Download Suppression of Leukocytic Mitosis by Sera of Hepatitis

Suppression of Leukocytic Mitosis by Sera of
Hepatitis-implicated Donors
BARBARA A. MEIXA, M.D.,
AND HOWARD F. TASWELL,
M.D.
The American National Red Cross, 17th and D Streets NW, Washington, D. C. 20006,
and Section of Clinical Pathology, Mayo Clinic and Mayo
Foundation,
Rochester, Minnesota 55901
Abstract. Mella, Barbara A., and Taswell, Howard F.: Suppression of leukocytic mitosis by sera of hepatitis-implicated donors. Am. J. Clin. Path. 53:
141-144, 1970. In a blind study, 21 specimens of serum from 19 donors
thought to be hepatitis carriers were incubated in cultures of normal leukocytes. Sera from 15 (79%) of the 19 donors suppressed mitosis. Immunoglobulin levels of 16 donors were determined; they were increased in 11
(69%). In 17 (89%) of the 19 suspect donors, either one or both tests were
positive. Twelve specimens of serum from patients during various periods
of infectious hepatitis were also tested. Mitotic suppression was found only
when the sera were from patients in the preicteric or early icteric phase of
the disease. Of the 23 specimens of serum used as controls, four caused mitotic
suppression.
from patients acutely ill with infectious hepatitis suppresses leukocytic mitosis
when incubated with normal peripheral
blood in vitro.a Immunoglobulin levels in
blood from donors implicated as carriers
of hepatitis have been reported to be above
the 97.5 percentile of those in a control
group. 1 Because these findings might provide a means of detecting the agent of
either serum or infectious hepatitis in
blood to be used for transfusion, a study
was designed to determine if mitotic inhibition occurred in leukocytes of normal
peripheral blood after incubation with sera
from donors suspected of being carriers of
hepatitis virus. If a correlation between increased immunoglobulin level and mitotic
inhibition existed, a two-step test might be
developed to detect carriers of the disease.
The first step, immunoglobulin assay,
would provide a simple, inexpensive, and
relatively rapid screening test which would
detect the approximately 5% of donors
who have increased IgA, IgG, or IgM. The
second, and perhaps more specific, test
would be analysis of the effect of these
sera on normal leukocytic mitosis.
SERA
Method and Materials
A modification of the method of Moorhead and co-workers8 for culturing leukocytes and preparing chromosomes was used.
The cultures were prepared with blood obtained from Red Cross staff members who
had not had clinically-detectable hepatitis.
The test serum (0.1 ml.) was added to the
medium, the system was incubated for three
days, cultures were harvested, and slides
were made. Every metaphase figure on the
slide was counted and karyotyped visually.
A serum specimen was considered to have
suppressed mitosis when there were five or
fewer metaphase figures on the slide, compared with 30 to 100 for the controls. Each
Received May 26, 1969; accepted for publication
August 22, 1969.
Supported in part by Research Grant HE-10225
from the National Institutes of Health, Public
Health Service.
141
142
Vol. 53
MELLA AND TASWELL
TABLE 1. Results with Donors Implicated after
Single-unit Transfusions
Donor
Interval
after
Implicated
Donation
Immunoglobulin
Level
Mitotic
Suppression
(Mo.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
48
48
72
3
24
48
12
1
1
4
4
20
3
4
Normal
Normal
Increased
Increased
Increased
Increased
Increased
Increased
Normal
Increased
Increased
+
+,+
+,+
+
+
+
+
+
* From time of recipient's transfusion to date a new
blood sample was taken from the donor.
specimen of serum was tested in this manner on three separate occasions. Controls
of whole blood without test serum added
were prepared with every experiment. The
leukocytes of the suspected carriers were
not cultured. Immunoglobulin assay was
performed by the method of Fahey and
McKelvey.2
A blind study of 42 coded specimens of
sera from the Mayo Clinic was done. Fifteen of these sera were obtained from blood
donors who had been implicated in the
transmission of hepatitis either on more
than one occasion or after a single-unit
transfusion. The other 27 sera were from
a variety of control donors and patients.
Twenty specimens of serum collected for
the Red Cross Blood Program by cooperating blood banks were examined in the
same way. Immunoglobulin assay of eight
of these 20 samples had been done; 3 (nos.
