D (70-80 microns) or less. Thus, in the area of ~peci- veil 37 red cells are forced to be in such close proximity to each other that they cluster and form rouleaux in a few seconds. For example, n~b~ ially all the red cells in the thinnest upper regions of veil 37 are believed to be in rouleaux in 15 seconds.

Obviously, however, the amount of the spe~i- in the upper reaches of veil region 37 is small as compared to the entire speni- 29. It is believed, however, that rouleaux are starting to form elsewhere in the srPc;
and particularly in small cross-sectinnAl horns 31 and 32 all along the rod. m is rouleaux formation is probably somewhat accelerated as compared to formation in a Nestergren tube, but it still would result in an undesirably slow lag phase. Nevertheless, Ap~Ci- 29 enters the decantation or linear AP~i- Lation phase almost immediately after rouleaux formation has been completed in the upper end of veil region 37 when ~pecl-- tube 21 is held in the 30 degree orientation shown in FIGURE 4.

It is hypothp~i7sfl that the rouleaux formed in very thin upper region of veil 37 are immediately free to gravitate downwardly along veil 37 and across rod 27, and as they do so plasma is pulled up along horned regions 31 and 32 between the rod and tube and into veil region 37.
The orientation of tube 21 is such that rouleaux can escape veil region 37 and plasma can enter the veil wos6/olggo 21 q4a70 ~ P~

region. There rapidly begins to occur, therefore, a circulation pattern in which denser rouleaux settle down through and "seed" the n~Pri - picking up more cells as they fall and yet not trapping plasma below. The plasma, on the other hand rises up the tube along horns and replaces the downwardly settl ~ng rouleaux. As the plasma rises, its motion is believed to cause free eLyLhL~yLes to agglomerate with partially formed rouleaux all along horns 31 and 32, thu~ causing further rouleaux formation ~nd s~ ion.

The exact -- -nics of acceleration are not known, but in a blood crPrir of high sP~i- Ldtion rate within ~bout 30 seconds flow of plasma upwardly along horns 31 and 32 toward veil 37 can be clearly seen in creci 29 and rouleaux clearly has formed in an amount sufficient to cause the entire ~pPcin to be in the decantation phase.

At the present time many aspects of this technique for accelerating rouleaux formation are unknown. It is known, however, that the same crPci tube assembly will require 15 minutes to complete the decantation phase when rod 27 does ~Qt extend above speci top surface 34 and only 5 minutes when it does extend above surface 34.
Thus, if the horn regions 31 and 32 are used alone the spPcir-n can be settled in 1/4 the time of a 1~ LelyL~..
8~; t~tion test, but iE the Cpecir cross section is further thinned by extending rod 27 beyond surface 34 to form veil region 37, the sP~i- L~Lion time can be reduced to 1/12 of the Westergren se~ir ~L~tion time.

It is believed that rod 27 should extend above uppor spPcir-r r- icc~c by at least 3 to 4 millir Lers for optimum acceleration effect, but any protrusion starts to provide a thinned capillary veil which should be useful. The maximum useful protrusion similarly is not ~ Wog6/olsso 2 1 ~ 4 8 7 0 known, but blood speci- will rise up to an inch or more above ;~cn~ ~urface 34 along thin c~pillAry-like ~hAnn~l~, guch as horns 31 and 32.

As will be appreciated, variou6 other ~peci tube configurations are po~ihl~ to produce a very thin capillary cross section. Converging planar walls and converging planar and curvalinear walls may provide the same effect, and such nLLu~Lu~ can be provided by lumen inserts or integrally formed tube wall configurations.
To achieve the best results, it 18 believed to be advantageous to employ rAri 11 Ary forces to form, with a portion of the tube lumen, a thin veil or film of the ~p~ir . Such a veil or film, however, also must be positioned, it is believed, 80 that the rapidly formed rouleaux are free to escape film or veil region 37 and seed the sreci- , preferably while plasma i8 free to enter the film or veil portion of the lumen.

At the present tine the orientation of tube 21 which is best suited for both accelerated rouleaux formation and gravity settling during the decantation phase is believed to be between about 20 degrees to about 35 degrees from the horizon. Nost preferably tube 21 is oriented at about 30 degrees from horizontal plane 36 during both accelerated rouleaux formation and gravity 8ettl ing during the decantation phase. As tube 21 is lowered to an orientation below about 25 degrees, circulation of plasma up along horns 31 and 32 is reduced and sP~i L~tion times begin to increase. Similarly, as tube orientation is increased to above about 35 degrees egress of plasma becomes impeded by settl i nq of red cell Eyy,~Les which, at these higher angles no longer seed the ~ Lation process but instead impede plasma egress .

Ra~id n~rAntation and SYneresis ~l;minAtion WO96/01990 2 1 9 4 8 7 ~ - 18-The next step in the process of the present invention is to gravity settle the nro~i , which is now through the lag phase of se~i- Lltion~ The middle or decantation phase tends to be relatively linear until the hematocrit in~leSases s~gnSfscslntly and the settled erythrocytes trap or impede upward migration of plasma sst the bottom of the ~re~i- tube, that is the SpDcl-Dnters the syneresis phase.

In the present process, Sr~~~ Q~tsl~sn~ 21 is oriented in a manner enabling 8ettl;nq of eLy~ syLe cellsthrough upwardly migrating plasma without trapping or s~ -'ing of the plasma as a result Or the relatively small transverse cross sectional area of the tube.
Nestergren tubes, and settling tuoe 21, both have relatively small rSS D~ in order that the amount of whole blood required to perform se~i Lation testing can be mSni~57Od, but it is the small diameter which causes the slowing of SD~.- SItion at the end of the settling period. In the present invention the time required for the linear de~sa~,L~ion phase is greatly shortened and the syneresis phase is substantially eliminated by orienting spoci- tube 21 with its longitudinal axis 55 of sp~ci- tube 21 in a near horizontal orientation, as shown in FIGURE 4. As used herein, "near horizontal" shall mean between about 35 degrees and about zero degrees from the horizon.
Orienting longitudinal axis 55 in a near horizontal plane cause the L~ veLDe area of SpD~i- tube 21 to be qreatly increased as compared to the L,~.. v~S~e area (FIGURE 5) Or a vertically-oriented tube 21.

Such an orientation of the spPcS~- tube 21 will cause the rouleaux in the specimen tube to simultaneously gravitate or fall across the relatively small diameter of the tube, but over a relatively large area of the tube. The resistance to downward movement of rouleaux ~ W096/01990 2 1 94 8 70 -19- ;~

and to upward migration of plasma in the tube as a result of the large horizontal area of the near-horizontal, elongated tube sllh~nnt;~lly ~l;m;nnt~ impeding, choking or slowing down of the se~i Lion rate. The syneresis phase, as a result of dramatically increasing ~ touLlt and resultant trapping of plasma by the eLyUIluuyL~6, is substantially eliminated. By orienting the settling tube in a near horizontal orientation, the rouleaux move or settle down through the plasma in a manner which i8 very close to or approximates settl;ng in a bottomless tube. The h Lo~-lt or blood cell packing density buildup has very little effect in trapping plasma because of the large LL~ veL~e or horizontal area and of the very short distance need for plasma travel to escape the ~ttl ;n~J eLyLIllu~:yLeS.

IIO1eUV~L~ since the distance across the tube ~ r is short, the time required for completion of the decantation or linear settling phase is much less. It has been det~nm;n~fl that, after about 3.5 to about 5.0 minutes of gravity settl ing with the sp~ci- tube in a horizontal orientation, the decantation or linear 8~ L~tion phase is completed in a degree which is substantially equal to, and correlatable with, 60 minutes of settl inq using the Westergren method.

S~ tion DeterminAtion The next step in the process of the present invention, therefore, is to determine the amount of set~l ;nq which has OC~uL ~ ~d during the period of time which the lag phase was accelerated and the decantation phase was taking place, for example, 5.0 minutes. In order to facilitate a ~ rminAtion of the amount of settl inq which has oc~u~.~d, it is preferable to reorient Bp~
container 21 to a near vertical position. Thus, tube 21 can be rotated to a near vertical orientation, for example, to about 90 degrees from the horizon. This wo96lol9so 2 1 9 ~ 8 7 0 causes the settled erythrocytes to 81ip down the bottom side o~ the ~reri~ - tube 21 and the plasma on upper side of lumen 23 to float up t:he opposite side of the tube to the top.

