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Erythrocyte survival in rheumatoid arthritis.

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Erythrocyte Survival in Rheumatoid Arthritis
By EDWARD
S. MONGANAND RALPHF. JACOX
Thirty patients with active rheumatoid
arthritis were transfused with chromium51 labeled erythrocytes from either normal or rheumatoid donors. Three sets of
identical twins discordant for rheumatoid arthritis had a cross transfusion
from a healthy donor to the affected
recipient. I n no instance was there any
significant shortening of erythrocyte survival which could not be attributed to
minor blood group incompatibility. The
presence of rheumatoid factor in either
the donor, the recipient, or both did not
effect erythrocyte survival.
Trenta patientes con active arthritis
rheumatoidee recipeva transfusiones de
erythrocytos a marcage con chromo-51
ab donatores normal o rheumatoidee. In
tres pares de geminos identic con discordantia pro arthritis rheumatoidee
esseva le subjectos de un experiment0
de transfusion cruciate a b un donator
normal a un recipiente afficite. Esseva
notate nulle caso de un reduction significative del durata vital del erythrocytos
gue non poteva esser attribuite a minor
incompatibilitates de gruppo de sanguine. Le presentia de factor rheumatoidee in le donator, in le recipiente, o
in ambes non afficeva le durata vital del
erythrocytos.
T
HE QUESTION OF whether there is a normal or a decreased survival
of erythrocytes in patients with rheumatoid arthritis is complicated by
the fact that some observors have found a normal s u r ~ i v a l ~
while
- ~ others
have found decreased survival.4.7 None of these reports have given full details
about serological identification of the rheumatoid disease, the effects of drug
therapy or other variables which might influence results of erythrocyte survival studies. In 1961 Richmond et a1.8 reported that rheumatoid patients
had a normal survival of erythrocytes transfused with their own cells, but
that a decreased erythrocyte survival occurred when normal, compatible
donor cells were transfused into rheumatoid patients.
This study raises a question of whether erythrocytes from rheumatoid
patients might have some unique advantage of survival over cells obtained
from normal individuals. With this question in mind, we have carried out an
investigation of erythrocyte survival to determine whether the serological
state of the rheumatoid patient might influence erythrocyte survival. Thus
our investigation includes not only cross transfusion of cells from seropositive
rheumatoid donors into patients with rheumatoid arthritis, but also transfusiod of normal donor cells and cells obtained from patients with seronegative
rheumatoid arthritis.
A standard labeling technic with radioactive chromium was employed and
cross-transfusion experiments were performed in thirty patients, including
three sets of identical twins discordant for rheumatoid arthritis. Only four of
From the Department of Medicint, The Uniuersity of Rochester School of Medicine G
Dentisty, Rochester, New York.
Supported b y Grant #CRMC 24, The National Foundation.
481
ARTHRITIS
AND RHEUMATISM,
VOL. 7, No. 5 (OCTOBER),1964
482
MONGAN AND JACOX
the patients had a slightly shortened erythrocyte survival time and in two of
these patiedts, minor blood group incompatibilities were demonstrated which
could explain the decreased survival observed.
METHODS
All patients investigated were individuals with rheumatoid arthritis who were actively
attending the Arthritis Clinic at the University of Rochester. They were typed for ABO
blood group system and for blood groups Cc, D, Ee, and Kell. In a similar manner six
normal individuals were typed. When suitable donors and recipients were found, fresh
blood specimens were drawn and cross matching was performed. An indirect Coombs
test was also done. All patients had active, definite rheumatoid arthritis of a t least
one year’s duration. Steinbrocker’s functional class and radiological stagings was known
on each paticnt. None of the patients had leukopenia or splenomegaly. Rheumatoid factor
was determined by the latex flocculation test of Singer and Plotz.10
Thirty milliliters of blood was obtained by venipuncture and gently ejected into a
sterile flask containing 7 i d . of sterile ACD mixture. The blood was then thoroughly
mixed by gently swirling the flask. Approximately 75 microcuries of high specific activity
sterile radioactive sodium chromate was added to the blood citrate mixture. The citrateblood-chromate mixture was allowed to sit for thirty minutes at room temperature. The
sample was then centrifuged in sterile centrifuge tubes and the snpernatant plasma was
removed. An equal volume of sterile isotonic saline was added. The cells were thoroughly resuspended in the saline, and centrifugation was repeated. Washing with sterile isotonic
saline was carried out twice. Following the second washing, a volume of isotonic sterile
saline was then added to the sedimented erythrocytes so that the hematocrit of the mixture
was approximately 40-50 per cent. This mixture of chromated erythrocytes was then
slowly injected intravenously into a recipient.
