In vivo non–imaging techniques are
diagnostic procedures wherein samples (such as blood or urine) are collected
from a patient to measure the amount of radioactivity concentrated using a
scintillation well counter.
HEMATOLOGIC
STUDIES
A simple hematocrit reading can
sometimes yield misleading results when estimating red cell volume or plasma
volume. If the patient is dehydrated and has a decreased plasma volume, the
hematocrit will be false elevated. If the patient has an increased plasma
volume, the hematocrit will be falsely low. Direct measurement of red cell
volume and plasma volume is the only method to determine these values.
A.
Total Blood
Volume Determination
Estimations of
total blood volume (TBV) are clinically useful in the diagnosis and management
of polycythemia or when changes in the venous hematocrit fail to reflect
accurately changes in TRCV or TPV. When the venous hematocrit is increased,
TRCV measurements distinguish absolute increases in red cell volume from
decreases in plasma volume. In cases of gross splenomegaly, red cell pool in
the spleen and increases in the plasma volume may give misleading venous
hematocrit readings. TBV measurements are calculated using the isotope dilution
principle equation.
V = O
C
Where: V =
volume
O = equals does
administered in counts per minute
C = equals
concentration of dose after dilution in counts
per
minute
The larger the
volume into which the label is mixed, the lower the counts in the sample
withdrawn and, conversely, the smaller the volume, the higher the counts in the
sample. The isotope dilution principle is only true when working with a closed
system, where no radiopharmaceutical is allowed to leak out the system being
measured. Also, the volume of the unknown must not change significantly during
the measurement. In most cases, red cell volume measurements are performed in a
closed system using radiolabeled red blood cells.
1.
Plasma Volume
Determination
Plasma volume
measurements are performed using radiolabeled albumin. Albumin does not remain
within the intravascular space but diffuses rapidly into the extravascular
compartments. Since this vascular space is now an open system. Use of the
closed system isotope dilution principle for calculations would cause errors in
the plasma volume results. If the injected radiopharmaceutical leaves the
open–system at a slower rate than the uniform mixing rate within that system
and samples are taken only after mixing has been completed, the volume can be
obtained by an extrapolation procedure.
Procedure of
the test
a.
Materials
(1) Patient
is administered with 10 µCi I–125 human serum albumin contained in 1.5 ml.
(2) Tc99m
human serum albumin can also be used for labeling efficiency at time of
injection must be performed and must be at least 98% bound.
(3) Heparinized
blood is the sample of choice.
b.
Patient
preparation
(1) Patient
should be at rest and supine for at least 15 to 20 minutes prior to starting
the study. Plasma volume decreases when a person is standing because venous
pressure increases in the legs and water moves from the intravascular to
extravascular space.
c.
Tracer
administration and sample collection
(1) Prior
to tracer administration, collect a background heparinized blood sample. Inject
the radiolabeled albumin intravascularly, taking care to avoid infiltration of
the dose. The exact volume injected must be known for the calculations;
therefore weighing the syringe on an analytical balance before and after
injection is recommended.
(2) The
difference between the pre–injection and post–injection syringe weight is
equivalent to the volume injected.
(3) Record
the exact time of injection.
(4) Do
not rinse the syringe with blood.
(5) Three
– 10 ml heparinized blood samples are collected at exactly 10, 20 and 30
minutes post–injection from a venous site different from the injection site.
d.
Sample
handling
(1) Centrifuge
the samples and pipette 1 ml aliquots of plasma into labeled counting tubes.
(2) Prepare
a standard by diluting a duplicate dose (10 µCi I–125 human serum albumin in
1.5 ml) into a 500 ml volumetric flask.
(3) Again,
the exact volume must be known, so the syringe should be weighed. Do not rinse
the standard syringe in water.
(4) Mix
well by inverting and shaking the flask.
(5) Pipette
duplicate 1 ml aliquots of the standard into labeled counting tubes.
e.
Counting
(1) Adjust
the pulse height analyzer of the scintillation spectrometer to count I–125.
(2) Count
each 1 ml sample for a time period that is long enough to assure a counting
error no greater than 1%.
