I. The
Fibrinolysin
System
in
Pathogenesis
and
Therapy
Clara M. Ambrus, M.D., Ph.D., David H. Weintraub, M.D., Donal Dunphy, M.D.,
John E. Dowd, Ph.D., John W. Pickren, M.D., Kenneth R. Niswander, M.D.,
and Julian L. Ambrus, M.D., Ph.D.
Children’s Hospital of Buffalo, Roswell Park Memorial Institute, Buffalo General Hospital, and University of Buffalo, Buffalo, New York
(Submitted July 30, accepted for publication December 3. 1962.)
Supported by grants from the National Institutes of Health, U. S. Public Health Service, and the
American Heart Association.
Preliminary Report: C. M. Ambrus et a!: Proc. Soc. Pediat. Res. No. 268, p. 182, 1961.
ADDRESS: (C.M.A.) Roswell Park Memorial Institute, 666 Elm Street, Buffalo 3, New York. Present
Address: (D.D.) Department of Pediatrics, University of Iowa.
10
PEDIATRICS, July 1963
T
HE CLINICAL PICTURE of the respiratorydistress syndrome (RDS) can be
corre-lated in the majority of cases at autopsy
with the histologic picture of pulmonary
hyaline membrane disease (HMD).1 This
disease in recent years has come to the
forefront of pediatric interest. It is
con-sidered to be tile most common single
causes of neonatal death.2 It is most
prev-alent in premature infants and in infants
of diabetic mothers.31’ Positive correlation
has been reported between the incidence
of HMD in prematures and bleeding of the
mother during pregnancy, particularly if
bleeding was due to placenta previa.3
Cesarean section seems to increase the
in-cidence of HMD;6’12 it has been suggested,
however, that this is not related to the
op-erative procedure but to the maternal
bleed-ing episode necessitating cesarean
sec-tion.3’ 13
HMD has been recently reviewed by
Driscoll and Smith,14 Smith,15 and Gregg
and Bernstein.16 Abnormalities reported in
the circulatory system, pulmonary function,
and metabolism of these infants seem to be
interrelated. The sequence of appearance
of these abnormalities however is not clear;
thus their relative importance in the
etiol-ogy of HMD can not be definitely
estab-lished.
The basic matrix of the hyahine
mem-brane (HM) material itself has been
identi-fied as fibrin, with the aid of localization of
fluorescent anti-fibrin antibodies by Gitlin
and Craig17 and with electron microscopy
by Van Breeman.18 Lieberman suggested
that the abnormal deposition of fibrin in
the alveoli may be the result of some
de-ficiency of the fibrinolytic system in infants
with HMD. He reported first the lack of
a plasminogen-activator in the lung of
in-fants with HMD”’ 20; later he modified his
concept and attributed the lack of activator
activity to the presence of an
activator-inhibitor.21’22 Phillips and Skrodelis,23 Quie
and Wannamaker,24 and Samartzis et al.25,26
have analyzed tile fibrinolysin system in
the blood of infants. All three groups of
in-vestigators found decreased plasminogen
levels in premature infants. A few infants
with HMD23-2#{176} had particularly low levels.
A simplified schematic representation of the interrelationship between the different
members of tile fibrinolysin system is shown
in Figure 1. This system has been described
in more detail elsewhere.2 It is evident
that in vivo development of fibrinolytic
ac-tivity depends on both plasminogen and
plasminogen activator. If a derangement or
deficiency in the fibrinolysin system is
in-deed involved in persistence of an alveolar
fibrin membrane, it would be logical to
treat affected infants with fibrinolytic
en-zymes. The enzyme of choice, however,
will depend on the nature of the deficiency.
Craig, Fenton, and Gitlin28 found that
ACTIVATORS
FROM
TISSUES
OR
BACTERIA
PLASMINOGEN
FIBRIN CLOT
4I
z w
0
a.
4)
-I
a.
-j
0 z
4%O
PLASMIN
I
ANTIPLASMINS
PROTEIN FRAGMENTS
C0
OTHER PLASMA PROTEINS
(e.g. FIBRINOGEN
PROTH ROMB IN
FACTOR V
FACTOR VII)
FIG. 1. Simplified scheme of the fibrinolytic system.
PROTEIN FRAGMENTS
Blood from infants in respiratory distress
of HMD were perfused with the fibrinolytic
enzyme plasmin, dissolution and
fragmenta-tion of the membrane became evident. It
is possible of course that under clinical
con-ditions dissolution of the HM would not
alter the course of the disease, since the
underlying factors or other secondary
ef-fects may remain. On the other hand,
resto-ration of adequate gas exchange may break
the vicious cycle of pathological processes
and enable the infant to overcome the
dis-ease.
In the past years our group has studied
the fibrinolysin system and gained
experi-ence in the clinical use of different types
of fibrinolytic enzymes (plasmins) in a
variety of thromboembolic conditions.23
While our experience was primarily with
adults, the lack of toxicity of appropriate
dosage schedules of purified plasmin
prepa-rations encouraged us to initiate a clinical
trial of plasmin in infants with HMD.
The present report includes two phases
of our study. In the first, fibrinolytic
fac-tors have been analyzed in the blood and
lungs of premature infants with and
with-out HMD using improved methods
de-veloped in our laboratory.3’34 It was hoped
that these studies would enable us to make
a proper choice among the available
fibrin-olytic agents. Our group found353 that,
depending on the process employed to
ac-tivate human plasminogen (using
strepto-kinase (SK), urokinase (UK), “spontaneous”
activation procedure, etc.) plasmin
prepa-rations of different biochemical
character-istics can be produced. Depending on the
status of the patient, different types of
preparations may be indicated. The second
part of this study is a preliminary report
on the plasmin treatment of infants with
RDS. Many problems encountered in the
process of the clinical trial and encouraging
initial results seem to make worthwhile
a report at this early stage.
MATERIALS
AND
METHODS
was taken from the umbilical vein
immedi-ately before starting and after completing
infusions of plasmin or placebo. Blood from
healthy infants was obtained at birth. Nine
milliliters of blood was drawn into a
silicon-ized syringe containing 1 ml 3.8% sterile
sodium citrate solution, using hemorepellent
needles. The sample was immediately
cooled and centrifuged in a refrigerated
centrifuge. The following factors were
de-termined: plasmin, plasminogen,
plasmino-gen-activator, and antiplasmin. Plasmin was
determined in plasma; the other
compo-nents were tested after preparing serum
with the aid of purified thrombin. In
addi-tion, plasmin, plasminogen, and activator
activity were analyzed in the euglobulin
fraction of plasma. Plasmin was measured
by its ability to lyse human fibrin clots made
with purified (plasminogen free) human
fibrinogen and purified human thrombin.
Both reagents were prepared by the
meth-od of link and McDonald.38 The time
required for lysis of the clot is converted
to units with the aid of a standard curve.