The reorientation of the ~peci- tube to a near verticai position will cause the settled cqlls to reach the bottom of the tube quickly, and a meaDuL- nt of the separation boundary or interface between the plasma nnd settled cells can be taken, for example, as soon as five seconds after reaching a vcrtical orientation. In the vertical orientation the location of the separation boundary between plasma and erythrocytes can be more a_~uL~tely determined than in the near horizontal orientation. The tube may, however, be read in a semi-vertical position with a modest increase in associated readiny error.
As described above, the steps of accelerated induction of rouleaux formation, gravity settling in an orientation shortening the decantation phase and detPrmining the amount of settling can be implemented by hand by a laboratory technician. By simply employing a rack or tube support structure which will hold tubes 21 in the orientation of FIGURE 4 and then a second rack which can be used to vertically orien,t the tubes, a tPrhn;ciAn can easily perform the p1o~eduLe of the present invention without automation or special P~li~ L.

SPmi--Autn P~7 SPr~ir i~n .~nnAratus Nevertheless, it is an i ~ L feature of the present invention that the method of the present invention can be easily semi-automated. FIGURES 1 through 3 show one form of se~i L~Lion tube r-nirnlating apparatus 20 which can be used to perform the present process.
SP~; Lation apparatus 20 preferably includes a base 41 having leveling assembly, such as manually PngAgP~hlp leveling screws 42 and a spirit level 43, so that the apparatus can be leveled for 1~LO~ C;h1e results on a ~ WO96tO1990 2, 9 4 8 7 0 --21--labo~LuLy bench top. Mounted on base 41 is a housing 43 which preferably in~lnA~s a translucent front panel 44 behind which a light source, such as two U-shaped fluoIeDc~tttl.L tubes 46 and 47 (FIGURE 3), are positioned.

In order to enable proper orientation and r~nlrlllAtion of s~i ' tion tubes Zl and 22, a rotatable tube holder assembly, generally designated 48, is mounted to extend from front panel 44 of housing 43. In the form of s~ir L~ttLion apparatus 20 illuDL.ctted, assembly 48 includes a central U-shaped frame member 49 mounted by collar 51 to a shaft 52 for rotation therewith. Secured by fasteners to U-shaped frame member 49 are tube holder members 53 and 54. Each of the tube holder houqingc 53 and 54 includes a slotted or windowed front opening 56 with s~ L~tion measuring indicia 57 positioned closely proximate thereto. As best may be seen in FIGURE

3, the back side 58 of housings 53 and 54 is open so that light may enter the housing and fall upon tubes 21.
Housings 53 and 54 further include two ~ing support felts (not shown) which receive and firmly hold the tubes in the housings without interfering with the passage of light through the tubes and out slots 56. As will be seen from FIGURE 3, the tubes 21 are mounted in hollqingc 53 and 54 with rod 27 on the same side of the respective h~llcingc so that when assembly 48 is stopped in the position shown in FIGURE 2, rods 27 will be oriented inside tubes 21 essentially as shown in FIGURE 4.

Shaft 52 extends through front wall 44 of housing 43 and completely through the housing and out back wall 59.
A first spur gear 61 is mounted on the end of shaft 52 and uuupe~tively engages a pinion 63 carried by shaft 64 of motor 66. Mounted interiorly of housing 43 is an in~Ying disk 67 which is fixed for rotation with shaft 52 by collar 68. Also mounted interiorly of housing 43 is a magnet 69 and a fe-.. , tic collar 71 coupled to W096lo~99o 2194870 ~ ' r ~ 3 shaft 52. Finally, an ;ns~Ying detent assembly 72 and light ballasts 73 also are mounted inside housing 43.

Operation of se~i Lation apparatus 20 to implement the method of the present in~ention can now be described.
In the preferred form, the first step of the present invention is to U~UL~Ugl-1Y mix sp~n~ 29 in sp~i~ ~~
tube 21. Mixing insures that there is no pre-formed rouleaux in the rpeci , and mixing thoroughly mixes the anti-coa~l ~nt material in the Qre~;- tube with the whole blood that has been drawn. Apparatus 20, therefore, preferably is constructed in a manner which will ronirul~te tube 21 so as to effect mixing.

As will be set forth in more detail below, one of the ~ub~La.,Lial advantages of the construction of specl tube 21 in which rod 27 is positioned in lumen 23 is that the gas bubble which will always be present in the tube can be used as a mixing de~ice. Thus, by inverting tube 21 the bubble will move from one end oP the tube to the other, with the blood/anti-coagulant moving in the opposite direction. One of the substantial problems in connection with mixing small liquid volumes is that in small diameter tubes gas bubbles or the like will bridge, rather than move up and down the tube, and prevent mixing. The ~Lesence of the rod in lumen 23 enables the blood/anti-coagulant to pass beyond the bubble in horn regions 31 and 32, because the bubble cannot enter into the thin transverse cross section horn regions.

As a first step, therefore, motor switch 74 can be turned on and the motor 66 will slowly rotate the holder assembly 48 when the shaft and gears are in the solid line positions shown in FIGURE 3. Thus, gear 61 is engaged with gear 63 and assembly 48 is positioned out away from front panel 44 of housing 43. The gearing and motor speed can be set so that rotation occurs at about ~ Wo96/0l990 2 ~ 9 4 3 7 0 ~ ' . I/L, -3 rpm or 6 inversions per minute. About three minutes of rotatlon will insure that the specir - in tube holder assembly 48 are ~.vLv~yl.ly mixed.

In a semi-l~ ted process the te~hn~iAn merely turns switch 74 off after about three minutes of mixing. In n more fully automated process, termination of the mixing cycle is controlled by a timer. The gears 61 and 63 can be retained in intela ,_, by ~-gn~tic member 69 which aLL~ collar 71 thereto and r-int~in~ shaft 52 and gear 61 in the solid line position of FIGURE 3.

Once mixing is complete, the t~rhniri~n can push the shaft inwardly to the dotted line position shown in FIGURE 3, freeing collar 71 from magnetic attraction of magnet 69 and freeing gear 61 from pinion 63. As tube holder assembly 48 is pushed inwardly towards panel 44, ~ n~Yi ng disk 67 also is moved to the dotted line position at which it is engaged by i n~ Yi ng detent assembly 72. Detent disk 67 can have two notches which receive detent element 76 when the notches are in indexed relation to element 76. The in~Ying disk 67 is fixed by a collar for rotation with shaft 52 and has a first notch at a location which will secure tube holder assembly 48 in the position shown in FIGURE 2, namely, at an angle of about 30 degrees to a horizontal plane.
A second notch is provided in in~Ying disk 67 which will hold tube holder assembly 48 in the position of FIGURE
1, namely, in a near vertical position.

Accordingly, in a semi-aut~ ~ed system, the t~hni~i~n turns motor switch 74 off, pushes tube holder assembly 48 inwardly to a position closely adjacent to translucent front panel 44 and rotates the assembly to the position of FIGURE 2, at which it is held in place by detent element 76 ~ng~qlng a notch in~Ying disk 67. The asse~bly is left in this position for five minutes. The 21 9~870 Wo96/01990 technician can then manually rotate assembly 48 from the FIGURE 2 position to the FIGURE 1 position, at which point detent 76 will engage a second notch ~nA~Y;ng disk 67, holding the asse~bly as shown in FIGURE 1. The technician can then read the location of the plnsma/~-y U~LuuyLe i,.Lu.race using indicia 57 on the front of tube holder hnll~ingq 53 and 54.

One Or the ; Lal-t and highly advA I--J~u aspects of the present invention is that each blood ~peci- 29 can repeatedly have its ~Ai- LaLion rate tested.
Accordingly, the terhni~iAn normally will complete the mea~ul~ L process and then return the tube holder assembly to the FIGURE 3 solid line position and turn on the motor to re-mix the spPci . It should be noted, of course, that light switch 77 should be turned on so that reading of the pla~ma/erythrocyte interface or boundary can be easily A~ h~d by back lighting of the spPci-~~ through opening 58.

Using the apparatus of FIGInRE 1, therefore, a te~hniciAn could easily obtain six or seven seAi~ Lation readings in the time period required to obtain one reading using the Westergren process, including the time required to re-mix the 8p~ri- after each s~Air Lation determination.