(3) After
counting, adjust all samples to net counts per minute by subtracting patient
background from the patient samples and room background from the standard
samples.
f.
Calculation
The net count
per minutes of each post–injection sample is plotted against time on semi–logarithmic
graph paper. The best straight line is drawn through these points. The zero
activity is estimated by extrapolation; this value is used to calculate the
total plasma volume (TPV) using the equation:
TPV = volume
injected x net standard activity
Net patient activity at time zero
g.
Source of
error
(1) If
the radiopharmaceutical is infiltrated, an erroneously high result will be
obtained.
(2) If
the patient contains a radiotracer from a previous diagnostic test and the count
rate of the post–injection samples is not corrected for this contamination, an
erroneously low result will be obtained. Inaccurate measurement of the injected
volume and the standard volume can cause errors in the results.
2.
Red Cell Volume
Determination
Radiolabeling
of red blood cells for red cell volume measurement is referred to as random
labelling. This process labels all cells in the sample, from youngest
to the oldest erythrocytes or erythrocytes of random age.
The most
commonly used radionuclides for random red cell labelling are Tc99m
pertechnetate and Cr–51 sodium chromate. Due to the high elution rate of Tc99m
pertechnetate from the labeled red cells, this tracer is not recommended for
red cell volume determinations.
Methods
used for Red Cell Volume Determinations
a.
Ascorbic Acid
Method
(1) 10
ml of whole venous blood is collected from the patient into a 20 ml syringe
containing 2 ml of the anticoagulant acid–citrate–dextrose (ACD) solution.
(2) After
inverting to mix, transfer blood into a sterile vial.
(3) Add
30 µCi Cr–51 to the vial.
(4) Gently
mix and incubate for 30 minutes at room temperature.
(5) During
this time, 80% – 95% of the chromate ion is transported across the red cell
membrane and binds to the beta chain of the hemoglobin molecule.
(6) The
hexavalent chromate ion is reduced to trivalent chromic ion.
(7) 50
mg of ascorbic acid are then added to reduce the free chromate to chromic ion.
This prevents extracellular chromate from labelling circulating red cells.
b.
Wash Method
(1) 10
ml of whole venous blood are collected from the patient into a 20 ml syringe
containing 2 ml ACD anticoagulant.
(2) After
mixing, transfer blood into a sterile vial.
(3) Centrifuge
this mixture at 1000 to 1500 g for 5 to 10 minutes.
(4) Remove
and discard the supernatant plasma, taking care not to remove any red cells.
(5) Add
30 µCi Cr–51 to the packed red cells and mix gently.
(6) Allow
this mixture to stand for 30 minutes at room temperature.
(7) Wash
the labelled red cells in 4 to 5 ml isotonic saline, then centrifuge at 1000 g
for 5 to 10 minutes.
(8) Remove
the supernatant saline, resuspend the labeled red cells in saline and
recentrifuge.
(9) After
the last centrifugation, resuspend the red cells in saline to the original
volume. This method removes all of the free chromate ions so that the
reinjection mixture contains only red cell bound to Cr–51.
Procedure of
the test
a.
Materials
Use red cells
labeled with 10–20 µCi of Cr–51 sodium chromate
b.
Patient
preparation
Patient should
be at rest and supine for 15–20 minutes prior to the start of the study. A
patient background sample must be collected.
c.
Tracer
administration / sample collection
(1) Inject
intravenously 5 ml Cr–51 labeled red cells.
(2) Record
the exact time of injection
(3) Do
not rinse the dose syringe with patient’s blood.
(4) Withdraw
10 ml blood into a heparinized tube from a vein other than the one used for
tracer injections. In seriously ill patients, a delay of up to 30 minutes for
complete mixing may be necessary.
(5) In
cases such as polycythemia vera and splenomegaly, serial post– injection
samples at 30, 60 and 90 minutes should be collected.
d.
Sample
handling
(1) Perform
a microhematocrit determination on the stock–labeled red cell mixture and on
each post–injection blood sample.
