Stabilized erythrocytes and methods therefor

A double aldehyde treatment process of erythrocytes is disclosed. Erythrocytes from various sources can be treated in a two-step process to render them stable and subsequently useful in antigen antibody detection systems. Glyoxal is used as a first treating medium followed by a second treatment step using formaldehyde or glyoxal as the fixative. Glyoxal is used in the first step in amounts ranging from 0.1 to 0.4 gm. per 0.8 ml. of Packed Cell Volume of erythrocytes, followed by the second treatment step in which at least 0.1 gm. of formaldehyde or glyoxal and preferably 0.1 to 0.6 gm. per 0.8 ml. Packed Cell Volume of treated erythrocytes is used. The reaction medium is preferably aqueous hypertonic and most preferably a sodium citrate medium. The treated cells can be used in detection of hepatitis associated antigen in a reverse passive hemagglutination test.

This invention relates to the treatment of biological cell materials and 
more particularly to the treatment of erythrocytes. Specifically, it 
relates to the fixation of erythrocytes using a sequential two-stage 
coating technique involving certain aldehydic materials. 
BACKGROUND OF THE INVENTION 
In the field of medical diagnoses, it is very often convenient to use 
erythrocytes to aid in the detection of either antigens or antibodies in a 
test fluid. The erythrocyte in this case is used as a carrier particle for 
an attached antigen or antibody. As is known, when an antigenic material 
is brought into contact with an antibody which is specific for that 
material, an antigen-antibody reaction takes place. In some systems, this 
reaction is visibly perceptible, resulting in an antigen-antibody complex 
which can be discerned either by the naked eye or with the aid of 
laboratory visual equipment. In other cases, however, while there is an 
antigen-antibody reaction, the reaction product is not discernible either 
to the naked eye or with the aid of auxiliary equipment at any convenient 
level. In situations such as these, it is very useful to provide a 
particle medium as a vehicle for either the antigen or antibody so that 
subsequent reaction with the complexing partner can be visualized because 
clumping or agglutination of the particles is effected. 
The art has used a variety of particulate materials as the base upon which 
to attach the antigen or antibody for subsequent reaction, including latex 
particles such as styrene, butadiene, acrylic polymers and various blood 
cells such as human and animal erythrocytes (red blood cells). 
Erythrocytes are a very fragile, delicate component of blood, constituting 
the basic medium upon which antigens are carried throughout the host 
system. For example, human red blood cells are known to carry a wide 
variety of various antigens, the nature and composition of which give rise 
to a fingerprint which is useful in determining what kind of blood a 
recipient could tolerate upon a transfusion. 
When red cells are used to detect antigen-antibody reaction, the resulting 
agglutination is termed hemagglutination, and when the red cell is used to 
carry an antibody rather than an antigen for detection of a suspect 
antigen in host serum, the phenomenon is called reverse passive 
hemagglutination. 
As illustration of a reverse passive hemagglutination is the well-known 
detection system for the presence of hepatitis associated antigen in a 
patient's serum. The difficulty with using red blood cells in such a 
hemagglutination system, or indeed in any antigen-antibody detection 
system, is that red cells are extremely fragile, delicate and unstable. 
If red blood cells are left suspended in an isotonic medium, they will lyse 
within about twenty-one days. That is, the supporting structure of the red 
blood cells will start to weaken and cause the leakage of hemoglobin into 
the environment. Lysis of the cell results, making the material wholly 
unsuitable for any use in an agglutination detection system. 
The present invention is concerned with treating red blood cells to improve 
their stability and permit their use in antigen-antibody reaction 
detection systems. The process of so treating red blood cells is called 
fixation. 
THE PRIOR ART 
Fixation of erythrocytes to improve stability is a well-known technique. Of 
course, in the selection of appropriate fixing agents, one has to be 
careful that the fixatives do not themselves contribute to lysis of the 
erythrocytes to any intolerable degree or have a substantial deleterious 
effect on the subsequent agglutination systems. While the phenomenon of 
cell protection is not fully understood, it is believed that the fixative 
causes a chemical reaction with protein components on the surface of the 
cell, resulting in a protected cell. 
