R. WENNESLANDET Al...
be used to determi the “true” Vebe, if desired
(Reeve 1952: Chapin & Molbison 1952; Ebuugh
et al. 1955; Gregensen & Rawson 1959), The om-
tents of the bubly ouat represent only 0.5—1.0 per
cent of the oc vohawe en tealthy peopic (Winteobe
1961), and we frave found the tap of the cell column
easier to read than the interface between red art
avay layers Therefore, we have followed the practice of Reeve (1952) ww our studies of normal nb
jects. Hematocrit readings can be corrected approprrately when the buffy coat exceeds | mm.
RESULTS AND DISCUSSION
Evaluation of sources of error and
ee ee ee ee
Errors of measuring a volume by the dilution technique can be considered in two categories. First, is the accuracy of estimating
the amount of indicator added, in this case
tagged cells. If the dose administered is
smaller than supposed, then the volume
appears larger than it really is, and vice
vorsa. Included in this category are a) meas-
urement of the volume of ceils injected, b)
the accuracy of counting the radioactivity of
the tagged cell suspension, and ¢) hidden
errors, such as loss of some of the tagged
cells from the circulation im vive, as might
occur if cells were damaged by the tagging
procedure and were phagocytized or other-
wise removed from circulation. The second
category of crrors relates to the measurement
will he “vy |
J
thé
“ut
» it
aecnnacy
ol
ne nethoc,
I.
Overnight storape ob taped cell
cler
to study
the
patients
before
We
ther
1.6 per cent (Nomofet al. 1954). Samples
and standards were placed in dishes, 42 mm
in diameter, for counting. Variations in the
distribution of the tagged cells in relation to
tion could be obviated either by twirling the
dish just before counting or by hemolyzing
gewhere (Wennesland et af 1957), we
powed that although some of the tagged: and
radioactivity. The counting error avetaged
the cells. We found the fatter ultnecessary,
Thirteen duplicate samples were counted:
one member of each pair was frozen and
thawed, the other agitated by hand. The
mean difference between the hemolyzed and
agitated samples was nil, and the standard
deviation of the mean of the differences was
0.18, or about 1.5 per cent of the average
counts per second of the 13 hemolyzed speci-
mens. This result also shows the approximate size of the variations that can be ex-
asspension. In animal experiments described
drigerated cells may be canght im the lung,
wer and spleen of recipient) animals, the
agree of such cell loss is} qisufficient to
fect blood volume determinations When
dis stored for I day were uyected into doy,
he total loss of tafeam vita and ot vive was
Table [. Loss of Radiochromuon (rit)
home the Tagged Cells to the Supernatant
ialime ‘ahd to the Infusion -lpparatus after
$6—20 Hours’ Storage.
pected from pipetting and counting ‘efrors:
combined.
|
re
Errors in measuring the administered dose
of tagged cells. Thd two most important
changes, we have made in the method are
* With ahe well4ype sciutiliation detector wow
beang used, the 10 ml of tagged coll suspeneon
yiokh 50,000--60,000 cuunts/secund
No of
Loss of Cr*¥ ta:
Supernatant saline
lalusson apparatus®
Synnge
Glass bulb
Tubing
less Than OG per cent:
(Wennesland et al
MS7). Phe boss of Cr8® fram the blood dur-
status will be as uniform as possible.
ageing the cells in the afternoon and storig the suspension overnyght is cohventent,
pecially for tests on hospitalized: patients.
fable I shows that after overnight refrigerawm, the supernatant sahine coutains less than
j2 per cent of the radivactiwity of the whole
the blood specimens and the background
of the dilution of tagged cells in the blood. a) tapping i célik the day before instead
Included here are a) the time and technique of the day o 4 hed periment, and b) using
the infusion apparatus and indwelling necile
of blood sampling, and 6) the accuracy [of
fer administration of tagged cells and for
estimating the radioactivity of the
les.
sampling. Tests were made to assure that
Since the measurement of lradigactivity
is
critical to both the estimat {i hq lose
delivered and its dilution ib}
pther innovation wipes Che
yielded a total of 20,000—30,000 Counts/ ,eakfast so that their metibohe ane encata-
second!, providing a good contrast betwee;
crystals of the counter caused by sediments.
comments on techmiquz.
see
_——
Errors im measurement of radtouctiyit,
With the plane scintillation counter Used
during collection of the data reported here
10 ml of the tagged cell suspension generally
[observe-
Jones & Mollison (1956) believed it was
unlikely that the early loss of Cré! was due
to the handling of the blood, because they
fuund equal rates of loss when blood was
tagged in wiro and ta vivo.
2. The infusion apparatus. The importance of a clean venipuncture and of accurate
incasurement of the amount of injected tag
has log heen recopnized (Price & Longmire
1942), The rachoactivity left in the tabing
and glass bulb adds about 50 per cent to the
very small amount Jeft in the syringe
(Table 1). Variation in radioactivity reManip ino the entire delivery system are
quite sinall (Vable TP).
Radioactivity en",
of total dow
fore
stan
os).
#2
W432
Uba4
15
meg
lw
1
ip the first 24 hours after injection of cells
tapped and stored by our method avetaped
S per cent in 8 healthy subjects (Nomof etal.
1954). Mollison & Veall (1955) found a
sinilar rate (G per cent) in 16 experituetts
where the blood was returned immediately
alter tagging. Overnight storage therefore
appears not tu have any disadvantage, at beast
when dealing with normal blood!. Hughes
aed
0K
O 026
wen
(7
‘In attra halk ad the blood, messary in
all the tahouctave celltagging methods, may cause
indcterminte errocs in cxtimading Vebc of patents
wrk henrulytec tendencies unbess checked by measurccmods of cell survival In a lacge climeal expence with dire method, we have had to abandon
Ie test on pare ovcaweons because of visible hemoIyses of the tagged cell
suspension
The patients
antially had real discave, aad the hemolysis occur"Determined by measurua: the tachoactiwity: of
Selnngs, which wore reposted ulil the Cons
dered when each portan of apparatie owas
ced directly to the scamillation counter dh tot
Hiker
segralecandly
wuvnty.
frou
the
bescheroonl
rade
nel carly oe the lager pre edure (overnight sorae was deat tvebved)
This obvervadson paises the
peesthilty that cave taanalysis may be accennoted atl be aia important source of error in pateats with hemolytn
poestular faclons.
tendetcies due to extracor-
—
358