Method of preparing C3-sensitized erythrocytes

A set of four cell suspensions containing erythrocytes monospecifically sensitized with IgG, IgM, C3, and C4. The suspensions provide control cells to determine the purity and strength of the antibodies to these immunoglobins or complement components contained in antisera used for clinical testing. Strict control of the pH, temperature, and EDTA concentration of a solution to which blood is added allows for the elective production of C3- or C4-sensitized cells. Selecting a serum with IgG or IgM antibodies and inactivating undesired proteins permits the production IgG- or IgM-sensitized cells. The resultant cells have agglutination reaction strengths of about 2-4+. Erythrocytes from humans or other animals may undergo the monospecific sensitization. Other human proteins, such as IgA or C5, and plasma proteins from other animals may similarly be attached monospecifically to red blood cells.

BACKGROUND 
Clinical tests using antigen-antibody reactions have become increasingly 
important in the diagnoses of various human ailments. This type of 
testing, of course, also proves invaluable in blood compatibility 
determinations performed prior to transfusions. It not only, in general, 
assures the blood's suitability for the recipient (patient), but also 
screens for unexpected antibodies in the donor and the recipient. 
Other conditions also benefit from the use of clinical tests based upon 
antigen-antibody reactions. These tests find important use for immunologic 
disease states such as immune hemolytic anemias due to drug-antidrug 
complexes. Other examples include autoimmune hemolytic anemia, 
erythroblastosis fetalis, and transfusion reactions resulting from 
incompatible blood. 
In direct testing procedures, an antiserum containing antibodies to human 
immunoglobulin(s) and/or complement component(s) receives the erythrocytes 
undergoing testing. Agglutinated cells in the test indicate the presence 
immunoglobulin(s) and/or complement component(s) on the erythrocytes. The 
testing procedure usually includes one or more controls to insure the 
proper reactivity of the reagents. 
In indirect testing, the patient's serum typically undergoes incubation 
with red blood cells. The incubation allows antibodies in the serum to 
attach to antigenic sites on the erythrocytes. The cells, after removal of 
the patient's serum by washing upon completion of incubation, undergo 
testing with a serum containing antibodies to immunoglobulin(s) and/or 
complement component(s). This step resembles the direct testing procedures 
discussed above. The agglutination of the erythrocytes in the last step 
indicates the presence of the corresponding immunoglobulin(s) and/or 
complement component(s) on the erythrocytes, and indicates the presence of 
antibodies to antigens on the erythrocyte surface. Again, the use of 
controls minimizes false results. 
The antiserum used in the tests may take one of two forms. The classical, 
or polyspecific, antiserum generally contains antibodies to a wide range 
of immunoglobulins and complement components. In either the direct or 
indirect procedure, if the erythrocytes have become sensitized (coated) 
with any of these immunoglobulins or complement components, agglutination 
will occur and give a positive test result. The specific immunoglobulin(s) 
and/or complement component(s) that produced the positive result, however, 
remain unknown with the use of this polyspecific type of antiserum. 
Most, if not all, of the tests employing polyspecific antiserum will 
provide additional pertinent information if repeated with monospecific 
antisera. In comparison to the polyspecific antiserum, these latter 
reagents contain an antibody for a single immunoglobulin class or 
complement component. Agglutination occurs, of course, only if the 
erythocytes' surfaces contain the specific immunoglobulin or complement 
component corresponding to the antibody in the antiserum. 
The reliability of the direct and indirect testing procedures depends 
heavily upon the concentrations of the antibodies in both the polyspecific 
and monospecific antisera. Moreover, the purity (degree of 
monospecificity) of the single antibody in each of the monospecific 
antisera represents an important criterion in assuring the accuracy of the 
results of analyses employing them. 
However, the antibodies in the antisera represent esoteric proteins which 
may readily undergo denaturation and degeneration. This can occur as a 
result merely of the passage of time or of even slightly improper storage 
conditions. These deleterious occurrences can unfavorably alter the nature 
of either the polyspecific or monospecific antisera. 
Thus, the strengths of the antibodies in the antisera must remain known in 
order for the results of tests employing the antisera to have validity. 
This required knowledge involves two separate aspects for any particular 
antiserum. The specific immunoglobulin(s) and/or complement component(s) 
with which its antibodies will react represents a first crucial aspect of 
the antiserum. Furthermore, the reactive strength of the antiserum towards 
its corresponding protein must be known in order to provide meaningful 
test results. And, the reactive strengths of a polyspecific antiserum to 
each of its corresponding immunoglobulins and complement components must 
remain controlled to avoid inaccurate results. 