Thirty minutes after injection, approximately 7 ml. of blood was withdrawn from
the patient and placed in two EDTA bottles. Duplicate hematocrits were determined.
Exactly 3 nil. of the well mixed whole blood from one of the hematocrit bottles was
placed in a shell vial whereupon subsequent determination of radioactivity was measured
in a well type scintillator connter. Another aliquot of whole blood was centrifuged and
1 ml. of plasma was removed by a Pasteur pipette and placed in a second shell vial.
The plasma was then diluted to a total volume of 3 ml. with sterile saline for subsequent analysis of radioactivity. Samples similar to the above were obtained at 24 and 72
hours and the plasma and whole blood radioactivity was measured. In later samples only
a 3 ml. volume of the well mixed whole blood was placed in a shell vial for counting.
Specimens were obtained at five or six intervals during the ensuing month.
All measurements of radioactivity on each of the samples obtained in a single experiment were determined at the end of the study in each patient. The emmission of each
sample was measured and corrected for plasma and background radioactivity. The radioactivity for each blood sample was expressed as counts/minute per milliliter of packed
red blood cells. The sample obtained 30 minutes after injection of the chromated erythrocytes was considered to be 100 per cent active. All subsequent samples were expressed
in terms of the percentage decrease from this baseline measurement.
The best fit on the disappearance curve was calculated by the method of least squares.
The one-half survival time oE chromium 51 was then determined from that curve. No attempt was made to correct for elution. In calculating the correlation coefficient, the 100
per cent value was excluded since the greatest amount of elution of chromium occurs
shortly after the cells are injected into the circulation of the recipient.
RESULTS
Six normal healthy individuals of various ages were given transfusions of
compatible donor erythrocytes. The erythrocyte survival in these normal
recjpients (table 1) reveals a range of half life survival time of the chromium
483
ERYTHROCYTE SURVIVAL IN RHEUMATOID ARTHRITIS
Table 1.-Results of Cr5I Studies in Healthy Individuals Who Received
Erythrocytes from Normal Donors
__- - _ _ _______
.~
Case
Number
T'
y Cr"
1
2
3
6
Mean %
76
____
44
42
42
43
46
47
4
5
__-_
Hematocrit
____._
(days)
23.0
32.2
26.3
22.2
29.4
27.2
I__._____
Cr51
Survival Time-26.7
days
Correlation
Coeffieient
_________.
-0.987
-0.955
-0.998
-0.991
-0.988
-0.979
~_____-
D
.001
.001
.001
.001
.001
.010
-+ 6.9 days.
51 labeled erythrocytes of 22.2-32.2 days, with a mean half life of 26.7 days.
The ncrmal half life value established for this study was then set at 26.7 days
* 6.9 days ( 2 standard deviations) with normal values ranging from 19.833.6 days.
Table 2 represents the results obtained in 16 patients with rheumatoid
arthritis who received chromium 51 tagged erythrocytes from normal donors.
The first three patients shown in table 2 were identical twins with sero-positive definite rheumatoid arthritis who received transfused erythrocytes from
their normal unaffected identical twin. Identity of the twins was proien by
establishing that each set had identical blood groups in 25 systems and by
a successful skin transplantation from the normal to the affected twin. Figure
1 depicts the survival time of the tagged erythrocytes in each of the three
sets of twins studied. I t will be observed that erythrocyte disappearance is
linear and that the half life in each instance is normal.