One RPMI (Roswell Park Memorial
In-stitute) unit of plasmin was defined as
the amount required to lyse in 2 minutes
0.6 ml of a 0.3% purified human fibrin clot
made with 1 NIH unit of purified human
thrombin at pH 7.2 and 45#{176}C.Plasminogen
was measured after being activated to
plas-mm by optimal concentrations of SK;
activa-tor activity was measured by the ability to
convert one unit of plasminogen to plasmin.
Antiplasmin is measured by its ability to
neutralize plasmin activity of spontaneously
activated human plasmin, a plasmin
prepa-ration devoid of activator activity. The final
step in all three determinations is the
meas-urement of the resulting or remaining
plas-mm
activity.These methods and definitions of the
units have been described in detail
pre-viously.31 Our methods differ from methods
used by several authors in the following
main points. (1) All reagents are of human
origin. This eliminates misinterpretations
due to species specificity phenomena. We
have shown previously33 that the various
synthetic substrates measure different
activi-ties of fibrinolytic enzymes and are not
al-ways true indicators of fibrinolytic activity.
(
2) Purified (plasminogen free) reagents areused, thus it is possible to distinguish
be-tween activator and fibrinolytic activities.
(3) Plasmmnogen is assayed by exposing it
to a series of activator concentrations. We
found previously34 that, e.g., increasing
con-centrations of 5K first increase then
de-crease plasmin activity of a given amount of
plasminogen. Assays have to be performed
at optimal 5K concentrations, which will
depend on the amount of plasminogen in
the sample. (4) Antiplasmin is assayed by
its inhibitory effect on a plasmin
prepara-tion which has no activator activity of its
own, thus avoiding errors due to activation
of plasmmnogen in the sample.
Lung samples were obtained at autopsy
under aseptic conditions from infants who
died from HMD and from infants who died
from causes not involving the lungs.
Bac-teriologic cultures were made of lung
sam-ples. Contaminated samples were not
in-cluded in the evaluation. Samples were
frozen as soon as removed at autopsy and
analyzed for plasminogen activator and
fibrinolytic activity. In Petri dishes of 9-cm
diameter fibrin plates were formed with the
following composition: 8.5 ml of 0.35%
hu-man fibrinogen, 3 NIH units of purified
human thrombin (in 0.3 ml), 0.2 ml 0.22 M
Ca Cl5, and 1 ml antibiotic solution
con-taining 50 mg neomycin and 5 mg
poly-mixin. This antibiotic mixture had neither
lysis inhibitory or lysis enhancing activity
in contrast to some other antibiotics tested.
Inclusion of antibiotics was found
neces-sary in this system since many bacteria
pro-duced proteolytic and plasminogen
activa-tor enzymes which may interfere with the
assay. The final concentration of fibrinogen
was 0.3%.
For the measurement of plasminogen
activator activity, human fibrmnogen
con-taminated with plasmmnogen was used (Plate
phenomenon this contamination was
esti-mated to be 8 units of plasminogen per
plate. When 50X of various concentrations
of UK solutions were pipetted on these
plates, areas of lysis measured 24 hours
after incubation at 37#{176}Cwere proportional
to the amount of UK used. Based on these
experiments a standard curve was
estab-lished. The area of lysis obtained by 10
Ploug U UK3#{176}was defined 1 RPMI unit
plate activator activity (PAA). For the
measurement of fibrinolytic (plasmin)
ac-tivity, plates were prepared from purffied
human fibrinogen (Plate F). UK in a variety
of concentrations (0.01-150 Ploug U per
50)) did not produce lysis in 24 hours at
37#{176}C,however, these same plates readily
liquified upon addition of plasmin. One
RPMI unit plate plasmin activity (PPA) was
defined as the amount producing an area
of lysis equal to that produced by 0.1 RPMI
unit of spontaneously activated human
plasmin in a volume of 50?.
Parallel assay on these two types of
plates distinguishes between fibrinolytic
activity and activator activity. About 3-gm
pieces of lung tissue were homogenized
with the addition of 9 ml saline solution,
using a motor-driven Teflon pestle in a
tightly fitting glass tube under
refrigera-tion. Connective tissue components
re-mained at the bottom of the tube. The
homogenized portion which collected above
the pestle was decanted and used for the
assay. Half milliliter homogenate was dried
in an oven and weighed. All manipulations
were performed under aseptic conditions.
Samples of the homogenates were
inocu-lated into culture broth for detection of
possible contamination. Fifty lambda
por-tions of the homogenates were placed with
a lambda pipette on the surface of plates
F and F-P (both in triplicate). Lysis zones
were measured after 24 hours of incubation
at 37#{176}C.The mean lysis areas were
con-verted into units and expressed as units per
gram of dry weight of the homogenate.
Our methods differ from those used by
several authors in the following: (1)
Au-topsies are performed as soon after death
as possible; lung samples are removed under
aseptic conditions and immediately frozen.
(
2) Tissue homogenates are used, thusavoiding problems arising from release of
enzymes from tissue slices. (3) All reagents
are of human origin. (4) All reagents are
either purified or quantitatively assayed for
plasminogen contamination. (5) Antibiotics
which do not effect the fibrinolysin system
are incorporated into the assay plates.
Clinical Studies
Infants with respiratory distress syndrome
were included in the study only if they
be-longed into any of the following categories:
(a) premature infants, (b) infants of diabetic
mothers, (c) mature infants born by
cesar-ean section if indication for section was
bleeding. According to our previous
experi-ence3 these criteria provide for a very high
incidence of pathologically proven HMD in
the group of infants with respiratory
dis-tress syndrome. Prematurity was considered
on the basis of birth weight and not on
gestational age.
The clinical diagnosis of respiratory
dis-tress rested mainly on the following criteria:
elevated Silverman Score (SS)#{176}and a chest
x-ray picture suggestive of HMD. The
origi-nal SS takes into consideration the
follow-ing typical signs: “see-saw” movement of
upper chest and abdomen, intercostal
re-traction, xyphoid retraction, expiratory
grunt, and chin descent. The maximal score
for each criterion is 2, resulting in a
maxi-mal total score of 10. We have found it
difficult to grade the descent of the chin
and have not included this sign into the
scoring. Accordingly the maximal SS in our
study is 8. More recently4l “chin descent”
has been deleted also by Silverman and
“flaring of alae nasi” has been substituted.
However this substitution was not adopted
by us.
The routine care of premature infants at
the Buffalo Children’s Hospital was
de-scribed in detail previously by two of the
incubator maintained at an environmental
temperature which varies between 88-90#{176}F,
and a relative humidity of 80-95%. Oxygen
is used only when cyanosis is evident, never
in higher concentration than 38%. Feedings
are omitted at least for the first 24 hours
of life. Antibiotics are used when there is
a history of premature rupture of
mem-branes or if the infant shows signs of
in-fection.