Determining the antibodies and their reactive strengths in the antiserum 
does not represent a classical chemical problem. An antibody molecule 
generally can react with only a specific single amino-acid group on an 
immunoglobulin molecule or complement molecule. Thus, different antibodies 
react with different amino-acid combinations and, therefore, different 
immunoglobulins or complement components. However, antibodies with 
different specificities have basic chemical structures which are very 
similar. Present chemical knowledge lacks the tools to differentiate 
between the different antibodies for the different immunoglobulins and 
complement components on the basis of molecular structure. Yet, the 
antibodies that correspond to the different proteins do, in fact, possess 
differencs. They display reactivity only with their single corresponding 
immunoglobulin or complement component. 
Thus, the only readily definable difference between the different 
antibodies centers upon the reactivity of each with their separate 
protein; the only indication of the presence of an antibody for a 
particular immunoglobulin, for example, requires the reaction of that 
antibody with that immunoglobulin. One commonly used method for detecting 
this reaction involves the agglutination of erythrocytes to which the 
particular protein or immunoglobulin has attached itself. For example, by 
this method determining the presence of the antibody for the IgG 
immunoglobulin in an antiserum requires reacting that antiserum with 
erythrocytes having IgG molecules attached to them. Any resulting 
agglutination and the strength of that agglutination will show the 
presence and reactive strength of the antibody in the antiserum. 
Thus, the need for erythrocytes having a single attached immunoglobulin 
class or complement component becomes clear. Such cell preparations would 
assume an absolutely crucial role in determining the presence and reactive 
strengths of antibodies in both polyspecific and monospecific antisera. 
These cell preparations would thus assure the suitability of these 
antisera for their important clinical tests. 
While the need for red cell preparations having a single immunoglobulin 
class or complement component has become clear, their preparation has 
eluded success. Various attempts at making red blood cell suspensions 
having a single attached protein have not achieved their objective. For 
example, the "low ionic-strength method" of J. A. Moore and H. Chaplin, 
Jr., in their article "Anti-C3d Antiglobulin Reagents. II. Preparation of 
an Antiglobulin Serum Monospecific for C3d" appearing in Transfusion, 
14:416 (1974), attempts to produce erythrocytes having only the C3 
complement component attached. However, as shown in the article of G. 
Garratty and L. Petz, "The Significance of Red Cell Bound Complement 
Components in Development of Standards and Quality Assurance for the 
Anti-Complement Components of Antiglobulin Sera" appearing in Transfusion, 
16:297 (1976), these procedures result in cells sensitized with detectable 
immunoglobulins as well as other complement components. Moreover, the 
reactive strength of the desired complement component is not always at the 
desired level. Thus, the need for cell suspensions having a single 
attached protein has remained unfilled. 
SUMMARY 
To provide a useful standard against which to measure antisera, the cells 
in a suspension should be sensitized with molecules of one human 
immunoglobulin class or one complement component. Moreover, the cells in 
the suspension should remain devoid of any other immunoglobulin or 
complement component detectable by erythrocyte agglutination. The proteins 
constituting the important class of immunoglobulins and complement 
components include the IgG, IgM and IgA immunoglobulins and the C3, C3b, 
C3d, C4, C4b, C4d and C5 complement components and subcomponents. 
Naturally, the cells should be suspended in an aqueous solution isotonic to 
the erythrocytes. This will prevent lysis of the red blood cells. 
Typically, the erythrocytes in the suspension should have received 
sensitization with exactly one protein from the group listed above. In 
particular, they normally will not display any substantial sensitization 
to any other protein in that group. Such monosensitized cells will provide 
a very specific test for the presence and reactive strength of a single 
antibody specificity in an antiserum. That antibody, of course, will 
correspond to the single immunoglobulin, complement component or 
subcomponent with which the cells have undergone sentitization. 
Frequently, these proteins have the ability to break down into 
subcomponents within or outside of the organism concerned. These cleavages 
probably represent normal reactions. An antiserum containing antibodies 
for the protein itself may also, consequently, display reactivity towards 
that protein's subcomponents. Accordingly, a suitable cell suspension, to 
act as a control for this antiserum, may include, attached to the 
erythrocytes, one or more subcomponents of the protein or the intact 
protein. Under these conditions, no other protein should sensitize the 
same cells. 