In U.S. Pat. No. 3,714,345, dated Jan. 30, 1973, the inventors describe a 
double aldehyde treatment process for coating erythrocytes using pyruvic 
aldehyde in a first treatment step followed by formaldehyde in a second 
step. This stabilization is stated by the patentee to be effective without 
apparent alteration of the capacity of the treated cells to react with 
anti-A or anti-B serum. That is, the patentee alleges that the 
stabilization is not effective to reduce this aspect of the antigenicity 
of red blood cells. 
Other publications have indicated various techniques of fixation. For 
example, Ling in Brit. J. Haemat. (1961) 7; p. 299, shows the treatment of 
red blood cells with formaldehyde, pyruvic aldehyde, glyoxal, glutaric 
dialdehyde and glyoxylic acid. The author reports that pyruvic aldehyde is 
the preferred aldehyde for fixing red blood cells. The use of large 
amounts of glyoxal resulted in a treated cell which could not be 
consistently and reliably used as a base for attachment of serum proteins 
(such as antigens and antibodies). The author concluded that the glyoxal 
technique was unsuitable. Poor stability after six (6) months was obtained 
with glyoxal as opposed to the pyruvic aldehyde treated cells. 
Red cells have also been treated with peroxy salt solutions (Chem. 
Abstracts [1961], p. 27495) and used in antigen-antibody agglutination 
systems (Chem. Abstracts [1961], p. 20672). 
DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, it has now been discovered that 
erythrocytes can be stabilized in a two-step treatment involving 
contacting the cells with a quantity of glyoxal in a first treatment step 
followed by contacting the treated cells with a quantity of formaldehyde 
or glyoxal in a second treatment step. Glyoxal is an aldehyde which may 
exist in the monomeric, dimeric, trimeric or polymeric state. The monomer 
may be depicted structurally as 
##STR1## 
The material is a solid and is available commercially as the solid, 
dihydrated trimeric form having three moles of glyoxal and two moles of 
water per mole of trimer as follows: 
##STR2## 
or as a 40% aqueous solution (based on the weight of monomeric free 
glyoxal). Formaldehyde is most often encountered as an aqueous solution of 
formaldehyde gas commercially available as formalin, 40% weight/volume (40 
gms. formaldehyde per 100 ml. of aqueous solution). 
The quantity of red blood cells in any volume of liquid whether the cells 
be from sheep, turkeys, rabbits, humans or any other animal, is 
conveniently referred to in terms of the volume that they occupy. A useful 
measure of the quantity of red blood cells in any liquid suspension of 
those cells is the volume of cells expressed as a percentage of the entire 
volume of the liquid suspension in a given sample. This parameter is 
termed Hematocrit or packed cell volume and gives a reliable 
representation of the red cells in terms of providing a common denominator 
for designating volumes. Hematocrit is a standardly determined parameter 
and is expressed as a percentage figure. Thus, a Hematocrit of 40% means 
that the red blood cells in 100 ml. of a liquid suspension of red blood 
cells occupy 40 ml. That is, the packed cell volume is 40 ml. It can be 
seen, therefore, that if the volume of the liquid suspension is doubled 
while the volume of the red cells in that suspension remains the same, the 
Hematocrit will be one-half the original, i.e. 20% in the case of the 
example given above. 
Hematocrit is determined conventionally by using the standard laboratory 
macromethod of Wintrobe, as described in Clinical Diagnosis By Laboratory 
Methods, 14th Edition, W. B. Saunders Company (Publishers), Edited by 
Israel Davidsohn, M.D., F.A.C.P., and John Bernard Henry, M.D. At page 146 
of that reference book, the macromethod is described as follows: 
"EQUIPMENT. The Wintrobe Hematocrit tube is a thick-walled glass tube with 
a uniform internal bore and a flattened bottom. It is graduated in 
millimeters from 0 to 105 and has a rubber cap to prevent evaporation 
during the long period of centrifugation. 
Of the various forms of filling pipettes available, a 2-ml. syringe with a 
needle long enough to reach the bottom of the hematocrit tube is probably 
as good as any and quite practical. 