As will be appreciated, displA~ L of assembly 48 between the solid and dotted line position shown at FIGURE 3 also can be fully automated, and it would also be possible to automate the d~t~rminAtion of the location of the plasma/~LyLl,IouyLe interface. Thus, automated optical reading A~ can be positioned proximate tubes 21 in more sophisticated systems so that the entire process, including registration/recording of 5~i r ' tion results can be automated.

~ WO96/0l990 2 t 9 ¢ 8 7 0 FIGURE 6 shows a graph of seA;- tation results obtained using a~aL~L~s 20 and the process of the present invention. These results have been compared to Westergren 8rAi tion rates for the same ~r~c;- -.
The sper;- - are whole blood sp~ci ~ which have been ;fjrA in the laboLa-~Ly to change their seA; tion rate in a manner well known and set forth in the blood 8~A; Lion lit~LnLuL~. As will be 8een, using apparatus 20 and the method of the present invention one can simply multiply the results of use of the present apparatu6 by 2.0 over the full range of seA;~ L~Lion and obtain Westergren seA; LaLion rates. The best fit line for data taken using the present apparatus and method is not significantly different from a line of identity with the Westergren rates, that is, the r2 variation is equal to 0.98.

S~i~ Cont~inrr One of the problems which i5 particularly acute with blood ~rer;- grA;- -Lation studies is that it is highly desirable to minimi~e the amount of the sperir which is drawn. If a small volume of blood Fr~rir-n is placed in an elongated Speri rnnt~inor, however, it is relatively difficult to effect mixing of the ~peci-even when diluted with anticoAgullnt. Long thin speri containers with low volumes of blood will not readily allow a bubble to migrate from one of the cont~in~r to the other. Even beads or balls are , ti - difficult to employ as gravity mixing devices in small Ai~ Ler elongated tubes and such mixing devices may damage red cells and cause a release of hemoglobin.

In an additional broad aspect of the present invention, therefore, a method and apparatus for mixing constituents of a small volume of liquid in a cnnt~in~r having a small cross section is provided by the preferred form of Erecim tube 21 and 121 of the present invention, as wos6/o199o 2 1 94870 ~ "~

shown in FIGURES 4 and 8 of the drawings. Srec;- tubes 21, 121 can be elongated tubular members having a central lumen 23,151 ~YtPn~ing along the length of the tube.
An elongated member 27, 152 is mounted in the lumen and preferably extends over a ma~ority of the length of the lumen. Elongated member 27,152 can be secured to nn interior surface of the tube along one side of the tube, for example, by an ultraviolet-activated adhesive 153, or by other means, such as fusing a glass member to the interLor of a glass tube, or ~~gnP~irAlly holding the rod to a side of the tube, which will be described in more detail hereinafter.

The mixing adv~--L~ges of tube 21, 121 will be described by reference to FIGURES 8 and 9 and tube 121, but it will be u~-dec~L~od that tube 21 is similarly cv.l~LLu~ed.
As best may be seen in FIGURE 9, the spPci tube wall 150 and member 152 define therebetween a lumen 151 having a transverse cross section configuration which will prevent a gas bubble from completely filling the cross 6ection of the lumen, which would prevent the liquid from passing beyond the gas bubble as it rises in the tube.
This can be accomplished, for example, by providing a wedge-shaped transverse area. As will be seen in FIGURE
8, the use of a tube 121 having a cylindrical bore with an elongated cylindrical rod 152 mounted therein will define th~c~be~ a lumen 151 which is crescent-shaped in transverse cross section and has two w_d~_ r~haped horn regions or converging areas 156 at onds of the ~L~C IIL.

When an air bubble 157 i5 present in a ~pec;r-- tube constructed as shown for tube 121, surface tension forces will prevent the air bubble from extending into the converging wedge-shaped ~cesc~"L ends 156 of the cross sPctinnAl area. As rr~ci- tube 121 is tilted, therefore, bubble 157 will migrate up the length of the tube in the widest portion of the cross section of ~ wos6lol99o 2 1 9 4 ,~ 7 o . . ~

cregcent 154 while the liquid, blood and Ant;r~A~-lAnt, will migrate down the tube past the bubble in the horn regions or . ~haped ends 156 into which the bubble cannot extend. Thus, even in volumes as low as 1.2 milliliterD in cre~i cnntAin~rs having a diameter of only 6 m; 11 ;~ ~~- D~ tilting of the Erec;- tube back and forth by E~~ Ation apparatus 120, or rotation by ~a-~Lus 20, will allow bubble 157 to be very effective in mixing the liquid constituents in the 10 rnntA;nor.

In the preferred form, therefore, spoc;- tube 121 are vacuum tubes which cnntA;nC a pro~torm;nod amount of 5por; n diluent, such as, about 0.25 milliliters ant;coa~lAnt material, suitable for mixing with a 1.0 milliliter volume of whole blood. Such vacuum tubes with anticoagulant and a vacuum which will draw a predetorm;nod known amount of blood crec; are well known in the art. These vacuum tubes do not have elongated member 27, 152 mounted therein. The vacuum tube container 21, 121 of the present invention, therefore, ;nrlll~oc a rubber end stopper 26, 158 through which a needle 159 can be inserted. The inner end 161 ofthe needle preferably should not contact the outermost end 162 of member 152. Thus, end 162 of member 152 i5 recessed by an amount (for example, one-half centimeter) to allow inner end 161 of needle 159 to clear member 152.

Since it is not possible to draw a perfect vacuum inside cpo~;r-n tube 121, there will always be some gas, usually air, trapped in the vacuum tube. When a spec; is drawn, therefore, it will be pulled into lumen 151 by the vacuum therein until the lumen between the tube and elongated member is substantially filled (about 1.25 milliliterD), with the exception of a small air bubble 157. Air bubble 157 can then be used to mix liquid after needle 159 is removed from rubber stopper 158.

WO96/01990 21 94870 ,-,t ;~ /L~

A major alva~lt~y~ of srDci~ - cnnt~in~r 21, 121 nnd the method and a~p~L~Lus of the present invention i8 that at all times the blood sp~ci- - is sealed in containQr 21, 121. Thus, the danger to ~hnic;An~ from h~n~l ing whole blood is greatly reduced. Il e~vel, the ~ame ~p~ci can be used in the sealed container to repeat the -~i ~ation proces~.

As will be seen from FIGURE 8, ~r~cin tube 121 includes a stopper 58 having a diameter greater than the tube body. This will result in a slight tilt to the ~r~ri-tube, for example, of 2-4 degrees, from the horizon when tube 121 is placed in a horizontally oriented trough 137.
This has the advantage that it ensures that bubble 157 will ~e up at stopper 158 so that the bubble will not cause remixing when the tube is reoriented to a near vertical orientation to d~t~rminp the amount of settling.
It is a feature of the present process, therefore, to effect gravity settling with container 121 oriented with upper end or stopper 158 tilted up from horizontal by about 1 degree to about 6 degrees.

Referring now to FIGURE 9A, still a further alternative 121a of the specimen container of the present invention is shown. An elongated plastic insert member 181 i5 positioned in lumen 151a of tube 121a. The plastic insert includes horn regions or wedge-shaped cross sectional areas 156a into which a gas cannot extend. Thus, the liquid in tube 121a will pass beyond the gas bubble in areas 156a as the tube is tilted. It is believed that a tube constructed as shown in FIGURE
9A, if oriented with one horn region 156a u~ L, and if a very thin section ~Yt~n~d upwardly beyond the ~p~ri--- upper surface 34, would accelerate rouleaux formation and be suitable for use in apparatus 21.

~ wog6/olsso 2 ~ 9 4 8 7 ~ "~ C

Still a further alternative tube ~ i8 shown in FIGURE 9B. Tube 121b has been formed to provide the narrow cross sectional areas 156b by, for example, deforming a heated glass tube to provide narrow areas 156b. Again, areas 156b are s~ 1c;Dntly narrow to prevent a gas from entering these areas, and the liquid can move past the bubble in these areas. Whether or not glass fabrication terhn~ will allow a ~-~ffiniDntly thin horn region 156 to be formed to allow use of the accelerated rouleaux formation t~rhni~-- described in conn~rti~n with FIGURES 4 and 5 is unknown.