Patients 4-13 (table 2 ) were patients with sero-positive, definite rheumatoid
arthritis who received chromium 51 tagged red blood cells from healthy donors. Eight of the ten patients had normal one-half survival times, whereas
patients 12 and 13 had a decreased erythrocyte survival. Patients 14-16 (table
2 ) were individuals who had sero-negative, definite rheumatoid arthritis who
recei\.ed chromated erythrocytes from healthy donors. Two of these three
patients had a normal red cell survival. Patient #16, had a slight decrease of
survival time.
Table 3 depicts the results of erythrocyte survival wherein both the donors
and recipiedts had rheumatoid arthritis. A total of 14 transfusions were given
in which various combinations of donors and recipients were used. Patients
17, 18, and 19 who had sero-positive definite rheumatoid arthritis receiired
erythrocytes from donors who also had sero-positive rheumatoid arthritis.
All three of these experiments revealed erythrocyte survival times within normal limits. In cases 20-23 erythrocytes obtained from patients with seronegative rheumatoid arthritis were transfused into patients with sero-positive
rheumatoid arthritis. Erythrocyte survival times in each instance were normal.
Cases 2 4 3 0 were patients with sero-negative rheumatoid arthritis who received erythrocytes from patients with sero-positive rheumatoid arthritis.
Six of these studies are within normal limits, but case 26 had a definitely
shortened half life of chromium 51 labeled donor cells.
Table 4 summarizes the laboratory data and drug therapy in the patients
484
MONGAN AND JACOX
Table 2.--Clinical Data and Results of Crsz Studies in Patients with Rheumatoid
h o_
Received
Erythrocytes
_ _ _ _ Arthritis
_ ~ _W_
_
____from Normal Donors
Case
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Age
(rrs.)
51
61
25
32
62
52
56
62
67
59
“7
72
69
61
59
31
______
Duration
of Disease
Sex
F
F
F
F
M
F
F
F
M
M
F
F
F
F
F
F
(JM.)
10
8
2
9
4
9
1.5
8
1.3
13
3
9
2
10
11
1
Hematocrit
%
36
36
40
42
38
37
41
40
36
46
39
36
39
38
32
31
Latex
Fixation
T% CrS1
1 :2560
1:320
1: 320
1:5120
33.5
27.2
23.9
21.0
35.3
26.0
24.6
31.8
25.1
26.8
23.4
1:80
1:2560
1 :320
1:2560
1 :5120
1:320
1:1280
1:2560
1:160
Neg.
Neg.
Nep.
Correlation
Coefficient
(days)
27.2
36.1
19.5
-
-0.994
-0.990
-0.989
-0.989
-0.957
-0.983
-0.996
-0.996
-0.999
-0.989
-0.994
See Figure 2
See Figure 2
-0.998
-0.999
-0.995
P
.001
.001
,010
.001
.001
.001
.001
.001
.001
.001
.001
.001
.001
,001
studied. No significant differences were noted between the patients with
normal survival times arid those patients with shortened survival times in
any of the variables studied.
Selen of the recipient patients were anemic (i.e. hematocrit below 40 per
cent in males, below 36 per cent in females) at the time they received donor
cells. Only one of these patients ( # l 6 ) had a shortened erythrocyte survival
tlme. When the seven anemic patients are compared as a group to the six
normal controls, no significant difference in results is found. The mean halftime of chromium 51 disappearance for normal cells introduced into the
circulation of normal subjects was 26.7 days, compared with the mean halftime for disappearance of donor erythrocytes in the anemic recipients of
27.3 days. By Student’s t test the probability of these mean values not being
significantly different was well above 0.1 ( t = 0.21; n = 11; p >> 0.1).
Analysis of Patients with a Shortened Erythrocyte Survival Time
Figure 2 represents the erythrocyte curves determined in patients 12 and
13. In each instance the survival time appeared to be relatively normal until
the donor erythrocytes had been in the recipient circulation for two weeks.