The essential requirement of a centrifuge is that it generate a centrifugal 
field of not less than 2500 G. at the bottom of the cup." 
"REAGENT. For an anticoagulant, dried heparin, balanced oxalate or EDTA is 
satisfactory. If an inadequate amount of blood is drawn into the tube, 
resulting in an excess of oxalate or EDTA, the erythrocytes will shrink 
and the hematocrit will be falsly low." 
"PROCEDURE. The oxalated or heparinized blood must be mixed thorougly by 
not less than 30 slow and complete inversions of the container. Rolling 
the bottle in inadequate, and shaking is still worse because it may damage 
the cells." 
"After adequate mixing, the hematocrit tube is filled using the filling 
pipette or a syringe, preferably in one operation. The tip of the pipette 
is introduced to the bottom of the tube. As filling proceeds, the tip of 
the pipette is raised, but it remains under the rising blood meniscus in 
order to avoid foaming. The level of the blood should be noted and the 
tubes capped to avoid evaporation during the required centrifugation for 
30 minutes at 2500 G." 
"Reading is done without disturbing the specimen. The result is calculated 
from the formula: 
EQU Hematocrit (percent) = 100 L.sub.1 /L.sub.2 
where L.sub.1 is the height of the red cell column in mm. and L.sub.2 is 
the height of the whole blood specimen (red cells and plasma). The 
gray-white layer of leukocytes and platelets above the erythrocytes is not 
included in L.sub.1." 
It has been found in accordance with the present invention that the amount 
of glyoxal and formaldehyde used in the treatment steps can be 
conveniently related to a unit of Hematocrit measurement using the 
technique described above. A Hematocrit value will tell one skilled in the 
art what the red cell packed volume is. This volume will not ordinarily 
change from sample to sample of blood to any significant degree as regards 
the present invention provided the conditions for centrifuging samples are 
substantially equivalent. Thus a Hematocrit obtained on, for example, 
sheep erythrocytes can be compared to a Hematocrit obtained on turkey 
erythrocytes or human erythrocytes as regards the determination of the 
amount of fixative to be used in practicing the present invention. As used 
herein, Hematocrit value signifies the packed cell volume of red cells 
using the macromethod of Wintrobe at a force of at least 2500 G. for 20-30 
minutes. Additionally, the term "Packed Cell Volume" when used herein 
means that volume of red cells obtained under the foregoing conditions, 
unless otherwise stated in the text. 
A convenient Hematocrit value for practicing the present invention is 8%. 
This corresponds to 0.8 ml. of Packed Cell Volume per 10 ml. of liquid red 
cell suspension. This concentration gives a conveniently handled liquid 
suspension of red cells which is far less viscous than whole blood yet 
concentrated enough to treat significant amounts of cells. In accordance 
with the present invention, the amount of glyoxal used in the first 
treatment step is within the range of 0.1 to 0.4 gms. and preferably 0.1 
to 0.3 gms. glyoxal per 0.8 ml. of Packed Cell Volume. It is most 
convenient to supply the appropriate amount of glyoxal in the form of a 
dilute solution thereof, of the order of 1 to 4% (1-4 gm. glyoxal [based 
on the free monomer] dissolved per 100 ml. solution). This is suitably 
obtained by diluting commercially available 40% glyoxal to the appropriate 
concentration. 
In carrying out the process of the present invention, red blood cells are 
selected depending to a large extent on the subsequent antigen-antibody 
reaction that they will be employed to detect. In many situations, human 
cells are desired, but of equal suitability are the erythrocytes of sheep, 
horses, chickens, turkeys and rabbits. The contacting of the erythrocytes 
by the glyoxal is conducted in the presence of an aqueous medium, which 
has a degree of tonicity substantially compatible with the integrity of 
the cells, preferably a hypertonic medium, such as sodium citrate 
solution, for periods ranging preferably from 18-24 hours. Shorter and 
longer periods do not usually result in added benefit. The temperature of 
reaction is usually 18.degree.-25.degree. C. with room temperature being 
preferred. 