Naturally, a clinical laboratory would require a suspension having a human 
serum protein attached to the erythrocytes. Such a suspension should 
include red blood cells sensitized with one of the human proteins in the 
class given above. The more important members of this class include the 
IgG, IgM, C3, and C4 proteins. Facilities for other animals would, on the 
other hand, make use of suspensions containing proteins appropriate to the 
species it deals with. 
Polyspecific, oligospecific and monospecific antisera must contain known 
reactivities to immunoglobulins and/or complement cmponents corresponding 
to the erythrocyte sensitization. The polyspecific serum will demonstrate 
reactivity towards immunoglobulin(s) and complement component(s). An 
oligospecific serum possesses reactivity towards either immunoglobulins or 
complement components, but not both. A monospecific serum, while 
possessing reactivity towards only one immunoblobulin or complement 
component, will display a lack of reactivity towards the others. 
Thus, obtaining an accurate determination of specifity(ies) of these types 
of antisera requires tests for their reactivities with several different 
proteins bound to erythrocytes. Each of these proteins, of course, must be 
attached to erythrocytes provided in a separate suspension from the 
erythrocytes sensitized with the other proteins. Thus, the lab typically 
will have need for a set of cell suspensions with each containing 
erythrocytes sensitized with a single immunoglobulin class or complement 
component. 
The set of several cell suspensions, with each suspension incorporating a 
different protein, would primarily find use in clinical laboratory 
facilities. Accordingly, each suspension would include erythrocytes 
suspended in a aqueous isotonic solution and sensitized with a single 
human protein. In almost all instances, the erythrocytes would have 
undergone sensitization with a globulin detectable by agglutination in the 
usual antiglobulin testing. For convenience, the sensitization of the 
erythrocytes should suffice to provide an agglutination reaction strength 
of about 2-4+. 
The more important human serum proteins include the IgG and the IgM 
immunoglobulins, and the C3 and the C4 complement components. With regards 
to the immunoglobulins, only IgG and IgM appear capable of directly 
activating the classical complement pathway upon binding to an antigen. 
Moreover, most erythrocytes' antigen-antibody reactions involve 
immunoglobulins of these classes. 
With regards to the complement components, the classical pathway of 
complement activation involves early participation of C4. Consequently, 
antisera have often included anti-C4 either as one of many in a 
polyspecific serum or as the only protein in a monospecific serum forming 
part of a set. However, the antibodies corresponding to C3 have greater 
importance. The complement pathway results in the activation of up to 1000 
C3 molecules for each C4 molecule involved in the process. Thus, a serum 
containing antibody to C3 would appear to have a significantly greater 
likelihood of detecting bound complement than the serum with anti-C4. 
Accordingly, polyspecific and monospecific sera, containing antibodies for 
the IgG and IgM immunoglobulins and the C3 and C4 complement components 
can serve an important function in clinical laboratories. The testing of 
such sera to demonstrate the continued predictable reactivity of the 
antobodies thus involves the use of four cell suspensions. Each suspension 
should include erythrocytes sensitized with one of these four important 
proteins. 
The preparation of these suspensions involves the attachment of the 
immunoglobulins or complement components to the surface of the cells. That 
in turn requires the presence on the cells of complement receptors and 
antigens to which the immunoglobulins, as antibodies, can attach. 
Accordingly, the erythrocytes may derive from any animal to which the 
normal human can make antibodies. Typically, a mammal will provide such 
erythrocytes. Particularly good examples, of course, include rabbits, 
goats, horses, and human beings, with the first and fourth members of that 
group finding most frequent use. 
Attaching the different complement proteins to the erythrocytes generally 
follow procedures that closely resemble each other. Strictly controlling 
the conditions under which the reactions proceed, however, allows for the 
sensitization of the erythrocytes with one complement component to the 
exclusion of others. 
Thus, for example, sensitizing erythrocytes with C3 or C4 should begin in a 
solution having approximately 8.0 to 10.0 w./v. percent sucrose and a 
predetermined concentration of EDTA generally falling within the range of 
about 0.10 to 50.0 millimolar. Normally, the EDTA would take the form of 
Na.sub.2 EDTA.2H.sub.2 O in an approximately 2.5 to 20 millimolar 
concentration. The temperature of the solution should fall between about 
0.degree. to 37.degree. C. The solution should also have a pH of about 4.5 
to 8.0. A buffer in an approximately 0.1 to 25 millimolar concentration 
will maintain the solution in this desired pH range. For the sensitization 
with C3 and C4 complement components, the pH should generally not go 
outside of the range of 5.1 to 7.0. Utilizing Na.sub.2 HPO.sub.4 
--NaH.sub.2 PO.sub.4.H.sub.2 O as the buffer in a 4.9 to 5.1 millimolar 
concentration should serve to maintain the pH at the value needed for the 
reaction. 