(2) Prepare
a standard from the stock–labeled red cells by diluting 2 ml of the labeled
cells into a 100 ml volumetric flask. Again, the exact volume must be known.
Therefore, the syringe must be weigh before and after dilution.
(3) Bring
the volume in the flask up to the line using distilled water. Mix well by
inverting and shaking.
(4) Pipette
duplicate 1 ml aliquots of the standard into counting tube labeled “standard
whole blood.”
(5) Pipette
1 ml whole blood from each post–injection sample into a counting tube labeled
“sample whole blood.”
(6) If
the ascorbic acid labeling method was used, centrifuge the remaining stock–labeled
red cell mixture and each post–injection sample at 1500 g for 10 minutes.
(7) Pipette
1 ml plasma supernatant from each sample into labeled counting tubes. The
standard counting tube should be labeled “standard plasma” and the sample
counting tubes should be labeled “sample plasma.”
e.
Counting
Set the
scintillation spectrometer to count Cr–51. Count each 1 ml sample for
sufficient time to ensure no more than a 1% counting error. Adjust all samples
to net counts per minutes by subtracting the patient background from the
patient samples and room background from the standard samples.
f.
Calculations
If ascorbic
acid labeling method was used:
Volume (net standard x dilution)
TRCV = injected
(counts factor)
Net
whole blood samples counts
x decimal hematocrit
If wash
labeling method was used:
volume [(net whole x
dilution) – (net plasma x standard) ]
TRCV = injected [(blood counts factor) standard counts plasmacrit)]
Net whole
blood sample counts – (net plasma
x sample
(Sample counts plasmacrit)
x decimal hematocrit
plasmacrit = 1
– decimal hematocrit
g.
Sources of
error
(1) Infiltration
of the labeled red cells will cause erroneously high results.
(2) Damaged
erythrocytes or excessive binding of the Cr–51 by leukocytes or platelets will
yield spuriously high values.
(3) Falsely
low results are caused by a failure to obtain a pre–injection blood sample in a
patient who has previously received radioactive tracers.
(4) All
blood samples must be mixed well prior to hematocrit determinations and
pipetting of samples.
(5) Cr–51
should not be added to the ACD solution before the patient’s blood is added to
the vial. Dextrose contained in the ACD solution acts as a reducing agent and
inhibits red cell binding.
h.
Normal ranges
Men Women
Total blood
volume 55 – 80 50 – 75
Total red cell
volume *† 22 – 35 20 – 30
Total plasma
volume 30 – 45 30 – 45
*95%
confidence limits
†Valid at sea
level only
3.
Technical
considerations
a. Even
in healthy normal individuals, there is considerable variation in blood volume
among persons with identical height and weight. In patients with diseases such
as cachexia, heart or renal failure with edema, ascites or obesity, an even
greater variation in lean tissue mass must be taken into consideration.
b. Blood
volume varies with body size, body type, sex, disease state, basal metabolic
rate, nutritional state and amount of physical work.
c. Blood
volume varies with season, arterial oxygen content and changes in body
position.
d. In
normal persons within the ideal weight range, lean body mass makes up a
constant fraction of total body weight. In these individuals, blood volume
correlates well with weight. When fat is excessive (obesity) or deficient (emaciation,
cachexia), corrections must be made to obtain an adjusted ideal weight. Since
fat has a vascularity of approximately 20% adjustments can be calculated to
permit a comparison of an individual’s blood volume with normal values.
e. From
a precise measurement of red cell or plasma volume and venous hematocrit, the
volume of the other compartments and therefore total blood volume, can be
calculated in normal individuals. Normally, there is a fixed relationship between
whole–body hematocrit and venous hematocrit, with ration of 0.89 to 0.92. The
whole body hematocrit is usually lower than the venous hematocrit due to
variations in blood vessel size throughout the body.
f.
In splenomegaly, the ratio of
whole–body to venous hematocrit may be unpredictably increased to greater than
1.0. In patients, with polycythemia, plasma volume abnormalities as well as red
cell volume abnormalities may be present. Therefore, in clinical situations,
total blood volume can only be reliably measured by performing simultaneous independent
measurement of red cell volume and plasma volume.