The actual concentration of sodium citrate in the final medium will depend 
upon several factors including the dilutions of the aldehyde, the 
particular erythrocytes used and the like. Suitably, the aqueous medium 
comprises sodium citrate (based on the dihydrate) in the range of 4.5 to 
5% weight/volume. 
The first treatment step of the invention is a critical event in the 
stabilization of the erythrocytes. The very delicate, fragile, untreated 
erythrocytes are converted in this step to a more stable form, capable of 
tolerating variations of environment and conditions that the untreated 
cell could not. In view of this result, the conditions of the second 
treatment step may vary more widely and more drastically than those of the 
first treatment step as will be seen below. 
Following treatment in the first stage, the cells are washed free of any 
hemolysed cells that may have resulted, usually with an isotonic saline 
solution, and then treated in the second step with either formaldehyde or 
glyoxal at levels of at least 0.1 and preferably ranging from 0.1 to 0.6 
gm. and most preferably 0.1-0.3 gm. per 0.8 ml. of Packed Cell Volume. 
This treatment is conveniently carried out under the same conditions as 
the first glyoxal treatment although concentrations of the second aldehyde 
at the high end of the ranges tend to require shorter treatment times. 
After the second treatment is completed, the cells are then washed using 
preferably a saline or a buffer wash medium and are then ready for coating 
with antigen or antibody for subsequent use in a detection system. 
Cells treated in accordance with the present invention have been stable at 
5.degree. C. for over eight months, have not shown any signs of hemolysis 
and are suitable for coating with antigen or antibody. This is to be 
contrasted to the situation obtained with untreated cells wherein 
hemolysis begins almost immediately and is usually complete in about 
twenty-one days. 
Additionally, the treated cells retain their ability to be coated with 
antibody or antigen and react specifically. For example, antibody or 
antigen can be attached to the treated cell in accordance with well-known 
techniques. Various antigens and antibodies such as Human Chorionic 
Gonadatropin, hepatitis antibody, fibrinogen, albumin, gammaglobulin and 
the like may be used. 
The conditions set forth above for determining Hematocrit were presented to 
(a) give a standard for all sources of red cells; and 
(b) to utilize the most commonly encountered laboratory procedures. If 
different determination conditions are employed or if electronic cell 
counters are used, resulting in a red cell packed volume different from 
that obtained utilizing the Wintrobe conditions referred to previously, 
one should relate that red cell packed volume to a Hematocrit obtained at 
the prescribed Wintrobe conditions for calculation of the amount of 
fixatives to be used herein. 
The buffers or diluents used herein may be any of the biologically suitable 
materials normally used in the art, which are substantially compatible 
with the integrity of the cells. The term "biologically suitable" includes 
compatibility with the antigens or antibodies encountered and with 
non-lysis of erythrocytes. Such materials as solutions of normal saline, 
sodium citrate and the like may be used. Sodium citrate solutions of about 
4.5-5% weight/volume are most preferred.

EXAMPLE I 
100 ml. of type O Rh negative blood was collected from a human donor in a 
standard acid-citrate-dextrose (ACD) anticoagulant medium. The cells were 
washed four times in 10 volumes of iostonic saline. The cells were 
resuspended in one of two buffers as indicated below at a level of 8% 
Hematocrit (using the Wintrobe macromethod). The buffers had the following 
composition: 
1. Citrate -- 5.0% weight volume aqueous sodium citrate . 2H.sub.2 O, 
having a pH of 8.7; and 
2. Phosphate -- 16.18 gm. Na.sub.2 HPO.sub.4 -anhyd 4.9 gm. KH.sub.2 
PO.sub.4 - anhyd pH 7.2 (0.15M) 
various dilutions of 40 g. % aqueous glyoxal (based on free monomer) were 
prepared as indicated in the table below using the buffer indicated. 10 
ml. of the buffered glyoxal solution were then mixed with 10 ml. of cell 
suspension. Thus, in each case, 0.8 ml. of red cell packed volume was 
contacted by the indicated amount of glyoxal. 
______________________________________ 
% gms. Ald. 