This carefully prepared solution then receives a small amount of a 
suspension of erythrocytes in a physiologic liquid. The erythrocytes 
incubate in this solution maintained at the specific preselected 
temperature. After the incubation, the erythrocytes are removed from the 
solution, washed, and resuspended in a red cell preservative medium. 
To sensitize erythrocytes with the C3 protein, the solution generally has a 
pH very close to 5.1, an 8.4 millimolar concentration of Na.sub.2 
EDTA.2H.sub.2 O, and a temperature of about 0.degree. C. Approximately one 
volume of whole blood having an added anticoagulant combines with 19 
volumes of the solution to initiate the sensitization reaction. 
In comparison, the attachment of the C4 protein usually proceeds in a 
solution having a pH of about 7.0. The Na.sub.2 EDTA.2H.sub.2 O has an 
approximately 2.8 millimolar concentration, and the solution has a 
temperature of about 37.degree. C. 
Rather than merely adding whole blood as with the C3 sensitization 
procedure, the solution receives a modified erythrocyte suspension. To 
prepare it, a physiologic saline liquid replaces approximately 4/5 of the 
blood's original plasma volume. Again, however, one volume of the modified 
erythrocyte suspension combines with 19 volumes of the C4-procedure 
solution to allow for the attachment of that protein. 
The preparation of the IgG and the IgM cell suspensions also proceed along 
somewhat similar pathways. The former begins by inactivating any IgM 
agglutinins present in an antiserum possessing an antibody of the IgG 
class. To facilitate this step, the antiserum should originally have very 
little, if any, IgM agglutinins present with specificities for the surface 
antigens of the erythrocytes selected for sensitization. 
A solution, devoid of interfering IgM proteins and at a temperature of 
about 37.degree. C., may then receive whole blood or washed erythrocytes 
suspended in a physiologic solution. To permit the sensitization to occur, 
the erythrocytes must possess the antigen corresponding to the selected 
IgG antibody. Conveniently, the physiologic solution containing the 
erythrocytes may simply take the form of blood having an added 
anticoagulant. The actual sensitization with the IgG protein involves 
incubating the erythrocytes in the antiserum at a temperature of 
approximately 37.degree. C. for a period of at least 15 minutes. 
Afterwards, the completion of the preparation involves separating the 
erythrocytes from the antiserum and washing them. 
Two procedures have generally proved acceptable for inactivating any IgM 
protein present in the antiserum. First, the antiserum may undergo heating 
for a period of about 30 to 60 minutes at a temperature of about 
56.degree. to 60.degree. C. Alternatively, the antiserum may be treated 
with a mild reducing agent. Suitable agents for this purpose include 
2mercaptoethanol, dithiothreitol, and dithioerythritol. 
The IgM sensitization procedure requires an antiserum containing an 
antibody of the IgM class and substantially no IgG red cell agglutinins 
with specificities for the surface antigens of the erythrocytes selected 
for sensitization. Any C3 or C4 proteins contained in the antiserum, 
however, should first undergo inactivation. 
Conveniently, adding to the antiserum a primary amine or a compound 
producing ammonia in the antiserum inactivates C3 and C4 proteins. 
Examples of suitable compounds include ammonium hydroxide, ammoniun 
chloride, and hydrazine. The selected compound should remain in the serum 
at a pH of about 8 for a period of at least 30 minutes. 
After the inactivation of C3 and C4 proteins, the antiserum, while at a 
temperature of about 37.degree. C., may then receive washed erythrocytes. 
The cells incubate in the antiserum at a temperature of 37.degree. C. for 
a period of about 15 to 60 minutes while receiving gentle stirring. The 
removal of the erythrocytes from the antiserum and a thorough washing 
follows the incubation to complete the sensitization procedure. 
In either the IgG or the IgM procedures, the washing of the erythrocytes 
involves their repeated suspension in and removal from a red cell 
preservative medium. Moreover, the appropriate antiserum, prior to 
receiving the erythrocytes, should have a concentration that can produce 
cells having a desired level of reactivity. Typically, the cells, when 
mixed with undilute serum having an antibody of the IgG or IgM class, as 
appropriate, should result in a 2-4+ reaction. No less than a 1+ 
reactivity should occur with a 1:4 dilution of the same serum. Where the 
initial reactant antiserum would produce overly reactive erythrocytes, it 
should undergo sufficient dilution prior to sensitization to produce the 
desired reactivity.