RED CELL
SURVIVAL
The red cell survival study determines
the mean survival time of 51Cr–labeled autologous red cells in
patients with hemolytic anemia.
1.
Materials
Chromium–51 is
labeled to heparinized red cells with the ascorbic acid labeling technique.
Chromium activity is adjusted to 1.5 µCi per kg body weight, with a minimum
activity of 50 µCi.
2.
Tracer
administration and sample collection
After
labeling, the red cells are injected and the first blood sample is obtained 24
hours later. This time period permits removal of cells that were damaged during
the labeling procedure from the circulation, as well as clearance from the
blood of any injected plasma activity. Heparinized blood sample are obtained
from the patient, every other day, for the next 3 weeks.
3.
Sample
handling
On the day of
collection, 5 ml well–mixed whole blood is pipetted into labeled counting tube.
A pinch of saponin powder is added to the tube and mixed to lyse the red cells.
These samples are then stored at 4oC until the last day of the
study. Hematocrit determinations also are made on each sample on the day of
collection.
4.
Sample
counting
On the last
day of the study, all samples are counted on a scintillation spectrometer with
setting of 280 to 360 keV for 51Cr. Samples are counted long enough
to give a counting error of 1% or less.
5.
Calculations
a. The
net count per minute of each sample is plotted on semi–logarithmic paper as a
function of time.
b. The
best straight line is drawn through all the points.
c. The
half–time is obtained by extrapolating the line to time zero, which is the Y
intercept. Divide the Y intercept value by 2.
d. At
this value on the y–axis, draw a straight line parallel to the x–axis until it
intersects the best straight line drawn through the data points.
e. Drop
a perpendicular line to the x–axis. This value is the mean survival time of the
labeled red blood cells.
6.
Sources of
error
Shorter red
cell survival time will appear if patient loses blood during the study. If the
patient receives blood transfusions during the study, the mean survival time
will appear shortened.
7.
Normal values
The mean half
time of normal 51Cr labeled red cells is from 25 to 35 days. As
normal red cells age, they are removed from the circulation at a rate of 1% per
day. Therefore, the true mean survival time of a normal random red cell
population is 50 to 60 days. If this normal 1% per day removal is coupled with
the 1% per day elution of the 51Cr from the red cells, the combined
mean half time is 25 to 35 days.
SPLENIC
SEQUESTRATION
This test determines if the spleen is
the site of red cell destruction in a patient who has evidence of increased red
cell destruction. A splenic sequestration study is routinely performed in
conjunction with a red cell survival study.
1.
Materials
Use 51Cr
red cells, 1.5 µCi per kg body weight
2.
Patient
preparation
a. Counting
begins 24 hours after injection of the 51Cr labeled red cells.
b. The
patient is placed on the examination table and the skin is marked with
indelible ink.
c. Transparent
tape is placed over each anatomical location and patient is instructed not to
remove or wash off marks.
d. Counting
continues every other day for 3 weeks.
3.
Counting
The following
anatomical locations are marked for counting:
a.
Precordium
With the
patient in the supine position, the detector is centered over the left, third
intercostal space at sternal border.
b.
Liver
With the
patient in the supine position, the detector is placed over the 9th
and 10th ribs on the right midclavicular line.
c.
Spleen
With the
patient in the prone position, the detector is placed two thirds of the
distance from the spinal process to the lateral edge of the body, at the level
of the 9th and 10th ribs.
The same
counting geometry must be present each time the patient is counted. Sufficient
counts should be collected to give a counting error of 5% or less.
4.
Calculations
Results are
expressed as the ratio of the net organ counts per minute to the net precordium
counts per minutes for each day. These ratios are then plotted on linear graph
paper as a function of time.
5.
Sources of
error
Inconsistent
counting geometry from day to day will cause spurious results. Blood loss or
transfusion during the counting interval also will affect results adversely.
6.