3, 5, and 6 in Table 1) were duplicates of
those sent from the Mayo Clinic. This was
not recognized until the latter samples were
decoded. Twenty-four specimens of serum
were either duplicates or serial samples
from 12 patients or donors.
A blood donor was considered to be a
hepatitis carrier when the transfusion recipient was thought to have clinical evidence of hepatitis on the basis of increased
serum transaminase and bilirubin levels 15
to 180 days after receiving a unit of blood.
Results
Table 1 summarizes the results of the
tests on sera from 14 donors implicated as
hepatitis carriers after single-unit transfusions. Of the 18 specimens tested, 12 suppressed mitosis. Results with duplicate
samples (donors 3, 5, and 13) agreed, except for an unexplained discrepancy with
donor 6. Sera from ten of the 14 implicated
TABLE 2. Results with Donors Implicated
More Than Once
Inter- Numval after ber
Impliof
cated Times
Dona- ImpliDonor
tion
cated
15
16
17
18
(Mo.)
30
1
1
10
?
1
Immunoglobulin
Level
Mitotic
Suppression
+
+
Normal
Increased
Normal
Increased
Increased
Increased
Increased
+
+
+
T A B L E 3 . Results with Patients Who
Infectious
Hepatitis
Patient
A
B
C
D
E
F
G
H
Interval*
Immunoglobulin
Level
(Days)
5
2
+ 1
+ 1
+ 4
- 15
-f- 48
+270
+ 3
- 70
+ 1
+ 6
Normal
Increased
Increased
Increased
Increased
Normal
Increased
Increased
Increased
Normal
Increased
Increased
Had
Mitotic
Suppression
+
+
+
—
+,+
+
—
—
—
+
+
+
* Interval between the appearance of icterus and the
withdrawal of the specimen; minus indicates that
sample was taken before icterus developed.
February
1970
HEPATITIS-INDUCED MITOTIC INHIBITION
donors inhibited mitosis. Of the 11 implicated donors whose serum immunoglobulin
levels were known, eight had increased levels. Sera from six of the 11 donors had
both increased immunoglobulin levels and
the ability to suppress mitosis.
Table 2 summarizes the results of tests
on eight serum samples taken from five
donors implicated as hepatitis virus carriers
after multiple-transfusion episodes. Suppression of mitosis was found with all but
one of these specimens, and, in this one
case (donor 18), two other specimens of
serum drawn at different times did cause
mitotic suppression. All three specimens of
serum from this donor had greatly increased levels of immunoglobulin, as did
the sera of two others of these five donors.
Thus, sera from 15 (79%) of the 19 donors
implicated in the transmission of hepatitis
suppressed mitosis.
Table 3 depicts the results of tests of sera
taken from eight patients at various stages
of infectious hepatitis. Suppression of mitosis was found in the first few weeks of the
disease only. Four specimens (from patients
A, D, and G) which suppressed mitosis
were drawn between two and 70 days before icterus developed and, with one exception, these had normal immunoglobulin
levels. Two of the ten specimens (from
patients B and F) which were drawn prior
to or during the first week of the acute
disease did not suppress mitosis. The other
two specimens (from patients D and E)
which did not suppress mitosis were drawn
seven weeks to nine months after the onset
of the symptoms. All specimens had increased immunoglobulin levels except those
drawn five days or more prior to the onset
of symptoms.
Table 4 lists the results of the studies
of 23 serum specimens used as controls.
Four specimens caused suppression of mitosis. They had been drawn from donors
who had given numerous blood donations,
none of which has been reported to have
143
TABLE 4. Results with Control Donors
Immunoglobulin
Level
Source
Normal
Normal
Normal
Normal
Cirrhosis
Infectious mononucleosis
Pulmonary nodule
Infectious mononucleosis
Hydronephrosis
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Increased
Increased
Increased
Increased
Increased
—
Normal
Normal
Increased
Normal
Normal
Normal
Normal
—
—
—
—
—
—
—
—
Mitotic
Suppression
—
+
+
—
—
—
—
—
+
+
—
—
—
—
—
—
—
—
—
—
produced hepatitis in the recipients. Three
of these were from donors deliberately selected because they had increased immunoglobulin levels.
