Nuclear Medicine Resources Manual

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CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

360

5.10.2. Blood volume

5.10.2.1. Principle

Total blood volume consists of separate plasma and cellular compart-

ments. Knowing the haematocrit, blood volume can be calculated from either

the plasma volume (PV) or the red cell volume (RCV). However, more

accurate results are obtained if the total blood volume is determined by

separate measurements of plasma and red cell volume. In clinical situations, the

ratio between total body haematocrit and peripheral haematocrit often varies

widely. A separate determination of plasma and red cell volume is thus strongly

advised.

Plasma and red cell volumes are determined using the dilution principle,

where the volume in question is calculated from the concentration of a tracer

added in an accurately measured amount, mixed homogeneously within the

compartment to be measured, using the following formula:

V = Q/C

where

V is the volume of the compartment;

Q is the quantity of tracer added;

and C is the concentration of diluted tracer after equilibrium.

The following conditions must be fulfilled for the formula to be valid:

—The tracer must be homogeneously distributed within the compartment.

—There must be no loss of the tracer during the study period, unless the loss

can be accurately compensated for.

—The tracer distribution must not change at equilibrium.

—The tracer must not affect the compartment in any way.

—The tracer must be easily and accurately measurable.

5.10.2.2. Clinical indications

The main indications for the test are in the diagnosis of polycythaemia

(erythrocytosis). This condition is diagnosed by finding elevated haemoglobin,

haematocrit and red cell counts, and may be absolute (increased red cell

volume) or relative (haemoconcentration).

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361

The classification of polycythaemias is as follows:

(a) Relative polycythaemia (haemoconcentration: RCV normal, PV

reduced):

(i) Dehydration:

—Reduced water intake;

—Vomiting;

—Diarrhoea;

—Alcohol;

—Diuretics;

—Burns;

(ii) Stress polycythaemia:

—Relative erythrocytosis;

—Pseudopolycythaemia;

—Gaisböck’s syndrome.

(b) Absolute polycythaemia:

(i) Primary (low erythropoietin):

—Polycythaemia vera;

—Erythraemia (pure erythrocytosis);

(ii) Secondary (high erythropoietin):

—Appropriate (O

saturation decreased):

● chronic lung diseases,

● living at high altitudes,

● smoking,

● cyanotic heart disease,

● massive obesity,

● certain haemoglobinopathies.

—Inappropriate:

● bronchial or renal cell carcinoma,

● hepatoma,

● cerebellar haemangioblastoma,

● uterine fibroids,

● phaeochromocytoma,

● renal ischaemia,

● adrenal cortical hyperplasia or adenoma,

● essential erythrocytosis.

—cirrhosis;

—nephritis;

—congestive cardiac failure;

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—marked splenomegaly;

—pregnancy.

Differentiating between these conditions has important therapeutic

implications.

The availability of erythropoietin determinations has decreased the use of

blood volume determinations, perhaps because of the uncertain accuracy of the

latter. To improve the reliability of blood volume determinations, it is

imperative to pay attention to technical details, including adjustment of normal

and/or reference values for the patient’s body build, especially in obese

patients.

5.10.2.3. Radiopharmaceuticals

Although other labels may be used, the most common are:

—For RCV,

Cr and

99m

Tc labelled red cells;

—For PV,

125

I radioiodinated human serum albumin (RISA).

For RBC labelling,

Cr offers the advantage of negligible elution loss

compared with

99m

Tc. The use of

Cr becomes essential when RCV determi-

nation is carried out in conjunction with red cell survival studies. In vivo

99m

labelling of red cells is a practical alternative if only RCV needs to be

determined.

5.10.2.4. Equipment

Although dedicated semi-automated counters can be used for

125

I-RISA

PV determinations, a simple well counter will normally suffice.

5.10.2.5. Patient preparation

Since radioactive iodine is taken up by the thyroid, 200 mg of potassium

iodide should be given orally per day for two days before and eight days

afterwards, in order to block thyroid uptake.

Owing to the postural effects on PV, the patient should be in the supine

position for about 30 min prior to the study and remain so throughout the

entire study.