By the sixteenth day, there was marked destruction of donor cells in both
patients. This abrupt destruction of donor cells suggested that the recipient
had become sensitized to the donor’s erythrocytes. The experiment was
repeated wherein the same donor’s erythrocytes were transfused into the
patient. It will be noted (fig. 2 ) that an accelerated and more rapid donor
erythrocyte destruction resulted. A minor blood group incompatibility existed
for case #12 with donor cells Ee being transfused into recipient ee. An
analysis of the recipient’s serum for anti-E antibody revealed no serologically
identifiable antibody after the first and the second transfusion made nine
months later.
The blood type in patient #13 was identical to that of the donor. A search
for other m nor blood group antibodies was made before the second transfu-
485
ERYTHROCYTE SURVIVAL IN RHEUMATOID ARTHRITIS
\
PATIENT # I
PATIENT #2
M PATIENT#3
0-0
0-0
TIME IN DAYS
Fig. 1.-Erythrocyte survival in three patients with rheumatoid arthritis who
received donor red blood cells from their identical twin.
sion eight months later. No detectable antibodies were found during or after
the repeat study. Despite our failure to demonstrate an incompatability, it is
felt that these data suggest that there was a sensitization of the recipient to
the donor red blood cells in each case.
The survival time in case #16 was 19.5 days which is less than 19.8 days,
the lower limit of normal established in this study. Since 95 per cent of
expected cases would fall within two standard deviations of the mean, it is
conceivable that this value of 19.5 days does not represent decreased erythrocyte survival. A repeat transfusion employing erythrocytes from the same donor
was made nine months later (fig. 3 ) . The half life on this occasion was
20.2 days, a figure almost identical to the first one. There had, however, been
a significant change in the patient’s illness between the two studies. At the
time of the first transfusion the patient had an active arthritis reflected by
an increased erythrocyte sedimentation rate and a reduction of hematocrit
to 31 per cent. When the second transfusion was made the patient was at a
stage of a marked clinical remission of arthritis. The hematocrit had idcreased
to 42 per cent. Since the survival times in both studies were almost identical,
it seems unlikely that erythrocyte hemolysis played any role in the genesis of
the patient’s anemia.
Patient #26 had a definitely shortened blood cell survival with a chromium
51 half time disappearance of 16 days. This patient had severe rheumatoid
arthritis (class IV, stage IV) of 25 years duration. The disease was still
486
MONGAN AND JACDX
Table 3.-CEinical Data and Results of Cr5' Studies in Patients with Rheumatoid
Arthritis Who Received Erythrocytes from Other Patients
with Rheumatoid Arthritis
___--__
Duration
Case
Number
Age
(yrs.)
17
18
19
20
21
22
23
24
25
26
21
28
29
38
59
71
68
52
61
56
55
57
64
29
61
53
62
30
of Disease
Hematocrit
Sex
(yrs.)
%
F
M
F
F
F
M
F
F
M
3
2
12
3
5
4
6
32
F
F
F
F
28
12
F
4
30
39
41
36
41
38
39
39
40
36
37
41
39
43
6,
1::
12
T1h C P
Latex
Fixation
(days)
22.8
24.9
24.6
23.5
36.4
26.4
27.8
26.4
32.3
16.0
22.1
32.4
21.7
30.8
1:320
1:320
1:80
1:1280
1:160
1:80
1:80
Neg.
Neg.
Neg.
Nep.
Neg.
Neg.
Neg.