FirQt Alternative Laq ph~ Acceleration Process An~
~nn:~ratns~
While the pLcr~ d form Or the method and apparatus of the present invention have been described in connection with FIGURES 1-6, it also has been di~cuv~ed that t_ere are alternative ways for accelerating the lag phase of blood 50~1- L~tion, and the principles of shortening the de~..Lation phase by orienting the speri tube in a near horizontal position can be combined with these various alternative lag phase shortening ~Pnhni~Q.
5uch alternative terhniq~l~Q are believed to have disadvantages in connection with the apparatus which i 1 L them, but they do produce accurate data in a short time which canbe linearly correlated to W Le~yL~n 5~i- LaLion ratcs. Accordingly, for some applications, these alternate: -'i Ls may have certain adv~.tay~.

Referring now to FIGURE 7, an alternate se~1- L~tion apparatus cu--~L~Led in accuLd~l-ce with the present invention is shown. ~ir ' ation apparatus 120 preferably employs a centrifuge assembly as a means for in~llcing rouleaux formation in the blood ~peri- . Thus, centrifuge assembly 122 includes a rotatable turntable 124 on which tube receiving head 126 is mounted. A
plurality Or elongated tube receiving notches 127 are formed in head 126 and a tube retAining means, such as brand lZ8, can be mounted over head 126 50 as to retain srPci tubes 121 in notches 126 during rotation of the centrifuge. Various other forms of tube ret~1ning ~LLU~LU~S are suitable for use with centrifuge 122.
Turntable 124 is mounted to a drive controller assembly 129. Power can be controlled through on-off switch 131, the spin rate by control ]cnob 130 and the spin duration through knob 135.

Centrifuge assembly 122 of the present invention can be provided by any one of a number of standard labc,l~t~Ly centrifuges as long as t:hey are capable of splnning elongated ~peni n tubes 121 with the longitudinal axis thereof generally parallel to the spin axis of the centrifuge at a rate high enough to induce rouleaux formation in an amount sufficient to cause erythrocyte settling to begin at substantially the decantation rate for the speci in a short period of time.

Centrifuge assembly 122 accelerates the spen;- with a centrifugal force that Ipreferably is in the range of about 5 to 10 times the acceleration of gravity, g. A
centrifuge operating at about 400 rpm with a distance from the spin axis to the center of the specir - tube of about 4 cPnti- will produce sufficient centrifugal force to cause the somewhat more dense eL~LhLo~yLes to migrate through the plasma to the outermost surface defining the spPci- tube bore or lumen. As the erythrocytes are driven to the tube side, they come in contact with e.ach other and begin to adhere together to form rouleaux which are held against the outer side of the lumen by the centrifugal force. After about 20 to 45 seconds, sufficient rouleaux will be formed on the outer side of the srPci - tube lumen so that, if the rouleaux are allowed to gravitate, they will ~-- W096/01990 21 9487G ~1' 8~ r~

begin settling of the er~c; at the decantation rate.

As above described, the next step in the process of the present invention is to gravity settle the ~peci which is now through the lag phase of se~; Lion.
Spe~i~ c~nt~;nPr 121 again is oriented in a manner for settling of eLyLhl~yL~ cells through upwardly migrating plasma without being constricted or impeded by the relatively small cross sect1~nAl area of tube 121. This can be accomplished using the preferred elongated, small ~i~ rp~c1- - tube by placing the speci- tube in ~-nir~lAtion apparatus 123 with the longitudinal axis 155 of 6roci n tube 121 in a near horizontal orientation, in this case essentially zero degrees or in a horizontal plane, as shown in FIGURES 7 and 8.
Orienting axis 155 in a substantially horizontal plane cause the LLan~veL~e area of the sr~ci- - tube to be greatly increased as compared to a vertically-oriented tube 121. Additionally, the outer side 160 of the 8p~clr tube, against whlch the rouleaux were formed by centrifugation, is oriented in an uu~ L position.
~hus, sreci- tubes 121 are LLan_reL.~d from centrifuge head 126 to a support member 136, which has a plurality of tube-receiving mounting DLLu~Lu-~s, such as troughs or grooves 137. cpeci~-~ container 121 is oriented with its longitudinal axis 155 horizontal and side 160, which was facing outwardly in the centrifuge, facing upwardly on tube support member 136. This results in rod 152 in tube 121 being on bottom side 150 of the tube, which is just opposite of the position of rod 27 in tube 21 during decantation.

Again, the distance across the tube diameter is short, and the time required for letio~ of the decantation or linear settling phase is much less than for the Westergren process. After about 3.5 minutes of gravity WO96/01990 2 1 9 4 8 7 0 1 '' ~

settling the ~pQC; tube 12 in a horizontal orientation, the de~nLation or linear sQ~1- L~tion phase is _letQ~ to a degree whioh i5 ~ub ~ l l y equal to, and correlatabl,e with, 60 minutes of settling using the Westergren method.

In order to facilitate a ~QtQrmin~tion of the amount of settling which has O~U~L I ~ it is preferable that sQ~ atiOn apparatus 120 reorient speci- cnnt~inQr 121 to a near vertical position. ~hus, over a time interval of approximately 15 to 40 seconds, support member 136 can be rotated to a near vertical orientation, for example, to about 80 degrees from the horizon. This causes the settled erythrocytes to slip down the bottom side of the srQc1 tube 121 and the plasma on upper side 160 of the lumen 151 to float up the opposite side of the tube to the top.

It has been found that reorientation of the =pQCi- tube 121 to a near vertical position must be accomplished more slowly than reorientation of tube 21 in order to avoid re-mixing of settled cells. Once in the vertical orientation, however, the location of the separation boundary between plasma and erythrocytes can be accurately detQrm;nQd using scale 138 next to troughs 137.

As will be apparent from FIGURE 7, container orientation assembly 123 preferably includes support axle 139 and control and motor assembly 141 which can be used to rotate tube support member 136 from a horizontal to a near vertical position in a slow but smooth reorienting step. Apparatus 123 also may include means (not shown) for retaining each of tubes 121 in troughs 137 during their orientation to a near vertical position, although since they do not go beyond vertioal, tubes 121 can be retained in troughs 137 by means of gravity.
-~ W096/ol99o 2 1 9~87 0 _33_ ;~~ 8 In the method of the present invention, it is preferablethat the step of mixing the blood ~roci be undertaken immediately prior to ;n~llr1nq rouleaux formation. Thus, s~ inn ~aL~u~ 120, and particularly c~n~Ain~r-orienting ~aL~tu~ 123, of the present inventionpreferably is formed to mix the blood srec1 Ll.~u~l-ly ~ust priorto the centrifuging or rouleauY intil~ring step.

Sa~i- tation apparatus 120 preferably inrl~lti~ a controller 141, which ;nrlllti~ a mixer input 142 that will cause tube support member 136 to oscillate about a horizontal axis, for example, about axle 139. Such oscillation or tilting can be used to cause a gas bubble or a glass bead (not shown) to migrate from one end of ~peri- container 121 to the other. It would also be possible to effect mixing by a rnntinlling rotating process, but then retention means for the ~rec;-cnntAin~r~ clearly would be required. In the preferredform, an air bubble in the specin container is allowed to migrate from one end of the container to the other by tilting speri container 121 by about 60 degrees to 75 degrees from the horizon in one direction and then by about the same amount from the horizon in the opposite direction. Such tilting can take place at about six inversions or full swings per minute for approximately two minutes. The number of tilt cycles can be ad~usted by knob 145.

Referring now to FIGURES 10 through 14, details of the process of the present invention and operation of the sP~iin ~Lion apparatus 120 can be more fully described.

FIGURE 10 schematically illustrates sp~ci~ tube 121 i_mediately after obtaining a speci~-~ of whole blood and removal of needle 159. r~tAin~r 121, a 110 mi 11 ir ~r long tube with a 6 mi ~ 3t~r internal diameter bore and a 4 millimeter diameter rod in it, is WO96101990 21 ~4870 1~ ' 5~ r- ~

filled with whole blood and diluent 171, as well as an air bubble 157, which 1~P1e~~ D the air left in the incompletely evacuated crPc;- tube. Speci tube 121 i5 placed in tube orienting apparatus 123 in one of the grooves or troughs 137 in tray 136. The te~hn;ciAn then cnn turn on the orientation device 123 by switching switch 140 to start the mixing process. Mixer light 142 will come "on" and the speci tube is tilted about axle 139 above and below the hcrizon by about 70 degrees, aa schematically illuD~L~ted in FIGURE 11. Bubble 157 migrates through ~rPCi- 171 along the member 152 as the liquid spori pass,es beyond the bubble in the uL~Doell~D created by the rod 152 positioned in bore 151 of tube 121. The mixing process is rontin-lQd at six inversions per minute for approximately 2 minutes, at which time controller 141 for the mixer brings tube supporting tray 136 to a horizontal stationary position and turns off mixing light 142. It may be nPcP~ry for controller 141 to slow the rate of tilting or even stop at the extremes of the cycle to allow effective mixing, particularly of contents proximate the tube ends.