DETAILED DESCRIPTION 
1. Preparation of IgG-Sensitized Erythrocytes 
The serum employed in the procedure should contain an antibody of the IgG 
class and little or no detectable IgM red-cell agglutinins with 
specificities for the surface antigens of the erythrocytes selected for 
sensitization. For example, the serum may contain anti-Rh.sub.o (D), which 
agglutinates Rh.sub.o (D) positive red cells in a high protein medium at 
37.degree. C. or at the antiglobulin phase. The agglutination should not 
normally proceed in saline solutions at room temperature. The techniques 
for selecting the appropriate serum and for many of the other steps in 
this and the following procedures appear in the recent editions of the 
standard reference work, Technical Methods and Procedures of the American 
Association of Blood Banks. 
Any IgM agglutinins that may appear in the antiserum should be inactivated 
prior to the sensitization of the erythrocytes. Two methods generally 
suffice to accomplish this task. First, the antiserum may undergo heating 
in a water bath to approximately 50.degree. to 60.degree. C. for a period 
of approximately 30 to 60 minutes. During that time, it should receive 
frequent or continuous stirring. Alternatively, adding reducing agents to 
the antiserum will destroy the undesired IgM proteins. Suitable examples 
of such agents include 2-mercaptoethanol, dithiothreitol, or 
dithioerythritol. 
In order to receive the erythrocytes, the antiserum should enter a 
container having a stirrer. The container should sit in a 37.degree. C. 
water bath for a sufficient period to bring contents to that temperature. 
The erythrocytes used in the procedure should derive from whole blood 
collected from a healthy donor, typically group O. They should test 
positively for the antigen corresponding to the antibody selected. Thus, 
for example, the erythrocytes should be Rh.sub.o (D) positive when 
employing anti-D. The blood should, of course, receive a suitable 
anticoagulant such as CPD or be defibrinated. The sensitization procedures 
should occur on the day of collection. Until that time, the blood should 
remain at a temperature of about 35.degree. to 37.degree. C. prior to its 
use to avoid the binding of cold autoantibodies such as auto anti-I and 
fixation of complement. 
While undergoing gentle stirring, the warm antiserum receives an 
approximately equal volume of the thoroughly mixed anticoagulated or 
defibrinated whole blood. The combined antiserum and blood then incubates 
for about 60 minutes at 37.degree. C. with constant, gentle stirring. 
Centrifugation at 2.degree. to 8.degree. C. will then pack the cells and 
permit the removal of the supernatant. The erythrocytes, now sensitized, 
should undergo resuspension in at least four volumes of cold (2.degree. to 
8.degree. C.) red cell preservative medium. Thorough mixing should then 
precede centrifugation and decanting or aspiration as above. This washing 
procedure should occur at least three additional times so that the cells 
will have experienced it a total of at least four times. 
The washed, sensitized erythrocytes are resuspended in sufficient red cell 
preservative medium to provide them in a final concentration of about 2 to 
5 percent. This final suspension of the monospecifically sensitized cells 
should be stored at a temperature of about 2.degree. to 8.degree. C. when 
not in use. 
The sensitized cell suspension then undergoes testing with monospecific 
antihuman sera. These test sera would normally include anti-IgG (q-chain 
specific), anti-IgM (.mu.-chain specific), anti-C3, and anti-C4. To 
conduct the test, properly labeled test tubes should each receive one drop 
of one of the antisera. One drop of the cell suspension also enters each 
test tube. The test solutions then undergo centrifugation at 1000 rcf for 
approximately 15 to 20 seconds. Negative reactions should occur with the 
anti-IgM (.mu.-chain specific), anti-C3, and anti-C4 antisera. The test 
with the anti-IgG (.gamma.-chain specific) should yield a 2-4+ reaction 
with undiluted antiserum. A 1:4 dilution of the anti-IgG antiserum should 
yield at least a 1+ reaction. 
The foregoing procedure presumed a sufficient familiarity with the 
particular reactant antiserum used in the sensitization procedure to 
produce cells having the reactivities given above. Where such knowledge 
about the antiserum is lacking, it should undergo testing to determine its 
appropriate concentration to provide these reactivities. The test 
procedure involves serially diluting the antiserum in a suitable red cell 
preservative medium containing EDTA such as DADE Reverse Cyte.RTM. Diluent 
manufactured by DADE Division, American Hospital Supply Corporation of 
Miami, Florida. Each of the dilutions undergoes the complete sensitization 
with erythrocytes given above. The dilution providing the appropriately 
reactive erythrocytes is then used to produce the final sensitized cell 
suspension. 