Correlation
Coefficient
P
-0.998
-0.980
-0.982
-0.980
-0.987
-0.993
-0.999
-0.988
-0.986
-0.344
-0.933
-0.990
-0.991
-0.989
.001
.001
.001
.001
.01
.001
.a01
.001
.001
-
.01
.001
.001
.001
Table I.-Correkztion of Severity of the Patient's Rheumatoid Arthritis
with Laboratory Studies and Drug Therapy
_______--Total number of patients
Number of patients with an
elevated sedimentation rate
Number of anemic patients
Number of patients with a
latex titer of 1:80 or above
Number of patients with a
positive antinuclear factor
Number of patients with a
positive LE prep
Number of patients with a
decreased red blood cell
survival as measured by
Cr.51 technic
Number of patients taking
salicylates
phenylbutozone
gold
chloroquine
prednisone
Class I1
___--
Class I11
Patients with
Decreased RBC
Class IV
Total
Grow
4
30
4
____-___
Survival
11
15
8
2
15
4
5
0
27
7
3
1
8
10
2
20
2
1
5
1
7
1
1
4
0
5
0
1
2
1
4
4
14
2
23
5
2
3
3
5
10
16
active as reflected by a Westergren sedmientation rate of 100 mm. per hour.
The serum contained no rheumatoid factor. The patient had previously received blood transfusions on sel-era1 occasions during surgical procedures. It
is possible that this patient, as patients 12 and 13, had developed a minor
blood group incompatibility.
DISCUSSION
The results of this investigation' clearly demonstrate that erythrocyte survival in rheumatoid patients is norma1 and is independent of the source of
the erythrocytes used in the transfusion. Thus normal donor erythrocytes and
ERYTHROCYTE SURVIVAL IN RHEUMATOID ARTHRITIS
487
PATIENT # I 2
PATIENT # I 3
REPEAT STUDY#I2
REPEA1 STUDY#13
I
0
0
.
lo'
'
'.'0
I 0 ' 20 ' 30 ' 40 '
TIME I N
Fig. 2.-Erythrocyte
normal limits.
DAYS
survival in two patients in whom the results fell outside
erythrocytes obtained from sero-negative and sero-positive rheumatoid donors,
when tran'sfused into rheumatoid patients have a normal life span. The most
critical analysis of this question was available when three sets of identical
blood types were studied. It was found that each normal twin's erythrocyte
had a normal survival when transfused into the corresponding twin with
rheumatoid arthritis. We are thus unable to confirm Richmond's* observation
that rheumatoid patien'ts have some unusual capacity to destroy normal
donor cells more rapidly than when isologous labeled erythrocytes are tranfused.
In the three instances in which definite decreased erythrocyte survival was
observed, the pattern of erythrocyte destruction was that of a minor blood
group incompatibility even though no serological identification of this
phenomenod was demonstrable. Mollisonll has shown in cross transfusion
studies that it is frequently impossible to identify antibodies when there is
diminished survival due to incompatibility.
The question must be raised as to why some observors have reported
decreased erythrocyte survival in rheumatoid patients while others have
not supported this observation. In order to define the limits of erythrocyte
survival it is important to define any variable which is due to either the labeling technics or to possible external factors which produce blood loss apart
from intravascular destruction.
The normal range for the half-time of chromium 51 disappearance in our
488
MONGAN AND JACOX
0-0 INITIAL STUDY
O--.
lo
L
'
I0
'
STUDY REPEATED
WITH THE SAME
DONOR
2 0 ' 3 0 ' 4 0 ' 50
TIME IN DAYS
Fig. 3.-Erythrocyte survival in patient Number 16.
study was 19.8-33.6 days and is greater than' that reported by other observors. The half life of chromium 51 labeled erythrocytes for normal cells
transfused into normal subjects was 26.7 days compared to a mean half life
of 27.2 days of normal erythrocytes transfused into patients with seropositive
rheumatoid arthritis. Student t test analysis reveals the probability of these
findings not being significantly different to be greatly in excess of 0.1 ( t =
0.21; n = 15, p = > > .01).
Any variablity due to technic was controlled as rigidly as possible by the
following methods. The control subjects were studied by doing these individuals randomly during the time of the patient studies. The same method of
erythrocyte labeling and transfusion was employed. In all experiments great
care was taken to idsure adequate mixing of labeled chromate ions with the
erythrocyte suspension. Only chromium of high chromium 51 specific activity was used. A mathematical calculation of the slope of labeled erythrocyte disappearance was made to insure the most precise estimation of the
point of half time survival. It is felt that these technics have minimized
most of the variables that exist in such a procedure.