Discussion
Although mitotic suppression has in the
past been related to viral infections other
than hepatitis, the exact cause of the suppression and why it is not always found
have never been adequately explained. 7
Other observers have thought that the suppression is due to a viral effect on the DNA
of the cell or incorporation of the virus
into the cell, leading to abnormal replication.9
The suppression of mitosis found in the
acute stages of infectious hepatitis is consistent with the results of previous serial
studies.5 After the first weeks of the disease,
the patients' sera have no effect on normal
leukocytes; however, their own leukocytes
have been shown to have aberrant chromosomal patterns for the following seven
144
Vol. 53
MELLA AND TASWELL
months of convalescence.4 Studies of human
volunteers have demonstrated a viremia
during the late preicteric and early icteric
stages of infectious hepatitis. 3 In contrast
to the serum effect on mitosis, increased
serum immunoglobulin levels were noted
in late as well as acute stages of the disease,
but not prior to the onset of symptoms. In
a wide variety of diseases, immunoglobulin
levels may remain increased for a prolonged
period after acute infection and after the
patient no longer harbors the infective
agent. In contrast, mitotic inhibition has
been found only when an acute viremia
was thought to exist.
Three specimens of serum were taken at
different times from the donor who had
been implicated as a hepatitis carrier on
five separate occasions. Because one specimen did not suppress mitosis and two did,
the factor causing this phenomenon may
be transient. Most of the specimens of
serum from the suspect donors were drawn
three months to six years after their blood
had been implicated in cases of post-transfusion hepatitis. A transient viremia could
have been missed, accounting for lack of
suppression of mitosis by six of the 21
specimens tested. Mitotic suppression could
be caused by viremia while the increase in
immunoglobulin could be a later immune
reaction to the viremia, which would explain the results of this study. A toxic factor transiently present in the sera of chronic
carriers could be responsible for mitotic
suppression. Although many of the samples
in this study had increased immunoglobulin levels, it is not likely that this alone is
the toxic factor which causes suppression
of mitosis.
Determination of the serum immunoglobulin level is a simple, inexpensive,
rapid test, readily adaptable to mass screening. T h e occurrence of mitotic suppression
by the sera of nine of 11 hepatitis-impli-
cated donors with increased immunoglobulin levels provides further evidence of the
value of determining these levels to screen
blood donors.
The test for mitotic suppression is tedious. T o use it for mass screening would be
impossible at this time. Attempts are being
made to determine the exact cause of the
phenomenon and to simplify the technics
of determining its presence. If this is accomplished, a prospective study using both
methods will be made.
Acknowledgment.
Some of the blood samples
used in this study were supplied by the Central
Blood Bank of Pittsburgh and the Atlanta Regional Red Cross Blood Center.
References
1. Bevan, G., Taswell, H. F., and Gleich, G. J.:
Serum immunoglobulin levels in blood donors implicated in transmission of hepatitis.
JAMA.
203: 38-iO, 1968.
2. Fahey, J. L., and McKelvey, E. M.: Quantitative
determination of serum immunoglobulins in
antibody-agar plates. / . Immunol. 94: 84-90,
1965.
3. Krugman, S., and Ward, R.: Viral hepatitis, infectious hepatitis, serum hepatitis. In Infectious
Diseases of Children. Ed. 3. St. Louis, C. V.
Mosby Company, 1964, pp. 102-118.
4. Mella, B.: Chromosomal damage associated with
infectious hepatitis and its possible teratogenic
significance. Neurology (Minneap.) 18: 741-744,
1968.
5. Mella, B„ and Lang, D. J.: Chromosomal aberrations and suppression of leukocyte mitosis induced in vitro by a circulating factor associated with acute infectious hepatitis. Presented at the New York Academy of Science,
November, 1966. Ann. N. Y. Acad. Sci. 155,
article 3.
6. Mella, B„ and Lang, D. J.: Leukocyte mitosis:
Suppression in vitro associated with acute infectious hepatitis. Science 155: 80-81, 1967.
7. Montgomery, J. R., South, M. A., Rawls, W. E.,
Melnick, J. L., Olson, G. B., Dent, P. B., and
Good, R. A.: Viral inhibition of lymphocyte
response to phytohemagglutinin. Science 157:
1068-1070, 1967.
8. Moorhead, P. S., Nowell, P. C , Mellman, W. J.,
Ballips, D. M., and Hungerford, D. A.: Chromosome preparations of leukocytes cultured
from human peripheral blood. Exp. Cell Res.
20: 613-616, 1960.
9. Nichols, W. W.: Studies on the role of viruses in
somatic mutation. Hereditas (Lund) 55: 1-27,
1966.