Infants in this study received treatment
as follows. As soon as the diagnosis was
established, 5 RPMI U/kg plasmin (or
placebo) in 50 ml 5% dextrose in water was
infused through a catheter into the
umbili-cal vein in 4 hours. Simultaneously an
aero-so! was started into the Isolette using a
No. 280101-63 National Cylinder Gas
Nebu-lizer (97% of the particles delivered by this
Nebulizer are 3 micron or less in diameter).
The aerosol is introduced into the Isolette
through a 33 inch diameter plastic funnel.
The infants face is turned toward the
fun-nel which is placed at a distance not
greater than 3 inch from the nose and
mouth of the patient. No bottled gas is
used to power the Nebulizer, but an
elec-tric pump. The pressure and airflow
de-livery of the pump is adjusted for each
individual Nebulizer to result in the
aerosol-ization of 12 ml plasmin or placebo solution
per hour. The concentration of the aerosol
solution was adjusted to deliver 60 RPMI
U/kg plasmin (or placebo) in 24 hours.
Aerosolization was continued until the
in-fant either recovered or died.
On the basis of a randomization schedule
the material for treatment was either
SK-plasmin, (commercial Thrombolysin
reas-sayed in RPMI units), UK-plasmin
(pre-pared by our group or supplied by Parke
Davis & Co. and assayed in RPMI units) or
placebo, consisting of 5% dextrose in water
with a few drops of human albumin to
simulate the color and frothing ability of
plasmin. The group selecting and evaluating
the infants did not know whether placebo
or one of the plasmin preparations was
em-ployed. As mentioned before, immediately
before inserting the catheter into the
umbili-cal vein and immediately after removing it,
blood samples were taken for analytical
studies. The SS and the infant’s general
condition was recorded every 4 hours
throughout the course of treatment.
Analytical Studies
RESULTS
The first tables show levels of members
of the fibrinolysin system found in the blood
of mature healthy infants (Table I), healthy
infants from diabetic mothers (Table II),
and healthy premature infants (Table III).
Normal adult values given as comparison
are based on our previous experience. Table
IV shows fibrinolytic enzyme levels
meas-ured in the blood of 34 infants with
respira-tory distress. Of 20 infants who died in
this group, the diagnosis of HMD was
con-firmed by histopathology in 16. The
aver-age SS before treatment at tile time of
sampling was 6.3.
Measurable plasmin activity and
plasmin-ogen-activator activity was not found in
TABLE I
COMPONENTS OF THE FIBRINOLYSIN SYSTEM IN TIlE BLOOD OF MATURE, NORMAL NEWBORN
INFANTS U’eight (kg) Piamin’ U/mi) PEu . Piaminogen (RPM! U/mi) S Eu Activator’ U/mi) S Eu Anti-. 3 u,’mi S 2.66 2.67 2.87 2.92 2.91 3 3.11 3.3 3.57 3.6 3.67 3.84 3.85 4.2 >2.5 >2.5 Mean SE. 0 ‘1’ 0 0 0 T 0 0 0 0 T 0 0 0 0 0 ‘1’ 0 T 0 T 0 T 0 0 0 T 0 0 0 0 .. .. 0.27 3.4 0 1.5 0.73 2.8 0.81 4.9 0.65 4.4 0.39 1.8 0.63 3.5 0.65 3.3 0.95 4.6 0.11 5.0 0.61 3.5 0.28 3.0 0.8 4.2 0 2.5 0.31 1.9 0.37 3.3 0.47 3.48 ±0.07 ±0.14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 T 0 0 0 0 0 0 6.9 8.6 11.4 7.1 7.1 6.1 8.9 6.3 8.3 7.6 11.1 5.8 9.5 7.0 6.0 6.7 7.79 ±0.44 Average
adultvalues 0 0 3-15 5-10 0 0 5-13
P=plasma; S =serum; Eu =euglobulin fraction of plaazn&;
TABLE III
COMPONENTS OF TILE FIBRINOLYSIN SYSTEM IN THE BLOOD OF hEALTHY PREMATURE INFANTS
Piasmin’ (RPM! U/mi) P Eu Pia.minogcn’ (RPM! U/mi) S Eu 0 Weight (kg) 1.4 1.5 1.6 1.9 1.1 2.1 <1.5 <1.5 <2.5 Mean SE. .4ctirator’ (RPM I U/mi) S Eu 0 0 0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 T 0 0 0 0 0 0 0 0 Inti-plasinin’ (RPM! U/mi) S 11.1 5.6 12.5 11.5 8.2 10.5 40.7 51 6.4 17.6! ±5.46 o 2.1
0 0 1.7
o o 1.3
0 0 1.9
T 0 2
0 0 2.5
0 0 7.4
3.19
±0.76
* P=plasnia; S=serurn; Eu =euglobulin frncton of plasma; T =trace (<0.01 U/mi).
TABLE II
COMPONENTS OF THE FIBJIINOLYSIN SYSTEM IN TIlE BLOOD OF HEALTHY NEWBORN INFANTS OF
DIABETIC MOTHERS l’ia.,minogen * (RPM! U/mi) .Iet,rntor’ (RPM I U/mi) JJ’eight (kg) 2.9 1.94 4.18 4.3 Mean SE. Piasrnin’ (RPM I U/mi)
P Eu S
0 0!0
0 0 1.1
0 0 0
0 0 0.76
0 T 0
0.87 ±0.11 Eu 4.6 1.7 1.18 4.1 1.1 2.9.5 ± 0.64 mu-pins snin’ (RPM! U/mi) 9.5 3.4 6.1 6.8 1.1 5.48 ±1.28 S 0 0 0 0 0 Eu 0 0 ‘I’ 0 T
* P=plasma; S serum; Eu =euglohulin fraction of plasma;
T=triue (<0.01 Unil).
the plasma or serum of infants, but
occa-sionally traces could be demonstrated in
the euglobulin fraction of plasma. Activity
expressed as “trace” corresponds to a lysis
time of 18-24 hours, which cannot be
quantatively expressed in our assay system
(2 minute lysis time corresponds to one
unit and 3 hours lysis time to 0.O1U). Trace
plasmin activity was found in half of the
mature, healthy infants. If all healthy
new-born infants are grouped together, 37% show
trace plasmin activity as compared to 18% of
the infants with HMD. Trace activator
activ-ity on the other hand was found in more
in-fants with HMD (25%) than in healthy ones
(13%). For both activities the difference
be-tween healthy and diseased infants is
sig-nificant at the 1% level (p = 0.01).
Plasminogen could not be demonstrated
in the serum of premature infants, whether
healthy or suffering from respiratory
dis-tress. In mature infants, serum plasminogen
levels were low and considerably below the
adult range.