2. Preparation of IgM-Sensitized Erythrocytes 
The serum used in this procedure should contain an antibody of the IgM 
glass and little or no detectable IgG red cell agglutinins with 
specificities for the surface antigens of the erythrocytes selected for 
sensitization. An anti-Le.sup.a serum that agglutinates Le.sup.a positive 
red cells in saline at room temperature could represent such an anitserum. 
However, the antiserum should not agglutinate these erythrocytes in a high 
protein medium at 37.degree. C. or at the antiglobulin phase when tested 
with anti-IgG (.gamma.-chain specific). 
Any C4 in the serum could become affixed to the erythrocytes by the 
classical complement pathway. Accordingly, the preparation of the 
antiserum for the sensitization procedure includes inactivating any C4 
present. Adding a source of ammonia, hydrazine, or similar compounds will 
accomplish the task. Ammonium chloride represents a suitable example. When 
using it, a 0.03 N. NH.sub.4 Cl solution should have its pH adjusted to 
about 8 with 1 N. NaOH. Four volumes of serum should then receive one 
volume of this NH.sub.4 Cl solution and be combined at room temperature 
for approximately 30 to 90 minutes. The treated serum should then receive 
approximately 0.1 to 1.0 volume of EDTA in a one to twenty millimolar 
concentration. A six millimolar concentration of dipotassium EDTA at pH 7 
performs as required. 
To proceed further with the erythrocyte sensitization, a container with a 
stirrer should receive the antiserum. It then sits in a 37.degree. C. 
water bath until the contents reach that temperature. 
A healthy donor, typically group O, should provide the whole blood used for 
the procedure. His erythrocytes should test positive for the antigen 
corresponding to the selected antibody in the antiserum. Thus, for 
example, the erythrocytes should incorporate the Le.sup.a antigen when the 
antiserum contains the anti-Le.sup.a antibody. The blood should receive a 
suitable anticoagulant, such as CPD or be defibrinated, and undergo the 
sensitization procedure on the day of its collection. Again, maintaining 
the blood at 35.degree. to 37.degree. C. prior to its use avoids 
complement fixation and a binding of cold autoantibodies. 
Centrifuging the anticoagulated or defibrinated blood, at 25.degree. to 
37.degree. C. then packs the cells. The supernatant is discarded. 
The volume of packed erythrocytes undergoes resuspension in at least four 
volumes of saline kept warm at about 35.degree. to 37.degree. C. The 
thoroughly mixed saline suspension of erythrocytes is then centrifuged and 
the supernatant removed and discarded. The volume of washed and packed 
erythrocytes is then combined with approximately 10 volumes of the warm 
antiserum prepared above. Incubation then follows for 15 to 60 minutes at 
37.degree. C., during which time the suspension receives constant but 
gentle stirring. 
By way of comparison, the procedure for sensitizing erythrocytes with the 
IgG protein involves placing anticoagulated whole blood into the antiserum 
containing the desired protein. The IgM sensitization procedure, however, 
removes and washes the erythrocytes prior to their addition to the 
reactant antiserum. This procedure gives stronger sensitization than using 
old blood. 
After the incubation, however, the procedure for the IgM suspension exactly 
follows that for the IgG product. Thus, the cells undergo four washings 
which involves packing by centrifuation at 2.degree. to 8.degree. C. 
followed by the removal and discarding of the supernatant. The washed, 
sensitized erythrocytes are resuspended to a concentration of about 2 to 5 
percent in a red cell preservative medium and stored at 2.degree. to 
8.degree. C. when not in use. 
Furthermore, the same procedure employed for the IgG-sensitized cells tests 
the IgM suspension. Each of four properly labeled test tubes receives one 
drop of an antihuman serum containing either anti-IgM (.mu.-chain 
specific), anti-IgG (.gamma.-chain specific), anti-C3, or anti-C4. Each of 
the test tubes also receives a drop of prepared sensitized-cell 
suspension. 
The test tubes with the drop of the cell suspension and each of the 
appropriate antihuman serums experience centrifugation at 1000 rcf for 15 
to 20 seconds. The tubes with the anti-IgG (.gamma.-chain specific), 
anti-C3, and anti-C4 proteins should yield negative reactions. The test 
with the undiluted anti-IgM antihuman serum should yield a reaction 
strength of 2-4+ and at least a 1+ reaction with a 1:4 dilution of the 
same antiserum. 