The technician then lifts tube 121 from tray 136 and places the same in one of the notche6 127 of centrifuge head 126. The ~eL~tur turns the centrifuge "on," using actuator button 131, and the tube will be spun about a spin axis 172, as schematically illustrated in FIGURE
12. The spin rate will typically be 400 rpm at a radius 173 of 4 centimeters. Centrifugation continues for approximately 20 seconds, which will cause a layer of rouleaux, schematically il~ustrated at 174, to form along the outermost side 160 of tube bore 151. Bubble 157 will be present at the top of the tube, and the tube will be oriented in centrifuge head 126 with the rod 152 closest to spin axis 172 so that the rouleaux will be induced to form and collect against the tube wall farthest from rod member 152.
-~ WO96/01990 2 1 ~ 4 ~ ~0 -35- ~' ' P~

Once the centrifuge step i8 completed, the te~hn;c;An wlll remove ~p~ci- - tube 121 from centrifuge 126 and place the same in troughs 137 of tube orienting assembly 123, namely, in a near horizontal orientation as shown S in FIGURE 13. Preferab~y, there i5 no more than about 6 degrees upward tilt of the stopper end induced by a combination of stopper 158 and the trough orientation.
Some upward tilt is adv~nt~g~uus in that it ensures that air bubble 157 Will be located just under the stopper.
It will be seen from FIGURE 13 that rouleaux layer 174 and side 160 which was ~uL~ - L in the centrifuge will be oriented in the .~, L position. Conversely, rod 152 is now in a 1_ .- L position. Air bubble 157 will be located at the top of the tube under stopper 158.
The technician can then press actuator button 171, and the tray orienting controller 141 Will hold the tray in a generally horizontal position for approximately 3.5 minutes.

FIGURE 13A shows the ~per;- in tube 121 at the start 20 of gravity set~l ;ng. ~ AllY 174 can be seen to be collerted proximate the ,,, L side 160 of the tube and the l~ ;n~r of the spec; 177 is comprised of a mixture of erythrocytes and plasma in suspension. The centrifugation step, however, has created snff;ciPnt 25 rouleaux that upon placement of the tube in the horizontal position on tray 136 rouleaux will begin to settle or gravitate downwardly through mixture 177 of plasma and red blood cells. In a manner analogous to cloud seeding, as the rouleaux begins to fall through 30 mixture 177, additional ~LyLh,~yLes adhere to the falling clusters and additional rouleaux are formed.
The sp~c;- - undergoes the relatively linear decantation phase in which set~l ;ng or se~; L~tion occurs relatively rapidly.

wo s~olsso 2 1 9 4 8 7 ~ -36- '~ i~ t~

At the end of a pre~t~rm~n~ gravity settl;ng period, for example, 3.5 minutes, the spec;- will have the appearance as schematically illustrated in FIGURE 13B.
The bottom of the uL~sc~..L~ of lumen 154 will be filled with s~ eLy U~L~Les, largely in rouleaux 174.
A middle area of the spec;~ will contain a mixture 177 of plasma and eLyL~L~Les in which rouleaux are less densely packed. Finally, a layer of plasma 178 will be present proximate ,~ L side 160 of ~per;- tube 121. A separation boundary 179 al~o will be present between plasma 178 and the r~ ;n~r of the ~r~r;
including ~LyLhL~yLes~

The final step in the method of the present invention, therefore, is to measure the amount of settling which has OC~ULL~d during the gravity settling period. This can be theoretically ao l;~h~ by measuring the location of separation boundary 179 while the Frec;
tube is still horizontally oriented. As a practical matter, however, determination of the precise guantity of settling is less accurate when specimen tube is horizontally oriented. Thus, it is preferable that the step of det~mm;n;ng the amount of settling be ac 1; Ch~d by reorienting the ~r~c; tube to the position shown in FIGURE 14, namely a near vertical orientation. Such reorientation is ac~ 1; qh~d automatically after 3.5 minutes of gravity settling by controller 141, which smoothly and gradually tilts tray member 136 to a near vertical position, for example, to 80 degrees above the horizon. As the ~r~ri tube is tilted, the rouleaux layer 174 sinks to the bottom end of the tube as does the mixture layer 177, while the lighter plasma layer 178 gravitates to the top and comes to rest just under bubble 157. The position of separation boundary 179 can now be - ~d by comparing the same to a measuring scale 138 on tray 136, or as shown in FIGURE 13, a measuring scale 138a on sp~c;-~ ~096~1990 2 ~ 94 ~70 t~ & t; t~ F~llu S

tube 121, to ~t~n~t~in~ the quantity of se~; -L~tion which has OOUULL_d during the centrifugation and gravity Eet~l; ng time period.

The entire process, as above described, can be ~ he~ in legg than 5 minutes. IIJL~U~- L ~ the guantity o~ inn which hag O~ULL~d can be linearly related to Westergren se~i- tion units by simply multiplying the se~i- tion result in m i 11 i- ' Qr~ of fall by a linear multiplier. Using a best fit analysis of the data, ~L.~ -ly high correlation with Westergren data can be obtained using the apparatus of FIGURE 7 and a multiplier of 1.88. Correlation using a 1.88 times the measured so~i t~tion values using the FIGURE 7 apparatus and Westergren s~Ai ~tion values from sperir from the same patient have been found to have a Pearson correlation co~ff~ni~nt, r, value of approximately 0.99 and r2 value of 0.95, which is an e~LL~ -Iy high correlation.

1lOL~ el ~ and very i La-l~ly, the game sr~ci can be tested again using the present process by simply turning on the mixer after the s~ L~tion has been measured to re-mix and le ~l~uend the erythrocytes. The process is then run again until a new s~i tion value is measured. Repeated runs on the same cr~ci allow an average value to be used. In 20-30 minutes, therefore, the same ~p~ri~ can be tested three times to produce a highly accurate Westergren s~i L~Lion rate. Achieving three sets of s~ii t~tion data using the Westergren method would require 3 hours, if it could be done, which is not currently possible because the Westergren tube is not sealed, and the ~perim would have to be removed, remixed and reinserted into the tube, which would inevitably result in some loss of sre~i requiring the addition o~ new ~r~ni~ - to make up for the 1088. Additionally, such a process is ~-L. 1Y

WO96101990 219487 ~ -38- ~ ~ r~

tediousandwouldreguirepot~ntiAllyd~l2.~c~Luu~ o..uLe to contact with the 8pPni ~~~.

FIGURES 15 and 16 illustrate the high correlation of the se~;- Lion process of the present invention using the ~ Lus of FIGURE 7 with the Westergren process. In FIGURES 15 nnd 16, the acronym "SSR" stands for "Speedy S~ ation Rate" and indicates the process of the present invention. The acronym "ESR" stands for "ELYUILU~Y~ s~ii Lion Rate" and indicates data taken using the Westergren process. FIGURE 15 is based upon blood spPci from patients known to have disease or in~l: toLy conditions. FIGURE 16 is based upon blood FpPni - taken from healthy patients, which Freci were modified, as is weLl known in the art, by the addition of various amounts of gelatin and/or saline solution to increase the sP~i- Lation rate of the speci- to simulate the s~Air Lation rates which would occur when a disease or infli toLy condition is present.

In FIGURES 15 and 16, the same blood croci from a single patient was divided into two sub-cr~ci and ~eair ~ Ation was measured with one ~ub ~ i using the Westergren process and the other sub-speci- using the present process. In the present process, a centrifugation of 400 rpm on a 4 centimeter radius for a time period of 20 seconds and a gravity settling time of 3.5 minutes was used in each case. The SSR data was compared to the ESR data and a multiplier of 1.88 was used in both FIGURES 15 and 16 with the SSR data, based upon a "best fit" analysis of the SSR data to the line of identity.