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363

5.10.2.6. Procedure

(a) Plasma volume (RISA)

The plasma volume can be found by the following procedure:

(1) Measure the patient’s weight and height.

(2) Draw 5 mL of blood (EDTA) for a 2 mL plasma background sample.

(3) Carefully inject a known volume containing 100–200 kBq of

125

I-RISA

intravenously. Care must be taken not to extravasate the injected dose.

Timed blood samples should be drawn from the opposite arm at exactly

10, 20 and 30 min post-injection. Centrifuge and pipette 2 mL of plasma

for gamma counting.

(4) Standard solution: dilute a known volume of

125

I-RISA with water to a

known and exact volume of 1000 mL. Use an identical injection-flushing

technique to that used for patient injection. Since albumin can adhere to

glass, siliconized glassware is recommended. Alternatively, carrier

albumin or detergent can be added before addition of the RISA and

mixed well. Pipette 2 mL for gamma counting.

(5) Count the patient and standard specimens in a well counter at

125

I settings

(20–80 keV), subtracting background counts to obtain net counts/min.

(6) Use a semilogarithmic plot of count rate of the serial 10, 20 and 30 min

plasma samples and extrapolate to time zero to obtain the count rate

before removal of the labelled protein from the circulation occurred. This

count rate (at t = 0) is used in calculating the PV value:

PV = (Net counts/min standard ¥ 1000)/(net counts/min plasma sample).

(b) Red cell volume

The red cell volume can be found by the following procedure:

(1) Measure the patient’s weight and height.

(2) Under sterile conditions, draw approximately 10 mL of blood. Add 6 mL

of blood to 2 mL ACD in a sterile tube and mix well. Anticoagulate the

remaining 4 mL with EDTA and pipette a 2 mL aliquot for background

counting.

(3) Aseptically add 1–2 MBq

Cr sodium chromate to the ACD blood tube.

Incubate for 45 min at room temperature with continuous gentle rotation.

(4) Centrifuge at 1000g for 10 min. Remove the supernatant with a fine

needle and discard as radioactive waste.

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(5) Resuspend the red cells gently in sterile saline, repeat step (4) and

resuspend to approximately the original volume.

(6) Measure exactly 1 mL of the labelled cells, dilute to 1000 mL with

distilled water and pipette two 2 mL aliquots for gamma counting.

(7) With the patient in the basal resting state for at least 30 min, inject exactly

5 mL of labelled cells. With the patient still in the supine position, draw

mL EDTA blood samples at 10 and 40 min from the opposite arm.

(8) Check the injection site with a hand radiation monitor to ensure that

there is no extravasated radioactivity compared with the opposite arm.

(9) From each blood sample, pipette 2 mL for gamma counting and perform

a haematocrit measurement on the remainder.

(10) Count the patient and standard specimens in a well counter at

settings (280–360 keV), subtracting background counts to obtain the net

counts/min. Use an adequate time to obtain valid statistics. All specimens

are counted for the same time (approximately 10 min).

(11) RCV for each sample can be calculated from:

RCV (mL) = [(net counts/min)/mL std] × Hct × 1000 × 5 / [(net counts/

min)/mL blood sample)]

where Hct stands for haematocrit.

(12) Total blood volume (TBV) can be calculated from:

TBV (mL) = (RCV/Hct) × 0.91.

(13) Plasma volume (PV) = TBV – RCV.

(14) If the 40 min RCV is significantly higher than the 10 min value and the

patient does not have active bleeding or haemolysis, the 40 min value is

considered to be more accurate — especially if the patient has marked

splenomegaly causing delayed equilibrium.

If repeat measurements of RCV are necessary, labelling with

99m

Tc can be

used with the in vitro technique. The disadvantage of

99m

Tc is its fairly high

elution from red cells, making this method unsuitable for delayed sampling as

in splenomegaly or congestive cardiac failure.

Simultaneous RCV and PV determinations can be done using dual

channel counting. Corrections have to be made for

Cr scatter into the

125

channel.

5.10. HAEMATOLOGY

365

5.10.2.7. Interpretation

Normal blood volume values (mL/kg) are given in Table 5.19.