As for the IgG procedure, the reactant serum sensitizing erythrocytes with 
IgM may have to undergo testing in order to produce the desired 
reactivity. Again, that would involve preparing serial dilutions of the 
reactant antiserum containing the antibody of the IgM class. Each of the 
dilutions should proceed through the above procedure. Testing the 
resultant, sensitized erythrocytes would reveal the appropriate dilution 
for producing erythrocytes having the desired reactivity. 
3. Preparation of C3-Sensitized Erythrocytes 
The C3 sensitizing procedure commences with the preparation of two separate 
solutions containing different amounts of a phosphate buffer. Adding a 
portion of the second of these solutions to the first allows the 
achievement of a sensitizing diluent having the requisite pH for the 
subsequent steps. 
For the first solution, 92.4 g. of reagent grade sucrose, 690 mg. NaH.sub.2 
PO.sub.4.H.sub.2 O and 1578 mg. Na.sub.2 EDTA.2H.sub.2 O dissolve in 800 
ml. of distilled water in a one liter volumetric flask. This solution then 
receives sufficient additional water to bring its final volume up to the 
one liter mark. 
To prepare the second solution, another one liter volumetric flask 
receives, in approximately 800 mililiters of water, 92.4 g. of reagent 
grade sucrose and 1578 mg. Na.sub.2 EDTA.2H.sub.2 O. The second solution, 
however, in distinction to the first, receives 710 mg. of Na.sub.2 
HPO.sub.4. The different phosphate salt gives the second solution a 
different pH than the first. After the dissolution of these ingredients 
into the water, the second solution receives sufficient distilled water to 
bring its total volume to one liter. 
The second solution is slowly added to the first to adjust the pH of the 
latter until it reaches 5.1. A properly calibrated pH meter allows for the 
determination. The solution having a pH of about 5.1 then finds further 
use in the sensitization procedure as the required sensitizing diluent. 
Nineteen volumes of the sensitizing dilient with the pH of 5.1 enters a 
container having a stirrer. The container should sit in an ice bath until 
its contents reach a temperature of 0.degree. C. 
As above, the blood providing the erythrocytes that will receive the C3 
protein comes from a healthy donor, typically group O, and will have 
received a suitable anticoagulant, again CPD for example, or have been 
defibrinated. Fresh, unclotted blood may also be used. As with the prior 
procedures, the blood should undergo the described procedure on the day of 
its collection and remain at about 35.degree. to 37.degree. C. prior to 
its use. 
With gentle stirring, one volume of the well mixed anticoagulated whole 
blood then combines with 19 volumes of the sensitizing diluent previously 
chilled to 0.degree. C. The blood and the diluent then incubate together 
for 15 to 60 minutes at 0.degree. C. with constant, gentle stirring. 
The steps subsequent to this incubation exactly follow those given above in 
the prior two preparations. Thus, the cells receive four washings which 
consist of centrifugation at 2.degree. to 8.degree. C. followed by the 
removal and discarding of the supernatant and their subsequent 
resuspension in at least four volumes of red cell preservative medium at 
2.degree. to 8.degree. C. After the last centrifugation and removal of the 
supernatant, the sensitized erythrocytes undergo resuspension to a 
concentration of 2 to 5 percent in the red cell preservative medium. This 
suspension should remain at 2.degree. to 8.degree. C. when not in use. 
The sensitizing diluent does not provide C3 complement proteins which 
attach to the erythrocytes through this procedure. Rather, the proteins 
appear in the noncellular (plasma) portion of the same blood which 
provides the erythrocytes. The sensitizing diluent and the conditions of 
the incubation simply favor the attachment of the C3 complement component 
to the exclusion of any other proteins present. The different sensitizing 
diluent and incubation conditions given below in Section 4 result in the 
attachment to the erythrocytes of the C4 complement component in the 
original blood to the exclusion of other proteins contained in it. 
The erythrocytes sensitized with the C3 proteins should undergo the same 
testing procedure given above in Sections 1 and 2 for IgG- and 
IgM-sensitized erythrocytes. The four test tubes will each have one drop 
of an antiserum containing either the anti-C3, the anti-IgG (.gamma.-chain 
specific), the anti-IgM (.mu.-chain specific), or the anti-C4 protein. 