Not considering the offspring of diabetic
mothers, the plasminogen level of the
eu-globulin fraction of plasma ranks as follows:
highest in the mature infants, second
high-est in the healthy prematures and lowest
in the prematures with RDS. Tile difference
was not significant between the mean levels
of the healthy and diseased prematures,
however, the two groups combined had a
signfficantly lower level of
euglobulin-plasminogen than mature infants (p <0.05).
Two of the healthy premature infants had
exceptionally high levels of
euglobulin-plasminogen (twice as high as the mean
level of the others).
In the few infants in the study who were
born to diabetic mothers, plasminogen
values showed considerable variation, and
did not differ significantly from any of the
other groups.
The level of antiplasmin in both healthy
and diseased premature infants was higher
than in mature newborns. The difference
of the mean levels was not statistically
sig-nificant because of the large variation in
tile premature groups. However,
exception-ally high levels of antiplasmin (more than
three times the mean) have been found in
22% of the healthy prematures and 25% of
the prematures with RDS, but in none of
the mature infants. No correlation between
high antiplasmin levels and low
plasmino-gen levels could be estabished. In infants
showing RDS (Table IV) fibrinolytic factors
did not differ whether the infants recovered
or died, and whether HMD was confirmed
at autopsy or not.
Table V shows activator activity in the
Weight
(kg)
Plasmin* pla.s,nj,wgen* .lctirator* A
nh-(RPM! U/mi) (11PM! U/mi) (RPMI U/mi) pia.smin* Oufroine
-
(RPM!.#{176}-P Eu S Eu S Eu U/mi) Duseasef
Nondiabehic Mothers !Iivto pathology of lungs 1.12 1.2 1 .69 2.05 2.1 2.18 2.2 2.3 2.5 2.5 2.7 3.1 1.14 1 .4 1 .57 1.6 1.77 1.77 1.8 1.88 1.9 1.9 2 2 ‘2.3 2.3 2.3 1 .08 2.0 2.14 Meati SE. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 T 0 0 0 0 T 0 0 0 0 0 0 0 0 0 T 0 0 0 T 0 0 0 0 0 0 T 0 T T 0 0 0 0 0 0 0 0 0 U 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.07 1.0 1.3 2.1 1.5 0.9 1.2 2.2 0 2.9 1.1 4.4 2.5 0.6 1.43 1.6 0.4 0.8 4.7 1.1 0.6 2.7 0.2 1.5 0 1.5 1.5 3 1 .56 1.63 0 0.9 1.50 0.20
T 3.7 R
T 4.5 R
0 8.3 R
0 4.8 R
T 7.0 R
0 6 R
0 4.2 R
0 27.3 R
0 28.8 R
0 9.7 R
0 4.8 R
0 48.3 R
0 3.6 D
0 5.0 D
0 6.5 D
T 8.0 D
0 4.8 D
0 9.5 D
0 24.6 D
0 5.1 D
T 1.8 D
0 29.0 D
T 1.1 D
0 7.7 D
0 50.0 D
0 8.5 D
0 4.1 D
T 32.1 D
T 5 D
0 5.4 D
0 24.0 D
0 2.4 12.36 2.27 D hIM HM HM HM H1i HM HM HM HM HM hIM hIM HM HM HM hIM no HM no hIM no HM no HM TABLE IV
COMPONENTS OF THE FIBRINOLYS1N SYSTEM IN BLOOD OF INFANTS WITh RESPIRATORY DISTRESS SYNDROME
Diabetic Mother8
0 0
‘3.3 0 0
0 0 0 0 0 T 0 49.7 4.8 R D HM
* P=plasma; S=serum; Eu*’eug1obu1in fraction of plasma; T=trace (<0.01 U/mI); R=recovery; D=death;
hIM =hyaline membrane by histopathology.
t Average Silverman Score of the infants who recovered was 6.3 at the time of sampling.
the first 48 hours of life. “Normal” samples
originated from infants who died from
causes not involving the lungs. Samples
with the histopathological picture of HMD
included both untreated and placebo
treated infants. Lungs from infants who
were treated with plasmin before death
are not considered here, since the
adminis-tered plasmin had both fibrinolytic and
quan-TABLE V
PLASMINOGEN ACTIVATOR ACTIVITY IN THE LUNGS OF
NEWBORN PREMATURE INFANTS
4-5
5-12
> 12
4 S 5
4 S
tities could interfere with proper
interpreta-tion of the findings.
Both normal and diseased lungs show
considerable activator activity by the assay
technique employed in this study, statistical
evaluation of the data indicated no
signifi-cant difference between the two groups.
Significant difference is found, however,
if one compares activator activity of lungs
from autopsies performed in less than 10
hours after death with that of lungs from
autopsies performed in more than 10 hours
after death (p < 0.005). None of the lungs
in these groups showed fibrinolytic activity
when assayed on plasminogen free human
fibrin plates, indicating the absence of
di-rect proteolytic or fibrinolytic activity.
Au-topsy was performed in most of the infants
within a few hours after death. If death
occurred at night, or on the weekend, or
if difficulties arose in obtaining parental
permission, autopsy was delayed until the
next day. The longest time interval between
death and autopsy in the first group was
9.5 hours (in one case), therefore 10 hours
was used as an arbitary time limit to
sepa-rate “early” and “late” autopsies.
Clinical Results
Before the randomized, double-blind
study was started, plasmin was
adminis-tered to 12 infants with severe respiratory
distress, after considerable period of
ob-servation, in several instances shortly
be-fore death. The purpose of this initial study
was to work out the technical problems of
plasmin administration in newborns,
ascer-tain the safety of the contemplated plasmin
dose, and co-ordinate diagnosis, treatment,
and evaluation among the different
mem-bers of the team.
Table VI shows the relationship of length
of therapy to survival rate of the treated
infants. There were no survivors among
in-fants treated for less than 12 hours. Of the
12 infants in this group, 9 died; HMD was
confirmed by histopathology in all 9.
Analy-sis of blood samples taken immediately
be-fore and immediately after the intravenous
.
Hzsto pathology of Lung
Time between Death and Autopsy
<10 hr >10 hr
Normal
(Mean) (S.E.)
687 565
279 226 210
393
±98
457 137 105 48 44
158 ±77
HMD
(Mean) (S.E.)
690 540 350 295 267 263
104
358 ±74
183 110 91 62 44
..
.. 98 ±24
* RPM! plate activator, U/gm dry tissue.
None of the lungs had fibrinolytic activity.
infusion of plasmin showed no or only
minimal changes in clotting factors.
In the randomized, double-blind study
infants were treated as soon as the
diag-nosis was made. A detailed description of
the individual cases treated will be given
in a separate publication (Weintraub et at.:
In preparation). A summary of the results
obtained so far is given in Table VII, with
a few pertinent data of the infants and the
mothers, to permit comparison of the
groups Of 33 infants, 11 received
pla-cebo, 8 received SK-plasmin, and 14
re-ceived UK-plasmin. Recovery rate was 45%
TABLE VI
RELATION OF LENGTH OF THERAPY WITH PLASMIN TO
SURVIVAL OF INFANTS WITH RDS-INITIAL STUDY
Length of Treatment (Fir)
Number
Treated
Number
in the placebo treated group, 25% in the who died, autopsy was performed in 12.