Using a fixed reference range in mL/kg does not take into account the

fact that obese individuals will have relatively lower values when expressed in

mL/kg. It is more accurate to use individualized reference values for each

patient, using tables based on the patient’s weight and height or body surface.

5.10.3. Red blood cell survival

5.10.3.1. Principle

A test is done to determine whether anaemia is caused by decreased

survival of RBCs.

5.10.3.2. Technique

The following technique is used to measure RBC survival:

(1) Collect 10 mL of blood with 1.5 mL ACD as anticoagulant.

(2) Centrifuge the blood and discard the plasma.

(3) Label the remaining packed cells with 0.5 mCi/kg

Cr-chromate.

(4) Wash the cells and recentrifuge them to remove any unbound

Cr.

(5) Restore the volume by adding isotonic saline. Re-inject the patient.

(6) Withdraw samples every other day for 21 days, beginning 24 hours later.

The sample taken at time zero cannot be obtained earlier than 24 hours,

because approximately 10% of the label is lost on the first day. This may

be due to normal accelerated elution and/or damage during labelling.

(7) Determine the microhaematocrit for each sample. The counting is

performed in a well counter.

TABLE 5.19. NORMAL BLOOD VOLUME VALUES AND RANGES

(in mL/kg)

Males Females

RCV 30 (25–35) 25 (20–30)

PV 40 (35–45) 40 (35–45)

TBV 70 (60–80) 65 (55–75)

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(8) Plot blood counts on semilogarithmic paper.

(9) Dividing the whole blood cell counts by the sample haematocrit improves

accuracy and corrects for daily fluctuations of haematocrit.

5.10.3.3. Interpretation

(a) Normal findings

The half-life of RBCs is 25–35 days. Normal RBCs have a 120 day

lifespan. Thus, 0.8% of the RBCs are lost each day through senescence. The

measured

Cr-RBC survival half-life is not 60 days as predicted, because of an

additional ~1% elution loss of the

Cr from the RBCs.

(b) Abnormal findings

An RBC survival time of less than 25 days is abnormal.

The patient must be in a steady state. Recent haemorrhage or transfusion

affects accuracy. If the haematocrit varies significantly (where the patient is not

in a steady state), a closer approximation of RBC survival is obtained by

plotting whole blood cell counts without haematocrit correction.

5.10.3.4. Protocol

(a) Red cell labelling procedure with

Cr (ICSH, 1980)

The following procedure should be used:

(1) Collect 10 mL of the patient’s blood in a sterile tube or plastic labelling

bag that contains 1.5 mL of ACD (NIHA) solution. Centrifuge the blood,

withdraw the plasma and add 0.5 mCi/kg (20 kBq/kg) of high specific

activity

Cr sodium chromate.

(2) Incubate the blood at room temperature for 15 min with occasional gentle

mixing.

(3) Wash the cells twice with isotonic saline to remove unbound

Cr.

(4) After the second centrifugation and decantation, restore the original

volume with isotonic saline.

(5) A measured aliquot of the labelled RBCs can be used for red cell survival

and sequestration studies.

(b) In vitro

99m

Tc red blood cell labelling (Brookhaven National Laboratories

method)

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367

The following procedure should be used:

(1) Draw 4 mL of the patient’s whole blood into a heparinized syringe and

transfer this to a Vacutainer kit with a lyophilized stannous citrate

mixture containing 2 mg of tin. The blood is incubated with these reagents

for 5 min.

(2) Add 1 mL of 4.4% EDTA (disodium or calcium disodium salt), mix and

centrifuge the tube upside down at 1300g for 5 min.

(3) Withdraw 1.25 mL of the packed RBCs, transfer to a vial containing 1–

99m

Tc-pertechnetate and incubate with gentle mixing for 10 min.

(4) The red cells are now ready for injection. Alternatively, they can be

heated at 49°C for 15 min and used for spleen scintigraphy.

99m

Tc-RBC labelling

The following procedure should be used:

(1) Constitute commercial stannous pyrophosphate containing 2–4 mg of

stannous ions with normal saline, and inject an aliquot, containing 10–

mg of tin per kg body weight, into the patient.