Glyoxal Glyoxal Buffer Cell Buffer 
______________________________________ 
A 4 0.4 Citrate Citrate 
B 4 0.4 Citrate Citrate 
C 4 0.4 Citrate Citrate 
D 4 0.4 Citrate Citrate 
E 6 0.6 Citrate Citrate 
F 1 0.1 Phosphate 
Phosphate 
G 3 0.3 Phosphate 
Phosphate 
H 5 0.5 Phosphate 
Phosphate 
______________________________________ 
Each sample was mixed on a magnetic stirrer at 20.degree.-25.degree. C. for 
18-24 hours. The fixed cells in each sample were then separately washed 4 
times in saline, then readjusted to 8% Hematocrit in the 5% citrate buffer 
described above. 
EXAMPLE II 
The indicated volumes of each of the samples A through H obtained in 
Example I were treated with the aldehyde shown below in the amounts 
indicated. 
__________________________________________________________________________ 
Treated Weight of 
Vol. of Treated 
Red Cell Aldehyde 
Aldehyde 
Aldehyde 
Sample 
Cell Suspension 
Suspension Buffer 
Aldehyde 
Volume 
Buffer 
% Present 
__________________________________________________________________________ 
A.sub.G.sup.* 
10 ml. Citrate Form..sup.1 
10 ml. 
Citrate 
1.1% 0.11 g. 
B.sub.G 
10 ml. Citrate Form. 10 ml. 
Citrate 
5.4% 0.54 g. 
C.sub.G 
10 ml. Citrate Glyoxal.sup.2 
10 ml. 
Citrate 
1.0% 0.1 g. 
D.sub.G 
10 ml. Citrate Glyoxal 
10 ml. 
Citrate 
5.0% 0.5 g. 
E.sub.G 
1.15 ml. Citrate Form. 1.15 ml. 
Citrate 
3.2% 0.037 g. 
F.sub.G 
6.3 ml. Citrate Form. 6.3 ml. 
Citrate 
3.2% 0.2 g. 
G.sub.G 
10 ml. Citrate Form. 10 ml. 
Citrate 
3.2% 0.32 g. 
H.sub.G 
10 ml. Citrate Form. 10 ml. 
Citrate 
3.2% 0.32 g. 
__________________________________________________________________________ 
.sup.* In each case G means glyoxal treated. 
.sup.1 40% w/v formaldehyde solution diluted to indicated aldehyde % with 
citrate buffer. 
.sup.2 40% w/v glyoxal solution diluted to indicated aldehyde % with 
citrate buffer. 
The cells were mixed with the aldehyde at room temperature for 18-24 hours. 
They were then washed four times in saline, adjusted to 8% Hematocrit in 
0.1 M phosphate buffer having the following composition: 
3.26 gm. KH.sub.2 PO.sub.4 (anhydrous) 
10.78 gm. Na.sub.2 HPO.sub.4 (anhydrous) 
1 gm. NaN.sub.3 
q.s. to 1 liter 
The cells were permitted to sit at +5.degree. C. for one week and then were 
inspected for hemolysis. The supernatant of samples F, G and H was yellow, 
indicating that the cells were not fully stabilized. 
EXAMPLE III 
Each of the samples A through H obtained in Example II was utilized in an 
antigen-antibody detection system as follows: 
One hundred twenty-five (125) .mu.l. of the 8% Hematocrit cells suspension 
in the 0.1 M phosphate buffer are dispensed into a test tube. The cells 
are washed once with isotonic saline, the saline decanted and 0.5 ml. of 
0.1 M acetate buffer.sup.1, pH 4 is added to the cells. To this is added 
5-20 micrograms of affinity purified hepatitis B.sub.s antibody from a 
chimpanzee, and the suspension mixed at room temperature for 75 minutes. 
The cells are then washed 4 times with isotonic saline, the saline 
decanted and cells suspended in 0.1 M phosphate buffer described in 
Example II and additionally containing 0.01 M EDTA, 1% normal human serum, 
and 0.1% gelatin. A 25 .mu.l. sample of each cell suspension was then 
introduced into a microtitre well containing 25 .mu.l. of a diluent and 7 
.mu.l. of either 
(a) human serum known to contain hepatitis B antigen (weakly positive by 
radioimmunoassay technique); 
(b) human serum known to be negative for hepatitis B antigen by 
radioimmunoassay techniques; 
(c) nothing in addition to the diluent (the control well). 