SK-plasmin treated, and 86% in the UK- Two infants in the placebo group and one
plasmin treated group. Of the 14 infants in the SK-plasmin group who died did not
TABLE VII
PLASMIN TREATMENT OF INFANTS WITH RESPIRATORY DISTRESS SYNDROME (RANDOMIZED, DOUBLE-BLIND
STUDY) Treatment . Weight (kg)
ss
X-ray Finding . Delivery Routet Bleeding during Pregnay Maternal Diabetes Out-conu4 Autopsy Data Lungs ICH Placebo (Mean) 2 2.1 2.18 2.44 2.5 1.14 1.4 1.88 ‘2 ‘2 2.14 6 7 8 5 6 6 6 6 7 6 8 65 -+ + -+ + + .. + -V V V V CS V V V V V V + -+ -+ -+ + -R It R R R D I) D 1) D D .. .. .. .. .. JIM JIM HM HM .. .. . .. #{149}#{149} .. + -+ + + #{182} SK-plasmin (Mean) 2.05 2.2 1.6 1.9 2 2 2.2 2.3 5 (; 5 4 6 7 .5 7 5.6 -+ -+ + + + CS V V V V CS CS CS + -+ + -+ + -R R I) D D D D 1) .. .. # IIM .. JIM hIM # .. .. .. -+ -UK-plasmin #{149} (Mean) 1.13 1.2 1.3 1.69 2.15 2.3 2.4 2.5 2.7 2.7 2.7 1.08 1.59 6 6 4 8 5 5 7 7 8 7 4 5 6 4 5.9 + + + + ++
-+ -+ + + ± + V V V V CS CS V CS V CS CS CS V V -+ + + + -+ + -+ -+ + -R R R .. It .. R .. R .. R . R .. R .. R .. It .. It .. D ± I) ± . .. .. .. .. .. .. .. .. .. .. .. + +* Silverman Score at the time treatment was started.
t V= vaginal delivery; CS= cesarean section.
R= recovered; D=died. In placebo group 45% recovered, 55% died; in the SK-plasmin group 25%recovered,
75% died; in the UK-plasmin group 86% recovered, 14% died.
§ ICH = intracranial hemorrhage; HM=hyaline membrane disease confirmed by histopathology (± HM ex-plained in text under Clinical Results).
#{182}Congenital anomaly of the brain.
TABLE VIII
MANIFESTATION OF FIBRINOLYTIc AND ACTIVATOR
ACTIVITY IN THE BLOOD OF INFANTS WIT!! RESPIRATORY
DISTRESS SYNDROME AYER TREATMENT WIT!! PLASMIN (5 RPMI U/KG, iv.; 10 RPMI U/KG, BY
AEROSOl., iN 4 I.IouIts)*
None of these infants exhibited free plasmin or activator activity
before therapy.
Only those cases considered, where JIM was found by histo-pathology.
show pulmonary HM’s; however, extensive
hemorrhage or congenital anomaly of the
brain was found, which could account for
the clinical picture of respiratory distress.
Both infants who died in the UK-P treated
group showed extensive intracranial
hemor-rhage; histology of their lungs showed some
eosinophilic fragments, few hyaline
mem-branes, and a general picture which could
be interpreted either as a beginning phase
of HM formation or as a possible phase of
resolution of HM’s. In Table VII these cases
were considered as ± HM. These cases will
be discussed in detail in the separate
publi-cation previously mentioned.
Table VIII shows plasmin and activator
activity in the blood samples obtained after
4 hours of treatment. Data of infants who
died and showed no HM’s by
histopathol-ogy were excluded from consideration. Of
the 19 infants treated with plasmin
prepa-rations, 10 exhibited circulating free plasmin
and activator activity after completion of
intravenous therapy. No relationship was
found between these factors and the
clini-cal course. The complexity of the
relation-ship between the presence of free plasmin
and thrombolysis has been discussed
else-where.32
COMMENT
Free plasmin activity was demonstrated
by several authors in parturient mothers
and in the cord-blood of newborn
in-fants.2343 If this activity is a result of the
delivery process, it can be expected to
de-crease with time. In our study, samples
were taken at delivery from the healthy
newborns; from the sick infants, however,
samples were taken only when the
syn-drome was fully developed, usually several
hours after birth. The decreased incidence
of fibrinolytic activity in the infants with
HMD may be in part a reflection of the
time delay in sampling.
The increased incidence of activator
ac-tivity in infants with HMD may result from
both exertion and hypoxia in these infants.
Tissue hypoxia has been reported to
pro-Number wit! .Vurnher with Type of Infant.’ (linical Trace or More Trace or More Plasmin Treate4l Result,’ of Plasmm
.4clirily
of .letirator .‘lclirity
SK-plasmin
1 Recovered J)ie.It
0 0
3
UK-plitsmin 1l Recovered a
l)iedf’ I
(I I
duce release of tissue activators.44
If we do not consider the diabetic groups
(where the degree of maturity varies widely)
it seems that the fibrinolytic system in
pre-mature infants shows a characteristic
pat-tern: plasminogen can not be demonstrated
in the serum; in the euglobulin fraction it
is below the level found in mature infants;
antiplasmin levels are generally high.
Plasminogen levels were found to be
rela-tively low in mature newborns also; adult
levels were reached only at 6 to 7 months
of age (data to be reported elsewhere). It
can be assumed, therefore, that the low
plasminogen level in prematures is a
reflec-tion of the developmental stage of the
in-fants. We have no explanation for the high
antiplasmin level found in many
prema-tures. In infants with HMD, the euglobulin
plasminogen level was lower than in
nor-mal, healthy prematures.
The low plasminogen level in newborns
was reported before,232* with the same
rela-tive difference among mature and
prema-ture infants as found by us; absolute levels
can not be compared due to differences in
methodology. These same groups did not
find increased antiplasmin levels; however,
they estimated antiplasmin activity from the
difference in the SK-activated plasminogen
level between serum and euglobulin
we have the choice of
sub-strate and the type of plasmin used for
neutralization greatly influences the results
of antiplasmin assay. When the plasmin
used has activator activity also (as
5K-plasmin does) this may seriously interfere
with the assay. In our study, neutralization
of a human plasmin preparation lacking
activator activity was used as a test
ma-terial and residual plasmin activity was
measured on purified human fibrin.
Analysis of activator activity in lung
homogenates demonstrated comparable
ac-tivities in normal lungs and lungs with
HM-s. Of interest is the finding that the
activator activity is apparently due to a
labile enzyme, thus delay in autopsy may
result in a significantly lower level of
lung-activator activity. Unfortunately the cases
studied are not distributed evenly along the
time scale to draw definite conclusions as
to time decay relationships. An interval
greater than 10 hours between death and
autopsy seems to result, however, in highly
significant decrease in activator activity.