Normal values
The normal
spleen to precordium ratio is between 0.5 to 1.0 and the normal liver to
precordium ratio is 0.5. An initial spleen to precordium ratio greater than 2.0
indicates an increased splenic blood pool. A progressive, gradual increase over
the course of the study indicates active splenic sequestration of the labeled
red cells.
VITAMIN B12
ABSORPTION TEST
The consequences of untreated Vitamin
B12 deficiency are extremely serious and include certain
hematological abnormalities such as anemia, neurological defects and, if the
condition is untreated, death. In the early stages, the symptoms of the
deficiency are vague and insidious.
Causes of Vitamin B12
deficiency
1. Inadequate
intake
2. Malabsorption
a. Absence
of intrinsic factor
(1) Congenital
(2) Addisonian
pernicious anemia
(3) Total
gastrectomy
(4) Subtotal
gastrectomy
b. Excessive
excretion of hydrochloric acid: Zollinger–Ellison syndrome
3. 2o
intestinal malabsorption
a. Destruction,
removal or functional incompetence of ileal mucosal absorptive sites.
b. Competition
with host for available dietary Vitamin B12
(1) Diphyllibotrium
latum (fish tapeworm)
(2) Small–bowel
lesions associated with bacterial stagnation (jejunal diverticula, strictures,
blind loops, etc.)
c. Pancreatic
insufficiency*
(1) Paraaminosalicylic
acid (PAS)
(2) Neomycin
(3) Colchicines
(4) Calcium–chelating
agents
d. Drug
therapy
4. Genetic
abnormality in transport protein transcobalamin II.
*Although any
of these agents may produce abnormalities in Vitamin B12 absorption,
only patients on long term PAS therapy have been reported to develop clinical
evidence of Vitamin B12 deficiency.
Procedure of the test
A.
Stage I
1.
Materials
The procedure requires
0.5–0.6 µCi labeled 57Co Vitamin B12. It is so important
that this oral dose be in the range of 0.25–2.0 µg of Vitamin B12,
the amount that might be present in a typical meal. Quantities above this level
may be absorbed by a mechanism not dependent on the presence of intrinsic
factor. Labeled urine containers large enough to hold a 24–hour sample are also
needed.
2.
Patient
preparation
a. The
patient should have nothing to eat after midnight, the night before the test
and should remain fasting for 2 hour following the oral dose of 57Co–
labeled Vitamin B12.
b. The
patient should not receive enemas or laxatives or be scheduled for a barium
enema or intravenous pyelogram for the duration of the study.
c. Due
to the effects of hepatobiliary recirculation, treatment doses of Vitamin B12
should be discontinued 2 to 3 days prior to starting Schilling test.
3.
Dose
administration / Sample collection
a. The
57Co Vitamin B12 capsule is administered orally with
water.
b. Two
hours later, 1 mg of stable cyanocobalamin (Vitamin B12) is given
intramuscularly.
c. Non–radioactive
Vitamin B12, sometimes referred to as the “flushing dose,” causes
temporary saturation of the normal binding sites in the plasma and results in
renal excretion of a portion of the absorbed tracer dose.
d. Excretion
of radioactive vitamin B12 is maximal between 8 and 12 hours after
administration of the flushing dose.
e. Two
24–hour urine collections are obtained, one for each 24–hour period.
f.
The patient should be
instructed to collect all urine during this period.
4.
Sample
handling
After mixing
each 24–hour collection well, the total volume and specific gravity of each
sample is measured. Two 5 ml aliquots are pipetted from each 24 hour urine
collection into labeled counting tubes. A standard is prepared from the
reference standard solution supplied with capsules.
5.
Sample
counting
All samples
are counted with a scintillation spectrometer set to count 57Co.
Each sample is counted for a sufficient time to ensure no more than 1% counting
error. All sample counts are adjusted to net counts per minute (cpm) by
subtracting the room background radiation.
6.
Calculation
The percentage
of the administered dose excreted is calculated for each 24– hour urine sample
using the following equation:
% excreted = net cpm urine
sample x urine sample/sample volume
net
cpm standard standard
dilution factor
7.