They will each then receive one drop of the sensitized cell suspension and 
be centrifuged at 1000 rcf for 15 to 20 seconds. The test tube with the 
anti-C3 monospecific antihuman serum should provide a reaction strength of 
2-4+. A 1:4 dilution of the same serum should provide a reaction strength 
of at least 1+. The other test tubes should display negative reactions. 
4. Preparation of C4-Sensitized Erythrocytes 
As with the procedure given above in Section 3 for the C3 sensitized 
erythrocytes, the present scheme also begins with the preparation of two 
solutions. For the first solution, 92.4 g. of reagent grade sucrose, 690 
mg. of NaH.sub.2 PO.sub.4.H.sub.2 O and 526 mg. Na.sub.2 EDTA.2H.sub.2 O 
dissolves in approximately 800 ml. of distilled water contained in one 
liter volumetric flask. Additional distilled water brings the total volume 
to exactly one liter. In comparison with the first solution for the C3 
protein, the present solution contains only 526 mg. of Na.sub.2 
EDTA.2H.sub.2 O while the prior solution contained three times as much, or 
1578 mg. 
The second solution for the C4 preparation also includes the same lesser 
amount of Na.sub.2 EDTA.2H.sub.2 O. Specifically, it has 92.4 g. reagent 
grade sucrose, 710 mg. Na.sub.2 HPO.sub.4 and 526 mg. Na.sub.2 
EDTA.2H.sub.2 O with a final volume of one liter. 
The sensitizing diluent results by adding the second solution to the first 
until a pH of 7.0 is reached. This pH compares to the value of 5.1 used 
for the attachment of the C3 protein. 
A container with a stirrer then receives 19 volumes of the resulting 
sensitizing diluent having the pH of 7.0. The container remains in a water 
bath until its contents reach a temperature of 37.degree. C. 
Whole blood from a healthy donor, typically group O, should receive an 
anticoagulant, such as CPD, or have been defibrinated. Fresh, unclotted 
blood may also be used. It then undergoes the remaining procedures on the 
day of its collection. As with all of the above preparations, it should 
remain at a temperature of 35.degree. to 37.degree. C. prior to its use. 
Before combining with the diluent, the blood with the anticoagulant should 
undergo centrifugation at 25.degree. to 37.degree. C. to pack the 
erythrocytes. The supernatant is removed and measured, with 4/5 of its 
original volume discarded. The remaining 1/5 volume of the supernatant 
then receives sufficient sterile physiologic saline solution, maintained 
at 35.degree. to 37.degree. C., to bring its volume back to the original 
volume of the supernatant. The packed cells then enter the diluted warm 
supernatant and are mixed with it to resuspend them. 
With gentle stirring, one volume of the well mixed suspension of the 
erythrocytes in the diluted supernatant then combines with the above warm 
sensitizing diluent. The mixture incubates for 15 to 60 minutes at 
37.degree. C. while the gentle stirring continues. Upon the completion of 
the incubation, the cells undergo centrifugation at 2.degree. to 8.degree. 
C. with the subsequent removal and discarding of the supernatant. 
Further comparisons with the C3 preparation follow from the listing of the 
above steps. The C3 sensitization procedure involves the use of fresh or 
defibrinated whole blood or blood merely having an anticoagulant added. To 
prepare the C4-sensitized erythrocytes, the blood's liquid is removed from 
the erythrocytes and 4/5 of its volume is replaced by the physiologic 
saline solution. Incubation then proceeds at 37.degree. C. for the C4 
cells rather than 0.degree. C. for the C3-sensitized erythrocytes. 
The succeeding steps, however, follow the procedures given above for all 
three of the prior preparations. Thus, the cells experience four washings 
which consist of resuspending one volume of sensitized erythrocytes and 
four volumes of red cell preservative medium maintained at 2.degree. to 
8.degree. C. followed by centrifugation. Subsequent to their resuspension 
to a concentration of 2 to 5 percent in a red cell preservative medium, 
the cells remain in storage at 2.degree. to 8.degree. C. when not in use. 
The resulting sensitized erythrocyte suspension should also undergo the 
same testing procedure as the other three preparations. Following the same 
steps as above, the anti-C4 antihuman serum, when undiluted, should give a 
reaction strength with the erythrocytes of 2-4+ and at least a 1+ reaction 
where the antiserum has undergone a 1:4 dilution. The reactions with the 
anti-IgG (.gamma.-chain specific), the anti-IgM (.mu.-chain specific), and 
the anti-C3 antihuman serums should produce negative reactions.