Interpretation of results without knowledge
of the time interval between death and
autopsy may therefore be misleading.
Freezing the sample immediately after
re-moval seems also to be important. The
dif-ference between our results and the
find-ings of LiebermanlO,20 can be due to
sev-eral factors. The most important one is
probably the difference in the substrate
used. Lieberman used bovine fibrin, we
have used purified human fibrin. It was
recently found in our laboratory6’ that
the human activator system is distinct from
that involved in the activation of bovine
plasminogen. The significance of a bovine
activator in an enzyme system of human
ori-gin is as yet unknown. Since human
plasmi-nogen activator activity was found in
abun-dance in the lungs of infants with HMD,
no search for an activator inhibitor was
undertaken. Nevertheless, in the future we
plan to assay for such inhibitors.
The analytical results obtained in our
study do not seem to indicate a qualitative
abnormality of the fibrinolytic system of
infants with HMD compared to normal
pre-matures. The deficiency of plasminogen,
however, may deprive premature infants of
a natural defense against abnormal fibrin
depositions, wherever they may occur and
for whatever reason. Plasminogen
activa-tor activity frequently found in the blood
of infants with HMD did not seem to
in-fluence the course of the disease. This may
be due to the fact that no substrate is
avail-able for activation to a fibrinolytic enzyme.
The two groups who reported recently on
the enzymatic treatment of RDS used
agents primarily with activator activity.
De Los Cobos, Garcia, and Sagaon45 treated
seven infants with HMD with Varidase (a
mixture of streptokinase and
streptodor-nase); four survived, three died. Ebner,
Solomon, and McMillan46 treated four
in-fants with RDS with Actase (SK-plasmin
with high activator activity); three infants
survived, one died. On the basis of results
obtained in the analytical studies our group
felt that the fibrinolytic agent of choice
should be a plasmin preparation primarily
with high fibrinolytic activity; preparations
with activator activity only, or with high
activator and low direct fibrinolytic activity
may be of little value.
The dose of plasmin used in our study
was based on the in vitro fibrinolytic activity
of the preparations. In addition, both
SK-plasmin and UK-plasmin have activator
ac-tivity, which was not taken into account
when determining the dosage. The dose of
plasmin administered intravenously was
constant for each infant on a weight basis.
The aerosolized plasmin, while delivered
at a constant dose into the Isolette, was
probably inhaled at different rates, due to
the different respiratory pattern of the
in-fants. This pattern of course varies even in
the same infant during the course of the
disease. In spite of the inaccuracy of this
method of aerosolization, we have preferred
to deliver the plasmin into the chamber, to
intratracheal insufflation or employment of
a mask; methods which may have serious
risks of their own.
of various thromboembolic conditions in
adults2933 as well as experimental studies
with plasmin29 have shown that there is a
time lapse of several hours between the
in-fusion of plasmin and the first signs of
effectiveness. Results in the initial series of
this study (Table VI) seem to indicate that
the same holds true in the treatment of
HMD. This fact also suggests that cases of
RDS with a fulminating clinical course and
cases with late diagnosis can not be
ex-pected to benefit from plasmin therapy.
The number of cases treated in the
double-blind study is relatively small. On
the basis of weight, diagnostic criteria, and
pertinent maternal factors, the placebo and
plasmin treated groups seem to be
compara-ble, except for two infants of diabetic
moth-ers who both happen to fall in the UK-P
treated group. In the placebo treated group
the survival rate as well as the number of
infants with pathological diagnosis agrees
with data reported in the literature.1
In the control group 5 of 11 infants (45%)
recovered, in the UK-plasmin treated group
12 of 14 infants (86%) recovered. Because
of the small number of patients in both
groups, this difference is not statistically
significant (p = 0.14). If birth weight is
taken into account then the difference
be-tween the placebo and the UK-plasmin
series becomes statistically significant at
the 1% level (Appendix A). If the SK-plasmin
treated group, which resembles the placebo
group in its response, is grouped with the
placebo group, the difference in recovery
rate (86% versus 37%) is significant at the
5% level. The three infants of the four who
were over the premature weight limit
hap-pened to be in the UK-plasmin treated
group. All three were born by cesarean
sec-tion; in one, indication was placenta previa,
in two, maternal diabetes. All showed a
pulmonary x-ray picture compatible with
HMD. The UK-plasmin treated group is
otherwise comparable to the placebo and
SK-plasmin treated groups. Elimination of
the three cases mentioned above reduces the
recovery rate of the UK-plasmin treated
group only slightly (from 86 to 82%). It
should be mentioned, however, that among
the 11 patients weighing less than 2 kg
re-covery occurred only in four of the six
UK-plasmin treated infants. All three in the
placebo group and both in the SK-plasmin
group died. For further discussion see
Appendix A.
The effectiveness of UK-plasmin in
con-trast to SK-plasmin was rather surprising;
in peripheral thrombophlebitis such a
dif-ference was not observed by us.2831 In
HMD of prematures with low plasminogen
and high antiplasmin levels the superiority
of UK-plasmin may be based on its
speci-ficity toward fibrin in the presence of
anti-plasmin. This specificity was reported
pre-viously to be higher for UK-plasmin than
for SK-plasmin.34
Survey of the autopsy cases at tile Buffalo
Children’s Hospital in recent years (but
be-fore initiation of this study) has shown that
in a group of 58 infants with HMD, 39 (67%)
had associated cerebral hemorrhage, and
31 (53%) had pulmonary or visceral
hemor-rhage. This frequent association of
intra-cranial hemorrhage with HMD is
men-tioned in several publications.’ Among
the cases that came to autopsy in this
study, the incidence of intracranial
hemor-rhage was about the same in the plasmin
and in the placebo treated groups. No
cor-relation was found between circulating
fibrinolytic and activator activity on one
hand and occurrence of hemorrhage on the
other hand. Of course no information is
available on hemorrhage without clinical
manifestations in the patients who
recov-ered. On the basis of available clinical and
autopsy data we assume that plasmin was
given by a safe dosage schedule.
In many of the infants with HMD,
hemor-rhage was probably a consequence of
pro-longed hypoxia due to the respiratory
dis-tress; in a certain number, however,
cere-bral hemorrhage might have been the cause
of respiratory distress. Careful clinical
eval-uation of the infant from birth may help
to distinguish between primary cerebral
damage and HMD in a certain number of
picture will not be clear enough to permit
such differentiation. Decision may rest in
many instances on the consideration of
as-sociated “predisposing factors” as known
from statistical studies. Should fibrinolytic
therapy prove effective in HMD,
knowl-edge of the statistical incidence of
hemor-rhage associated with the clinical picture
of respiratory distress should make one
aware of the limitation of such therapy.