In FIGURES 15 and 16, therefore, each data point represents two SP6i- Lation rate mea~uL~ . Data point 91 on FIGURE 15, for example, is a SSR mea~uL~

~ W096/01990 2 1 ~ ~ 8 7 0 _39_ times 1.88 which yielded a 5~ i ' tion value of 26 mi11 i ~ L while the r. L~LYLen ESR value for the same patient was 29 mill; ' D. Data point 92 is a SSR x 1.88 value of 48 milli- r8 and a i'~ Le~L~n ESR of 39 m;ll i ~ t, ;.

Second ~ltern~tive T~ Ph~R~ Acceleration Process ~n~
~nr ~rat~l~
A second alternative ~ of the lag phase acceleration process of the present invention ha~ been found to achieve accelerated blood ~e~i- L~tion rates which can be linearly related to Westergren rates.
FIGURE 17 ill~LL~Les an apparatus suitable for use with this ~ i of the present process.

A spe~i tube 221 is provided which includes an elongated lumen 222 having an elongated member 223 mounted therein. Member223, however, is a f ~L L I , tic member, such as a glass or plastic tube 224 having a feL. i~ material 226 ln~erted therein and end seals 227. Nounted proximate tube 221 is an ele~LL -_ L
device 228, and the 8p~Cir~ tube 221 i8 supported on horizontal surface 229, although stopper 231 provides a slight inclination so that bubble 232 is proximate the stopper end of the sp~~i tube.

As described above, the rp~ci- 233 is first thoroughly mixed, for example by tilting or rotating tube 221 about a horizontal axis to cause bubble 232 to migrate from one end of the tube to the other. During mixing, if in a magnetized holder, the feLLI Lic rod is fixed to one wall and, if not, feLL~ tic rod 223 is free to translate from side to side inside lumen 222. After mixing tube 221 is placed on surface 229 and the ele~LL ~ -t energized to pull member 223 to the upper side 235 of lumen 222. The rod is held against upper side 235 for 20 seconds, and it is believed that during WO96/01990 21 ~ ~8 7 ~ _40~ ?~

that time period rouleaux begin forming in the horn regions between the rod and tube. If this t~hn i ~1~ were used alone, however, rouleaux forr~tion would still be undesirably slow.

After about 20 seconds, ele~L~ L 228 is turned off and feLL, _ ic rod 223 drops from upper side 235 of the tube lumen to the lower side 240 of lumen 222. The rod motion has a stirring or mixing ~ffect, but it is hypo~h~; 70~ that this motion of rod 223 through Cpeci-233 is sufficient to create further rouleaux andeffectively start the decantation phase.

After 3 to 4 minutes of gxavity settling, the gp~ri-can be slowly raised to a vertical position and ~e8i- Lion measured. One disadvantage of this alternative : _ ir ' of the present invention is that erythrocytes will remain in SncrPnCinn in the plasma to a degree giving the plasma a cloudy appearance. This may be the result of the rod's motion through the ~pPni- . Nevertheless, with strong har~lighting the plasma/eLyLh~yLe interface can still be located and the s~ir Ition rate det.ermined. It is believed that iocation of the interface may be most accurately d~t~rmin~ by using automated optical reading apparatus.

S~i Lation rates which linearly correlate to Westergren rates have been obtained in only 4 minutes using this ~i f i~Ci: ' - ' i - ' o~ the invention. AB will be appreciated the ad~..Lay~s of shortening the decantation phase by settling across the settling tube are again employed, and syneresis is essentially eliminated.

In describing the preferred 'i Ls of process and ~pparatus of the present imvention, it will be Ulld~. ~Lood that many of the parameters can be varied within the ~ wogfil0l9so 2 1 ~ 4 8 7 0 -41 scope of the present invention. More particularly, gravity settling time periods of 3 to 5 minutes have been found to produce ~ettllng rates in a 100 m~ll; Ler tube whic_ merely requires multiplication by a factor of 1.75 to 2.25 to produce equivalent Westergren ~ tion rates with an ~LL- ly good correlation. As will be appreciated, however, shorter and longer gravity settling times can be employed. Similarly, in the centrifugation ~ t shorter or longer centrifugation times, and higher or lower centrifugation forces, can be employed in combination with differing linear mUl~;rl ;~rc, For example, based upon somewhat limited cpe~; numbers using the centrifugation process to accelerate rouleaux formation, it appears that the best fit multiplier for a 3.0 minute gravity settling period for correlation with Westergren e~ tion rates is a multiplier of 2.12 in a 100 milli Ler tube. Similarly, a 4.0 minute gravity 8et~l;ng step appears to correlate with Westergren se~i;~ L~tion rates using a 1.80 multiplier.
The most signiricant aspect of all three : -'; tJ of the present process and apparatus is not the exact multiplier value, but that the relationship is linear 80 that a single multiplier can be found for the present process once the various duration parameters are set.
It will be appreciated, however, that as the length of the gravity settling step is increased, the erythrocyte packing density can become a factor driving the multiplier into a non-linear range. Thus, the preferred time period for the gravity settling step in a 6 mi 11; Ler by 100 m; 11 ;~ Ler tube with a 4 mi 11 ;~ ter rod insert member is between about 2 to about 6 minutes.

Changes in the length and diameter cr~c; container 21 are also poRc;hle and have somewhat less of an effect - on the measured se~i- Lation rates. Since set~l ing 35 occurs when the tube is on its side, the length ~ n wos6/ovgo 2194870 42 '~ h~S~ I ~OI/L

has very little effect, otller than increasing the amount of blood that must be drawn. Diameter effects are greater, both in terms of increasing the qpe~;~ size and the distance over whidlplasma must upwardly migrate as the red cells settle. ~ lumen height between the rod and tube when the tube i~ horizontal in the range of about. 1 m; 11 i- ~ ~r to about 6 ml 11 i ' ~rs is believed to be optimum. The volume of blood, however, will increase very rapidly with A i: Ler increases.

With respect to length of time of mixing, certain minimum mixing is rec~uired and there is no downside to additional mixing within r~A~nnAhle time limits such that the cells in the blood sp~c; are not damaged. There is, however, an overall increase in the time to process a sp~

The centrifuge time will effect results significAntly.
As the time is increased, more rouleaux than would be formed during the lag phase will be -formed. Settling would accordingly be too rapid or cause the se~i Lntion multiplication factor to c:hange. A maximum of about 45 seconds of centrifugation can be tolerated, but 20 to 30 seconds at 5-lO g's is preferred. As the spin radius and spin rate are changed, the centrifugal force also will be varied, which will increase or decrease the ~mount of rouleaux formation.

The rate of the reorientation step to enable ~
is not sensitive in the thin veil . -'i- and only slightly more sensitive in the centrifugation and magnetic rod i- Ls. If it is too fast, mixing can occur and if it is too slow, additional settling occurs.
Reorientation in the range of about 2 to 4 seconds is p~rmi~Fihle for the thin veil : ~i L and in about 15 to 45 seconds for the centrifugation and magnetic rod ~ ~096~1990 2 1 9 4 870 i3 ~ P ~

: -~; produces substantially the same results, with 20 seconds being preferred.

Once reorientation is ~ hQd, the ~~'i- L~Lion meaDuL- can be made almost immediately, for example, within 5 seconds. Waiting too long will have some effect on the correlation of data, even though the EpQri-LouLlt will be high as the spQrl-- will be in the final or syneresis phase. The ef~ect of waiting too long to measure can be ~{gni~ie~nt because of the cnnt;
10 ~1 _ ing of e~yU~Lu~yLes.

Claims (66)

WHAT IS CLAIMED IS:

1. A method for accelerated determination of the erythrocyte sedimentation rate of a blood specimen comprising the steps of:
accelerating rouleaux formation, as compared to Westergren lag phase rouleaux formation, in said specimen in an amount causing said specimen to begin sedimentation substantially at a decantation rate for said specimen;
thereafter gravity settling said specimen in a specimen container oriented to shorten the time required for substantial completion of specimen decantation; and thereafter determining the amount of settling of erythrocytes in said specimen.

2. The method as defined in claim 1 wherein, said accelerating step is accomplished by causing a portion of said specimen to have a very thin transverse cross-sectional area in a region of said container positioned for movement of rouleaux from said region into a remainder of said specimen.

3. The method as defined in claim 2 wherein, said region is positioned for the movement of specimen into said region as rouleaux moves out of said region.

4. The method as defined in claim 1 wherein, said accelerating step is accomplished by centrifuging said specimen to form rouleaux against an outward side of said container.

5. The method as defined in claim 1 wherein, said accelerating step is accomplished by containing a portion of said specimen in a narrow transverse cross sectional region of said container and thereafter moving a member away from said region.