SUMMARY
Thirty patients with active rheumatoid arthritis were tradsfused with
Chromium 51 labeled erythrocytes from either normal donor or rheumatoid
donor which were either sero-positive or sero-negative for the rheumatoid
factor. Some of the rheumatoid recipients were sero-negative and received
ERYTHROCYTE SURVIVAL IN RHEUMATOID ARTHRITIS
489
donor erythrocytes from normal or rheumatoid patients who were sero-positive for the rheumatoid factor. Three sets of identical twins which were discordant for rheumatoid arthritis had cross transfusion of labelled cells from
the healthy donor to the affected recipient. In r ~ oinstance was there any significant shortening of erythrocyte survival which could not be attributed to
minor blood group incompatibility.
ACKNOWLEDGMENT
We are indebted to Dr. Anthony Bowdler and Dr. Robert Weed for advice in the
use and calculations of the chromium labeling technic and to Miss Norma T r a b l d for
performing the blood typing and cross-matching.
REFERENCES
1. Lewis, S. M., and Porter, I. H.: Erythrocyte survival in rheumatoid arthritis. Ann. Rheum. Dis. 19:54-58,
1960.
2. Biechl, A. Stapleton, J. E., Woodbury, J.
F., and Read, H. C.: Anemia in
rheumatoid arthritis. I. Red cell survival studies. Canad. Med. Ass. J.
86:401-405, 1962.
3. Roberts, F. D., Hagedorn, A. B., Slocumb, C. H., and Owen, C. A.: Evaluation of the Anemia of Rheumatoid
Arthritis. Blood 21:470-477, 1963.
4. Ebaugh, F. G., Jr., Peterson, R. D.,
Rodman, G. P., and Bunim, J. J.:
Symposium on rheumatic diseases:
Anemia of rheumatoid arthritis. Med.
Clin. North America 39:489-498,
7.
8.
9.
1955.
5. Alexander, W. R. M., Richmond, J.,
Roy, L. M. H., and Duthie, J. J. R.:
Nature of anaemia in rheumatoid
arthritis. II. Survival of transfused
erythrocytes in patients with rheumatoid arthritis. Ann. Rheum. Dis. 15:
12-20, 1956.
6. Freireich, E. J,, Ross, J. F., Bayles, T.
B., Emerson, C. P., and Finch, S . C.:
Radioactive iron metabolism and ery-
10.
11.
throcyte survival studies of the
mechanism of the anemia associated
with rheumatoid arthritis. J. Clin. Invest. 36: 1043-1058, 1957.
Weinstein, I. M.: A correlative study of
the erythrokinetics and disturbances
in iron metabolism associated with
the anemia of rheumatoid arthritis.
Blood 14:950-969, 1959.
Richmond, J., Alexander, W. R. M.,
Potter, J. L., and Duthie, J. J. R.:
The nature of anaemia in rheumatoid
arthritis V. Red cell survival measured by radioactive chromium. Ann.
Rheum. Dis. 20:133317, 1961.
Steinbrocker, O., Traeger, C. H., and
Batterman, R. D.: Therapeutic criteria
in rheumatoid arthritis. J.A.M.A. 140:
659-662, 1949.
Singer, J. M., and Plotz, C. M.: The
latex fixation test. I. Application to the
serological diagnosis of rheumatoid
arthritis. Am. J. Med. 21:888-892,
1956.
Mollison, P. L.: Measurement of survival
and destruction of red cells in haemolytic syndromes. Brit. Med. Bull.
15:59-66, 1959.
Edward S. Mongan, M.D., Senior Instructor in Medicine,
Rochester School of Medicine and Dentistry, Rochester,
New Ymk.
Ralph F. lacox, M.D., Professor of Medicine, Rochester School
of Medicine, and Rentistry, Rochester, N e w York.
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