Normal values
Day 1 ≥ 9%
Day 2 ≤ 1%
8.
Sources of
error
a. The
loss of just one urine sample may cause falsely low results.
b. In
patients with abnormal urinary retention, such as benign prostatic hypertrophy,
the amount excreted in the first 24 hours will be reduced.
c. Significant
amounts of radioactivity will continue to be excreted for the next 24–48 hours,
so that the total excretion will eventually be within normal limits. For this
reason, it is recommended that two separate 24 hour urine specimens be
collected following the “flushing” injection of cyanocobalamin.
d. Excretion
of other previously administered radionuclides can cause erroneously high
results.
B.
Stage II
If the
absorption of 57Co Vitamin B12 is low when compared to
normal individuals, then a second absorption test is performed by giving hog
intrinsic factor along with oral dose of labeled Vitamin B12. This
test determines if the cause of the malabsorption is due to an absence of
intrinsic factor.
1.
Materials
In addition to
the materials described in Stage I, a 10 mg dose of intrinsic factor is needed.
2.
Patient
preparation
Same as Stage
I
3.
Dose
administration / sample collection
The 57Co
Vitamin B12 and intrinsic factor are administered orally with water
simultaneously. The remainder of the test is the same as described for Stage I.
4.
Normal values
Day 1 ≥ 9%
Day 2 ≤ 1%
5.
Sources of
error
a. When
the radioactive Vitamin B12 and the intrinsic factor are given in
separate capsules, there may be incomplete binding of the two in the stomach,
which gives a false–negative Stage II Schilling test. It is recommended that
prior to administration, the radioactive Vitamin B12 and intrinsic
factor be mixed together in water.
b. If
hog intrinsic factor is used, a negative result does not completely rule out
intrinsic factor–dependent malabsorption, since some patients who have been
previously exposed to hog intrinsic factor (present in many multivitamin
preparations) may have antibodies against it.
c. Vitamin
B12 deficiency can produce small bowel megaloblastosis with atrophy,
which may cause ileal malabsorption. If a Stage II study is performed before
Vitamin B12 therapy is started and the ileum is allowed to heal, a
false–negative result will be obtained.
C.
Stage III
1. Having
determined that the Stage II Schilling test is truly abnormal, other causes of
malabsorption must be investigated.
2. If
bacterial overgrowth is suspected, treatment with a broad spectrum antibiotic
followed by a repeat Stage I Schilling test is performed.
3. If
pancreatic insufficiency is suspected, a repeat Stage I Schilling test can be
performed after the administration of pancreatic extract.
D.
Dual Isotope
Method
1. The
conventional Schilling Test of Vitamin B12 absorption just described
is done in two stages, requiring at least 1 week for a final result if Stage I
is abnormal.
2. A
modified Schilling Test is available in which Vitamin B12 labeled
with two different isotopes of Cobalt, is administered. One form, 57Co
labeled Vitamin B12 is already bound to intrinsic factor by prior
incubation with normal human gastric juice, and other contains non–protein bound
58Co–labeled Vitamin B12.
3. The
two capsules are ingested orally, followed by injection of non– radioactive Vitamin
B12 and two–24 hour urine collections.
4. Differential
isotope counting is then performed to determine the percent administered dose
of each isotope that has been excreted in the urine. A patient with normal
Vitamin B12 absorption will excrete the same amount of both
isotopes, while a patient lacking intrinsic factor will excrete greater
quantities of the intrinsic factor bound 57Co–labeled Vitamin B12.
In patients with bacterial overgrowth or small bowel lesions resulting in
Vitamin B12 malabsorption, both isotopes will be excreted in
abnormally low quantities.
Advantages of
dual isotope method
(1) The
practical advantage of taking only 2 days, instead of 1 week to obtain result
leads to earlier diagnosis and treatment and saves money.
(2) The
use 57Co bound to human gastric juice eliminates the use of hog
intrinsic factor preparations, against which some patients may have developed
antibodies.
(3) While
an incomplete urine collection is undesirable, some information can often be
salvaged from 57Co:58Co ratio.
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