6. The method as defined in claim 5 wherein, said containing step is accomplished by holding a rod magnetically against an upper side of said container, and said moving step is accomplished by removing the magnetic force to release said rod for gravitation away from said region.

7. The method as defined in claim 1, and the step of:
prior to said accelerating step, mixing said specimen while in said container.

8. The method as defined in claim 1 wherein, said accelerating step, said gravity settling step and said determining step are all accomplished while said specimen is in an elongated container having an elongated lumen.

9. The method as defined in claim 8 wherein, said gravity settling step is accomplished by orienting a longitudinal axis of said container in a near horizontal orientation.

10. A method for accelerated determination of the erythrocyte sedimentation rate of a blood specimen comprising the steps of:
inducing rouleaux formation in said specimen in a time period substantially less than the Westergren lag phase time period for said specimen and in an amount sufficient to cause erythrocyte settling in said specimen to begin to occur at substantially the decantation rate for said specimen;
thereafter gravity settling said specimen in a container oriented to substantially complete the decantation phase of sedimentation in a time period substantially less than the Westergren decantation time period for said specimen; and thereafter determining the amount of settling of erythrocytes in said specimen.

11. The method as defined in claim 10 wherein, said inducing, gravity settling, and determining steps are all accomplished in an elongated specimen tube having an elongated lumen and having an elongated member mounted in said lumen, said member having a transverse dimension less than a transverse dimension of said lumen.

12. The method as defined in claim 10 wherein, said inducing step is accomplished by forming a very thin transverse cross-sectional region of a portion of said specimen in said container in a position suitable for gravitation of rouleaux out of said region and movement of an additional portion of said specimen into said region.

13. The method as defined in claim 12 wherein, said forming step is accomplished by forming a region having a transverse cross sectional dimension less than about 10 erythrocyte diameters.

14. The method as defined in claim 10 wherein, said inducing step is accomplished by placing said specimen in an elongated container having an elongated lumen with an elongated member mounted in said lumen, said member having a diameter less than a diameter of said lumen and having a length sufficient to protrude beyond a top surface of said specimen when said container is inclined to a horizontal plane.

15. The method as defined in claim 14 wherein, said inducing step further includes orienting said container in an inclined near horizontal orientation with said elongated member proximate an upper side of said lumen and said container inclined by an amount causing an end of said elongated member to protrude beyond a top surface of said specimen.

16. The method as defined in claim 15 wherein, said orienting step is accomplished by orienting said container at an angle between about 20 degrees and about 35 degrees to a horizontal plane.

17. The method as defined in claim 16 wherein, said orienting step is accomplished by orienting said container at an angle of about 30 degrees to a horizontal plane.

18. The method as defined in claim 15 wherein, said gravity settling step is accomplished by orienting said container in a near horizontal orientation with elongated member at bottom of said container.

19. The method as defined in claim 10 wherein, said inducing step is accomplished while said specimen is held in an elongated container having an elongated lumen with a rod member secured to an upper side of said lumen, and said inducing step and said gravity settling step are both accomplished by orienting said container with a longitudinal axis of said lumen at an angle between about 20 degrees and about 35 degrees from a horizontal plane.

20. The method as defined in claim 19 wherein, said orienting step is accomplished by orienting said container with said longitudinal axis at an angle of about 30 degrees to a horizontal plane.

21. The method as defined in claim 19 wherein, said determining step is accomplished after said gravity settling step by reorienting said container to place said longitudinal axis in a near vertical plane.

22. The method as defined in claim 19, and the step of:
prior to said inducing step, mixing said specimen while in said container by an amount sufficient to insure that substantially all erythrocytes are suspended as individual cells in plasma in said specimen.

23. The method as defined in claim 10 wherein, said inducing step is accomplished in less than about two minutes; and said inducing step and said gravity settling steps are accomplished in less than about 10 minutes.

24. The method as defined in claim 10 wherein, said inducing step is accomplished by placing said specimen in a container having a cylindrical lumen with a cylindrical elongated rod of lesser diameter than said lumen positioned inside said lumen and held against a side of said lumen.

25. The method as defined in claim 24 wherein, said rod is positioned on an upper side of said lumen; and said inducing step is accomplished by orienting said container in a near horizontal orientation and releasing said rod for gravitation to a lower side of said lumen after a predetermined time period less than about two minutes.

26. The method as defined in claim 10 wherein, said inducing step is accomplished by centrifuging said specimen;
said gravity settling step is accomplished with an upper end of said container inclined at an angle between about 1 degree and about 6 degrees from a horizontal orientation for a known period of time less than 10 minutes; and said determining step is accomplished by reorienting said elongated container from a substantially horizontal orientation to a substantially vertical orientation over a period of time minimizing mixing of settled erythrocytes with plasma in said specimen prior to measuring the amount of erythrocyte settling.

27. The method as defined in claim 10 wherein, during said determining step, multiplying the amount of settling by a multiplier determined from a plurality of specimens by a best fit linear analysis of data as compared to Westergren settling data for said specimens.

28. The method as defined in claim 10 wherein, said inducing step is accomplished by centrifuging said blood specimen about a centrifugal axis in an elongated container having a longitudinal axis thereof oriented substantially parallel to said centrifugal axis until sufficient rouleaux are formed along an outer side of said container for said specimen to begin the decantation phase of settling;
after said centrifuging step, said gravity settling step is accomplished by orienting said container with said longitudinal axis substantially horizontally oriented and said outer side of said container facing upwardly;
after said gravity settling step, determining step is accomplished by reorienting said container until said longitudinal axis is substantially vertically oriented over a period of time sufficiently long to prevent significant mixing of settled erythrocytes with plasma in said specimen; and promptly after said reorienting step, measuring the amount of erythrocyte settling occurring while said container is substantially vertically oriented.

29. A method as defined in claim 28 wherein, said centrifuging step is accomplished at a spin rate and radial distance producing between about 5 to about 10 times the acceleration force of gravity.

30. A method as defined in claim 29 wherein, said centrifuging step is accomplished in a period of time between about 10 seconds and 40 seconds.

31. A method as defined in claim 28 wherein, said gravity settling step is accomplished with an upper end of said container inclined upwardly from a horizontal plane by about 1 degree to about 6 degrees for a known time period in the range of about 2 to about 5 minutes.

32. A method as defined in claim 10 wherein, said gravity settling step is accomplished in a tubular container having a lumen diameter of about 6 millimeters.

33. A method as defined in claim 10 wherein, said container is tubular and has a cross sectional area configuration of a lumen over a length thereof preventing entry of a gas bubble into a portion of said cross sectional area to enable mixing of said specimen with said gas bubble.

34. A method as defined in claim 33 wherein, said container has a rod mounted in said lumen and on one side of said lumen, said rod having a diameter in the range of about 1 to about 4 millimeters less than the diameter.

WHAT IS CLAIMED IS:

35. A method as defined in claim 10 wherein, prior to said inducing step, collecting said specimen in a vacuum tube having a rod with an external diameter less than the lumen of said vacuum tube mounted inside said vacuum tube and secured to one side thereof;
and prior to said inducing step, mixing said specimen in said vacuum tube by tilting said vacuum tube back and forth to cause a bubble of gas in said tube to move from one end of said vacuum tube to the other end.

36. A process for acceleration of rouleaux formation in a blood specimen comprising the steps of:
(a) placing said specimen in a container; and (b) subjecting a portion of said specimen to one of:
(i) formation of a very thin transverse cross-sectional region inside said container; and (ii) confinement of said portion in a narrow transverse cross-sectional region inside said container followed by movement of a member defining said narrow transverse cross-sectional region away from said narrow transverse cross-sectional region.

37. A method of mixing the constituents of a small volume of liquid in an elongated tube having a lumen with a length dimension a width dimension and a transverse cross sectional area, said method comprising the steps of:
placing a small volume of liquid to be mixed in an elongated tube with sufficient gas in said tube to form a bubble in said liquid, said placing step being accomplished by placing said liquid in a tube having a transverse cross section over a substantial portion of said length dimension which varies across said width dimension from a narrow region to a transversely interconnected wider region over said substantial portion of said length dimension, said narrow region being sufficiently small to prevent entry of said bubble therein and said wide region being sufficiently large to permit entry of said bubble therein; and tilting said tube to cause migration of said bubble in said lumen between opposite ends of said tube in said wide region while said liquid passes beyond said bubble in an opposite direction in said transversely interconnected narrow region.