FNT .sup.1 2.45 gms. NaC.sub.2 H.sub.3 O.sub.2. 3H.sub.2 O 
FNT 4.7 ml. acetic acid (glacial) 
FNT q.s. to 1,000 ml. with distilled water 
In each case, the antibody coated cells were mixed by shaking and allowed 
to stand undisturbed for 2 hours at room temperature. The wells were 
thereafter examined for the presence of an agglutination reaction. 
Examples A through D gave reactions for the positive serum and no reaction 
with the known negatives. The control gave no reaction. 
Sample E was prepared by using the 0.6 gm. glyoxal/0.8 ml. Packed Cell 
Volume, and gave non-specific reactions with negatives. 
Samples F through H were found not to react specifically indicating that 
they were unsuitable for use in a hepatitis B.sub.s antigen detection 
system. 
Summarizing the previous examples, the following shows the amounts of 
glyoxal and formaldehyde or glyoxal on the treated cells, Gx. indicating 
glyoxal, F. is formaldehyde, and the numbers preceding each signify the 
grams in tenths, per 0.8 ml. of Packed Cell Volume. 
______________________________________ 
A 4 Gx. 1 F. 
Specific Reaction 
B 4 Gx. 5 F. 
Specific Reaction 
C 4 Gx. 1 Gx. 
Specific Reaction 
D 4 Gx. 5 Gx. 
Specific Reaction 
E 6 Gx. 3 F. 
Non-Specific Reaction 
F 1 Gx. 3 F. 
Non-Specific Reaction 
G 3 Gx. 3 F. 
Non-Specific Reaction 
H 5 Gx. 3 F. 
Non-Specific Reaction 
______________________________________ 
EXAMPLE IV 
Example I was repeated to produce appropriately treated red blood cells 
using the following amounts of materials in the first treatment step. 
______________________________________ 
Glyoxal Treatment in Citrate Buffer - 
Red Cells in Citrate 
Sample % Aldehyde Gms. Aldehyde 
______________________________________ 
1 1 (Glyoxal) .1 
2 1 (Glyoxal) .1 
3 1 (Glyoxal) .1 
4 1 (Glyoxal) .1 
5 5 (Glyoxal) .5 
6 5 (Glyoxal) .5 
7 5 (Glyoxal) .5 
8 5 (Glyoxal) .5 
9 3 (Glyoxal) .3 
10 3 (Glyoxal) .3 
11 3.2 (Formaldehyde) 
.32 
12 3.2 (Formaldehyde) 
.32 
______________________________________ 
The second fixing stage was carried out following the procedure of Example 
II using the aldehyde indicated below: 
______________________________________ 
Gms. Aldehyde/ 
Sample % Aldehyde .8 ml. Packed Cell Vol. 
______________________________________ 
1 = 1 Gx. 1.1 Form. 0.11 
2 = 1 Gx. 5.4 Form. 0.54 
3 = 1 Gx. 1 Gx. 0.1 
4 = 1 Gx. 5 Gx. 0.5 
5 = 5 Gx. 1.1 Form. 0.11 
6 = 5 Gx. 5.4 Form. 0.54 
7 = 5 Gx. 1 Gx. 0.1 
8 = 5 Gx. 5 Gx. 0.5 
9 = 3 Gx. 3 Gx. 0.3 
10 = 3 Gx. 3.2 Form. 0.32 
11 = 3.2 Form. 
3.2 Form. 0.32 
12 = 3.2 Form. 
3 Gx. 0.3 
______________________________________ 
Samples 1-4 and 9 and 10 showed specific reactions for HB.sub.s Ag 
following the procedure set forth in Example III and gave no reactions for 
controls and known negatives. 
Samples 5-8 give non-specific reactions for HB.sub.s Ag in the negative 
samples. 
Samples 11 and 12 give no specific reaction for positive HB.sub.s Ag and 
are therefore unsuitable.