(2) After 30 min, inject the required quantity of

99m

Tc-pertechnetate (usually

10–25 mCi or 370–925 MBq). RBC labelling occurs almost immediately.

(3) It is important to inject the stannous pyrophosphate shortly after recon-

stitution to avoid oxidation of the tin.

(d) In vivo

99m

Tc-RBC labelling

The following procedure should be used:

(1) Inject 0.5 mg of stannous ions intravenously from a reconstituted

commercial stannous pyrophosphate kit.

(2) Insert a 19 gauge butterfly infusion set into an appropriate vein. Attach a

four way stopcock and flush from a syringe containing ACD.

(3) Approximately 20 min following injection of the tin, withdraw 3 mL of

blood into a 5 mL shielded syringe containing 370–925 MBq (10–25 mCi)

99m

Tc-pertechnetate.

(4) Flush the tubing with ACD solution. After 10 min of incubation with

gentle agitation at room temperature, the labelled RBCs are re-injected

through the infusion set.

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368

5.10.4. Splenic sequestration

A test is performed to determine if destruction of RBCs, which shortens

RBC survival, is caused by splenic destruction of RBCs.

5.10.4.1. Technique

The following procedure is used:

—Usually performed in conjunction with an RBC survival study.

—Imaging or regional counting (using non-imaging detectors) should be

performed on the same every-other-day schedule as the RBC survival

study.

—Activity is measured over the precordium, anterior liver and posterior

spleen.

5.10.4.2. Interpretation

Normal and abnormal findings can be characterized as follows:

(a) Normal findings:

—The spleen-to-liver ratio is 1:1.

—This ratio remains approximately 1:1 during the course of the study.

(b) Abnormal findings:

—Increased spleen-to-liver ratio.

—The spleen-to-liver ratio increases over the course of the study.

Splenomegaly itself, without pathological sequestration, can yield

spleen-to-liver ratios of between 2:1 and 4:1. For this reason, a rising

ratio is the best evidence of significant sequestration.

5.10.5. Schilling’s test

5.10.5.1. Physiology

Vitamin B

is not synthesized by plants or animals, but is produced by

microorganisms found in the soil and in the intestines and rumens of animals.

Dietary vitamin B

comes from meat and dairy products.

Vitamin B

is primarily stored in the liver. Total body stores are high and

daily excretion low. It takes three to five years to develop vitamin B

deficiency if dietary intake is halted or malabsorption occurs. Thus, vitamin B

5.10. HAEMATOLOGY

369

deficiency caused by diet is rare (unlike a deficiency of folate), occurring only

in strict vegetarians.

For absorption, vitamin B

must be complexed with intrinsic factor (IF),

which is a protein secreted by parietal cells in the gastric fundus. Vitamin B

-IF

complex moves down the bowel and is absorbed in the terminal ileum. Its

absorption requires an alkaline pH and calcium. The vitamin B

-IF complex

dissociates and vitamin B

diffuses or is actively transported across mucosa.

Vitamin B

enters the portal vein, bound to transcobalamin-II transport

protein and is delivered to the liver. Over the next 8–12 hours, a portion re-

enters the circulation, binding to a larger transport protein, transcobalamin-I.

When the storage capacity of transcobalamin-I is exceeded, vitamin B

excreted.

Vitamin B

deficiency is caused by several mechanisms:

(a) Decreased intrinsic factor:

—Pernicious anaemia (usually caused by autoimmune disease);

—Gastrectomy.

(b) Intestinal malabsorption in the terminal ileum:

—Short bowel syndrome;

—Sprue;

—Regional enteritis;

—Lymphoma.

—Chronic pancreatitis;

—Cystic fibrosis.

(d) Fish tapeworm infestation.

5.10.5.2. Background

The following conditions are clinical manifestations of vitamin B

deficiency:

(a) Megaloblastic anaemia – this may be absent early in the disease. Because

of the close metabolic relationship of vitamin B

and folate, folate

administration can correct anaemia. Unfortunately, the neurological

changes are unaffected and can progress. For this reason, it is important

to differentiate folate from vitamin B

deficiency.

(b) Thrombocytopenia.

(d) Subacute combined degeneration of the spinal cord.