38. A method as defined in claim 37 wherein, said placing step is accomplished by placing said volume of liquid in a tube having a lumen with a rod mounted in said tube, said rod having a smaller external transverse dimension than the internal transverse dimension of said lumen to define a wedge-shaped transverse cross sectional area therein varying from said narrow region to said transversely interconnected wide region.

39. The method as defined in claim 38 wherein, said placing step is accomplished by placing said volume of liquid into a vacuum tube having a cylindrical lumen and a cylindrical solid rod mounted to one side of said lumen.

40. The method as defined in claim 38 wherein, said step of placing a volume of liquid is accomplished by placing a blood specimen in said tube.

41. A method as defined in claim 37 wherein, said placing step is accomplished by placing said specimen in a tube having a side thereof inwardly protruding into said lumen to define said transversely interconnected narrow region and wide region.

42. A specimen container for use in determining the erythrocyte sedimentation rate of a blood specimen comprising:
a container body having a wall defining a lumen formed to hold said blood specimen therein, said lumen including a lumen portion in fluid communication with a remainder of said lumen and defined by opposed portions of said wall spaced apart by a distance less than a distance required for entry of a liquid mixing bubble into said lumen portion, and said opposed portions of said wall further being sufficiently close together to accelerate the formation of rouleaux, as compared to the rate of formation of rouleaux in a Westergren sedimentation tube, during a lag phase of sedimentation.

43. The specimen container as defined in claim 42 wherein, said container is an elongated tubular container, and said opposed portions of said wall defining said lumen portion are spaced apart by a distance not greater than a distance producing capillary flow of said specimen in said container.

44. The specimen container as defined in claim 42 wherein, said opposing portions of said wall are provided by two converging portions of said wall of said container body.

45. A specimen container for use in determining the erythrocyte sedimentation rate of a blood specimen comprising:

an elongated tubular member defining an elongated lumen formed to hold a blood specimen in said tubular member; and an elongated rod having a transverse dimension less than a transverse dimension of said lumen, said rod being mounted in said lumen and secured to said tubular member along a side of said lumen to define with said tubular member a region of thin transverse cross-sectional area in said lumen for acceleration of the formation of rouleaux in the specimen.

46. The specimen container as defined in claim 45 wherein, tubular member is a vacuum tube having a lumen with a substantial circular transverse cross section and a measured amount of blood diluent therein, and said rod has a substantially circular transverse cross section.

47. The specimen container as defined in claim 46 wherein, said rod has a length dimension sufficient to extend above a top surface of said specimen when said tubular container is oriented at an angle between about 20 degrees and about 35 degrees.

48. The specimen container as defined in claim 46 or 47, and a support assembly supporting said tubular container with a longitudinal axis of said lumen positioned at an angle in the range of about 20 degrees to about 35 degrees to a horizontal plane with said rod secured to an upper side of said lumen.

49. The specimen container as defined in claim 48 wherein, said support assembly supports said tubular container at about 30 degrees from said horizontal plane.

50. The specimen container as defined in claim 48 wherein, said support assembly is further formed for manipulation of said tubular container to effect mixing of said specimen.

51. The specimen container as defined in claim 48 wherein, said support assembly is further formed for manipulation of said tubular container to a near vertical orientation to enable a sedimentation measurement to be taken.

52. A specimen container for use in determining the erythrocyte sedimentation rate of a blood specimen comprising:
a container body having a wall defining a lumen formed to hold said blood specimen therein; and an insert member positioned in said lumen, said lumen including a lumen portion in fluid communication with a remainder of said lumen, said lumen portion being defined by a wall on said insert member and an opposed portion of said wall of said container spaced apart by a distance less than a distance required for entry of a liquid mixing bubble into said lumen portion, and the opposed wall on said insert portion and portion of said wall of said container further being sufficiently close together to accelerate the formation of rouleaux, as compared to the rate of formation of rouleaux in a Westergren sedimentation tube, during a lag phase of sedimentation.

53. A specimen container comprising:

an elongated tube defining a lumen extending along a length thereof, said lumen having a transverse cross-sectional configuration formed for movement of a mixing bubble therealong in a liquid specimen contained in said lumen, and said transverse cross-sectional configuration of said lumen further including a longitudinally extending lumen portion in fluid communication with a remainder of said lumen, said lumen portion being defined by opposed wall portions of said tube formed to extend inwardly into said lumen and spaced sufficiently close together to prevent entry of the mixing bubble into said lumen portion during movement of the mixing bubble along said remainder of said lumen.

54. An apparatus for determination of the erythrocyte cell sedimentation rate of a blood specimen comprising:
an elongated specimen tube having a lumen formed to contain a blood specimen therein;
a centrifuge assembly formed to receive and spin said specimen tube with a longitudinal axis of said specimen tube oriented substantially parallel to a spin axis of said centrifuge assembly at a rate sufficient to cause significant rouleaux formation in said specimen against an outwardly facing side of said lumen of said specimen tube; and specimen tube orienting apparatus positioned proximate said centrifuge apparatus and formed to receive and hold said specimen tube for gravity settling of erythrocytes with said longitudinal axis of said specimen tube oriented in a substantially horizontal orientation and said outwardly facing side of said lumen during centrifuging oriented in a substantially uppermost position.

55. The apparatus as defined in claim 54 wherein, said specimen tube has a wedge-shaped transverse cross sectional area extending over substantially the entire length of said lumen.

56. The apparatus as defined in claim 55 wherein, said specimen tube has a rod having an external diameter less than the diameter of said lumen mounted in and secured to a side of said specimen tube opposite said outwardly facing side of said lumen during centrifuging to define said wedge-shaped transverse cross sectional area.

57. The apparatus as defined in claim 54 wherein, said specimen tube is a vacuum tube having a measured amount of blood diluent therein and a vacuum sufficient to draw a known amount of blood specimen into said specimen tube.

58. The apparatus as defined in claim 54, and said specimen tube orienting apparatus is formed to manipulate said specimen tube to repeatedly produce migration of a gas bubble in said specimen tube back and forth between opposite ends of said specimen tube to effect mixing of said specimen.

59. The apparatus as defined in claim 58 wherein, said specimen tube orienting apparatus is further formed to slowly change the orientation of said specimen tube after gravity settling from a substantially horizontal orientation to a near vertical orientation.

60. The apparatus as defined in claim 54 wherein, said specimen tube orienting apparatus is further formed with a movable tube holding tray mounted for tilting of said specimen tube for mixing of said specimen and for reorientation of said specimen tube to a near vertical orientation for determination of the amount of erythrocyte sedimentation.

61. A specimen container comprising:
an elongated tube defining a lumen extending along a length thereof, said lumen having a transverse cross-sectional configuration formed for movement of a mixing bubble therealong in a liquid specimen contained in said lumen, and said transverse cross sectional configuration of said lumen further including a longitudinally extending narrow region of said lumen and a longitudinally extending wide region of said lumen, said narrow region and said wide region being transversely interconnected over a majority of said length of said lumen, said narrow region being defined by opposed wall portions spaced sufficiently close together to prevent entry of the liquid mixing bubble into said narrow region during movement of the liquid mixing bubble along said lumen, and said wide region being defined by said opposed wall portions of said lumen which are sufficiently far apart for movement of said mixing bubble therealong whereby a significant amount of said specimen in said narrow region can flow past said mixing bubble in a direction opposite the movement of said mixing bubble in said transversely interconnected wide region of said lumen upon inverting said container to enable mixing of said specimen.

62. The specimen tube as defined in claim 61 wherein, said tube has a cylindrical lumen, and said lumen has a cylindrical rod secured to a side of said lumen to define said transverse cross sectional configuration as two horn-shaped narrow regions and a crescent-shaped wide region.

63. The specimen tube as defined in claim 61 wherein, said tube is a vacuum tube; and a specimen diluent contained in said vacuum tube.

64. The specimen tube as defined in claim 62 wherein, said lumen is less than about 10 millimeters in diameter, and said rod is less than about 8 millimeters in diameter.

65. The specimen tube as defined in claim 64 wherein, said lumen is about 6 millimeters in diameter and said rod is about 4 millimeters in diameter, and said lumen is about 100 millimeters in length.

66. The specimen tube as defined in claim 61, and, an elongated tubular insert member mounted in said tube and forming with a lumen said transverse cross sectional configuration.