On the basis of the preliminary results
we feel encouraged to continue this study.
It is possible that dosage could he
de-creased and administration simplified to
aerosolized plasmin alone; we feel,
how-ever, that these possibilities should be
ex-plored only if effectiveness of UK-plasmin
can be proven under uniform conditions on
a larger case material.
SUMMARY
In the serum of normal prematures and
premature infants with respiratory distress
syndrome, plasminogen was absent. In
ma-ture newborns plasminogen levels were low,
as compared to adults. In the euglobulin
fraction of plasma, plasminogen level was
highest in mature newborns, lower in
healthy prematures, and lowest in
prema-tures with respiratory distress syndrome.
Antiplasmin level was exceptionally high in
about a fourth of the premature infants with
or without respiratory distress syndrome.
Plasminogen activator activity was found
more often in the blood of infants with
respiratory distress syndrome than in
nor-mal infants. This may be due to the
libera-tion of tissue activators as a consequence of
hypoxia. Because of the absence of the
substrate (plasminogen), this activator level
may have no significance. Tissue activator
activity was found in the lungs of
prema-ture infants whether they died of hyaline
membrane disease or from other causes.
Forty-five infants with respiratory distress
were treated in a therapeutic study. Twelve
were treated in a preliminary series and 33
in a randomizd, double-blind investigation.
Of the latter, 11 were treated with placebo,
and 5 (45%) survived; 8 were treated with
streptokinase activated human plasmin and
2 (25%) survived; 14 were treated with
urokinase activated human plasmin and 12
(86%) survived. Among the infants who died,
no definite hyaline membrane disease was
found by histopathologic examination in
two of the placebo group, one in the
strep-tokinase-plasmin treated group, and the two
who died in the urokinase-plasmin group.
No significant side-effects of plasmin
ther-apy were seen. Although considerable
fibrinolytic and plasminogen-activator
ac-tivity was generated in many treated
pa-tients, there was no significant fall in blood
coagulation factors. Intracerebral
hemor-rhage, which appears to occur often in
pa-tients who die with hyaline membrane
dis-ease, was not more frequent in the
plasmin-treated group than in the placebo group.
REFERENCES
1. Bauman, W. A.: The respiratory distress
syn-drome and its significance in premature
infants. PErnAmics, 24:194, 1959.
2. Levine, S. Z., et al.: Respiratory difficulties of
newborn infants. New York J. Med., 58:
372, 1958.
3. Cohen, M. M., Weintraub, D. H., and Liien-feld, A. M.: The relationship of pulmonary hyaline membrane to certain factors in
pregnancy and delivery. PEDIATRICS, 26:
42, 1960.
4. Avery, M. E., and Oppenheimer, E. H.:
Re-cent increase in mortality from hyaline
membrane disease. J. Pediat., 57:553, 1960.
5. Skogrand, A., and Harnaes, K. R.: Causes of
death in premature infants. Acta Path.
Microbiol. Scand. 49:321, 1960.
6. Latham, E. F., Nesbitt, R. E. L., and
Ander-son, G. W.: A clinical pathological study
of the newborn lung with hyaline-like
membranes. Bull. Johns Hopkins Hosp.,
96:173, 1955.
7. Blystad, W., Landing, B. H., and Smith, C.
A.: Pulmonary hyaline membranes in new-born infants: statistical, morphologic and experimental study of their nature,
occur-rence, and significance. PEDIATRICS, 8:5,
1951.
8. Miller, H. C., and Jennison, M. H.: Study of
pulmonary hyaline-like material in 4117
consecutive births: incidence, pathogenesis,
and diagnosis. PEDIATRICS, 5:7, 1950.
w.
: Neonatal deaths among infants ofdiabetic mothers. Amer. J. Dis. Child., 100:
818, 1960.
10. Winter, W. D., and Gellis, S. S.: Pulmonary
hyaline membranes in infants of diabetic
mothers. Amer. J. Dis. Child., 87:702, 1954.
11. Rose, V. : Infants of diabetic mothers : clinical
and pathological features in a series of 25
cases. Canad. Med. Ass. J., 82:306, 1960.
12. Snyder, F. F.: Pulmonary hyaline membrane
disease : origin in premature infants
de-livered by cesarean section during labor or
after placenta previa and abruptio
pla-centae. Obstet. Gynec., 18:677, 1961.
13. Strong, L. B., Anderson, C. S., and Platt, J.
W.: Neonatal death and elective cesarian
section. Lancet, 1:954, 1957.
14. Driscoll, S. G., and Smith, C. A.: Neonatal
pulmonary disorders. Pediat. Clin. N. Amer.,
9:325, 1962.
15. Smith, C. A.: Circulatory factors in relation to
idiopathic respiratory distress (hyaline mem-brane disease) in the newborn. J. Pediat., 56: 605, 1960.
16. Gregg, R. H., and Bernstein, J.: Pulmonary
hyaline membranes and the respiratory
distress syndrome. Amer. J. Dis. Child.,
102:123, 1961.
17. Gitlin, D., and Craig, J. M.: The nature of
the hyaline membrane in asphyxia of the
newborn. PEDIATRICS, 17:64, 1956.
18. Van Breemen, V. L., Neustein, H. B., and
Burns, P. D.: Pulmonary hyaline
mem-branes studied with the electron
micro-scope. Amer. J. Path., 33:769, 1957.
19. Lieberman, J.: Clinical syndromes associated
with deficient lung fibrinolytic activity: I.
A new concept of hyaline membrane
di-sease. New Engl. J. Med., 260:619, 1959.
20. Lieberman, J., and Kellogg, F.: A deficiency
of pulmonary fibrinolysis in hyaline mem-brane. New EngI. J. Med., 262:999, 1960.
21. Lieberman, J.: The nature of the fibrinolytic
enzyme defect in hyaline membrane disease.
New Engl. J. Med., 265:363, 1961.
22. Lieberman, J., and Kellogg, F.: The
fibrino-lytic enzyme defect of hyaline membrane
disease. Calif. Med., 95:278, 1961.
23. Phillips, L. L., and Skrodelis, V.: A
compari-son of the fibrinolytic enzyme system in
maternal and umbilical-cord blood.
PEDI-ATRICS, 22:715, 1958.
24. Quie, P. C., and Wannamaker, L. W.: The
plasminogen-plasmin system of newborn
in-fants. Amer. J. Dis. Child., 100:836, 1960.
25. Samartzis, E. A., and Cook, C. D.: The
re-lationship between age and fibrinolytic
activity of serum. Acta Pediat., 49:724, 1960.
26. Samartzis, E. A., Cook, C. D., and Rudolph,
A.
J.
: Fibrinolytic activity in the serum ofinfants with and without the hyaline
mem-brane syndrome. Acta Pediat., 49:727,
1960.
27. Ambrus, C. M. : Clot formation and
dissolu-tion: basic considerations. New York J.
Med., 62:3738, 1962.
28. Craig, J. M., Fenton, K., and Gitlin, D. :
Ob-structive factors in the pulmonary hyaline
membrane syndrome in asphyxia of the
newborn. PEDIATRICS, 22:847, 1958.
29. Ambrus, J. L., et al.: Clinical and
experi-mental studies on fibrinolytic enzymes. Ann.
N.Y. Acad. Sci., 68:97, 1957.
30. Sokal,
J.
E., Ambrus, J. L., and Ambrus, C. M.: The treatment of thrombosis withplasmin. J.A.M.A., 168:1314, 1958.
31. Ambrus, J. L., et al.: Clinical pharmacology of
various types of fibrinolytic enzyme
prep-arations. Amer. J. Cardiol., 6:462, 1960.
32. Ambrus, J. L.: Fibrinolytic agents in the
therapy of thrombo-embolic disorders.
Pro-ceedings of NIH Hematology Study
Sec-tion Conference on Thrombolysis, Chicago,
Ill., 1960. p. 200.
33. Ambrus, J. L., et al.: Clinical and
experi-mental studies with fibrinolytic enzymes:
in Anticoagulants and Fibrinolysins.
Phil-adelphia, Lea & Febiger, 1961, pp.
413-428.
34. Ambrus, C. M., and Markus, G.: Plasmin-antiplasmin complex as a reservoir of
fibrin-olytic enzyme. Amer. J. Physiol., 199:491,
1960.
35. Markus, G., and Ambrus, C. M.: On the formation of different types of plasmin by
streptokinase activation. J. Biol. Chem.,
235:1673, 1960.
36. Werkheiser, W. C., and Markus, C.:
Distri-bution between the proactivators in human plasminogen for the activation of human and bovine plasminogen. Fed. Proc., 21:65,
1962.,
37. Markus, C., and Werkheiser, W. C.:
Distri-bution between esterase and protease
ac-tivities derived from plasminogen. Fed.
Proc., 21:65, 1962.
38. Hink,
J.
H. Jr., and McDonald, J. K.:Prepara-tion of profibrinolysin-free fibrogen and
thrombin from human plasma. Nature, 194:
1080. 1962.
39. Plough, J.: Urokinase an activator of
plas-minogen from human urine. Biochim.
Bio-phys. Acta, 24:278, 1957.
40. Silverman, W. A., and Andersen, D. H.: A controlled clinical trial of effects of water
trial data it led to the following answer: If
birth-weight is taken into account then the difference between the placebo and the UK plasmin series becomes statistically significant at the 1% level.
The basic rationale for COVAST is simple.
Suppose there was just one baby in each series.
Under what circumstances would we have
rea-sonably clear-cut evidence in favor of a given
treatment? If the outcome is the same for both
babies, this does not provide such evidence. If
the outcomes are different, but the baby with
the higher weight survives, then this again is equivocal evidence. The survival might be due
to the treatment or to the more favorable
prog-nosis. However, if the surviving baby had the lower weight, then this would be a fair piece of
evidence in favor of the treatment that was given to the survivor.
Suppose that the chances of survival are better
on UK plasmin than on the placebo. Then we
would expect to find instances where a baby that
was given UK plasmin and survived was lighter
than a baby that was given the placebo and died.
If the babies are ordered by weight, then we find 21 instances in line with the first hypothesis
(noninversions = NI = 21). There are no instances
to the contrary (inversions = I = 0). But if there
was no differential chance of survival, we would
have expected about as many inversions as
non-inversions.
The significance test here is a member of the
Sign Test family:
total observations +4’
COVAST= (21_0)2 =8.69;
which exceeds the critical value for the 1% level
(6.64).
The statistical test serves to call attention to
the survival of low weight babies (under 2 kg) in
the UK plasmin series (something that doesn’t
occur in the other series). However, note that
no arbitrary decisions concerning weights are needed to apply the test-no babies are arbitrarily
omitted and no arbitrary “break points” in the
weight scale are used. Although COVAST is only
slightly more complicated than the usual
chi-square procedures, it seems to offer some
import-ant statistical advantages. However since
mathe-matical derivations for COVAST have not yet
been published in the statistical literature, it was
deemed more appropriate to put the results with
this procedure in an appendix rather than in the text of the paper.”
rate and necropsy findings among prema-ture infants. PEDIATRICS, 17: 1, 1956. 41. Silverman, W. A. : Dunham’s premature
in-fants. New York, Hoeber, 1961, p. 144. 42. Weintraub, D. H., and Dunphy, D. : The care
of premature infants. Quart. Rev. Pediat., 14:155, 1959.
43. Ratnoff, 0. D., and Donaldson, V. H. : Phy-siologic and pathologic effects of increased fibrinolytic activity in man. Amer. J.
Car-diol., 6:378, 1960.
44. Kwaan, H. C., and McFadzean, A. J. S.: On plasma fibrinolytic activity induced by
is-chemia. Clin. Sci., 15:245, 1956.
45. DeLos Cobos, C. H. A., and Gracia, E. J.,
Sagaon, J. L. : The treatment of “pulmonary
hyaline membrane syndrome” in the
pre-mature newborn infant. Bol. Med. Hosp. Infantio., 16:41, 1959.
46. Ebner, S., Solomons, C., and McMillan, H. J.:
Treatment of Respiratory distress of the
newborn with human fibrinolysin. Rhode
Island Med.
J.,
44:89, 1961.47. Bross, I. D. J.: Taking a covariable into
ac-count. Submitted for publication.
Acknowledgment
We are grateful for the encouragement, advice,
and assistance of Drs. E. Neter, M. I. Rubin and
K. L. Terplan; and for the help in various phases
of this study to Drs. P. K. Birtch, I. D.
J.
Brossand B. Eisenberg. We wish to acknowledge the
assistance of our Fellows: I. Berkel, M.D., and (NI __I)2
N. Yanai, M.D., and the devoted help of our CO VAST = K NI +1
technicians: H. B. Lassman, BA.; I. B. Mink, B.A.; 12
F. S. Szymanski, BA.; and research nurses: N. K
-Edwards, RN.; R. E. Golebiewski, B.A., R.N.;
A. Krafchak, RN.; and V. S. Pawlak, RN.
APPENDIX
A
Irwin D. J. Bross, Ph.D., Department of
Biostatistics, Roswell Park Memorial Institute
It will be noted from Table VII that while the distributions of the weight of babies in the placebo
series is fairly similar to that for the babies given UK plasmin, there seem to be some differences.
For example, there are babies in the UK plasmin
series that are lighter (and also some that are heavier) than any of the placebo babies. So the
question arises: if the birthweight were taken
into account, what would a comparison of sur-vival in the placebo and UK plasmin series show?
Since standard statistical techniques are not
entirely satisfactory here, a new statistical tech-nique was developed especially for this problem.
It was called COVAST, since it is a COVariable
