Nuclear Medicine Resources Manual

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CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

390

5.11.5.3. Procedure

The following procedure should be adopted:

(1) Add 1 mL of 0.9% NaCl to the stannous tartrate vial and shake well, to

dissolve the powder.

(2) Aseptically draw up 20–25 mCi of

99m

TcO

, ideally from a previously

eluted generator (within 24 hours).

(3) Carefully open the Infecton vial and draw up the contents into a 3 mL

syringe with a 25 gauge needle or insulin syringe to minimize the

formation of metal complex.

(4) Add the Infecton to the stannous tartrate vial and place the vial in a

suitable lead shield.

(5) Immediately add the

99m

TcO

to the Infecton through a 25 gauge needle.

Shake the contents of the vial.

(6) Leave the contents of the vial to incubate at room temperature for 10–

min.

(7) Mark a syringe ‘

99m

Tc-Infecton’ and draw the dose (370 MBq (10 mCi))

with a 25 gauge needle. Measure the dose in a dose calibrator and place

the dose inside a lead syringe shield for injection.

(8) Using an insulin needle or a TB syringe with a 25 gauge needle, apply a

spot on a Whatman No. 1 strip. Develop the strip in MEK.

(9) Cut the strip into two at two thirds of the length from the origin and count

each half. The

99m

Tc-Infecton remains at the origin, while the free

99m

TcO

moves to the solvent front, i.e.:

Percentage of radiochemical purity

5.11.5.4. Quality assurance log

The following forms should be completed:

Material Supplier & log No. Expiry date Quantity

Infecton ___________________ _______________ ______________

Stannous tartrate ___________________ _______________ ______________

99m

TcO

___________________ _______________ ______________

0.9% NaCl ___________________ _______________ ______________

counts of the lower half

¥ 100%.

counts of the upper half + lower half

5.11. INFLAMMATION AND INFECTION

391

Quality control of

99m

Tc-Infecton

—Stationary phase: ITLC-SG;

—Mobile phase: MEK;

—Counts of upper half = ______;

—Counts of lower half = ______;

—Percentage of radiochemical purity = ______ ¥ 100% = ______%.

Prepared by _____________________________________________ Date____________

5.11.6. Interpretation

(a) Interpretation of white cell images

Normal white cell images show transient lung uptake, persistent normally

increased spleen uptake with some uniform liver uptake and widespread red

bone marrow uptake. Four hours after injection there is evidence of some

urinary and caecal activity. A poor white cell preparation may show persistent

lung uptake and a more equal spleen-to-liver uptake ratio. The thyroid does

not need to be blocked, but some thyroid uptake may be seen if there is a

delayed injection. Blood pool activity is persistent and decreases slowly with

time.

White cell uptake will persist during the healing phase of osteomyelitis

even when the bacterial infection has been treated successfully. Lack of white

cell uptake may occur in chronic inflammation when a more monocytic or

lymphocytic infiltration is present or when there has been prolonged antibiotic

therapy for resistant infection. This may be due to loss of leucotaxin and

related cytokine stimulation. Technetium-99m HIG or

Ga-citrate uptake may

persist in such situations.

(b) Interpretation of HIG images

HIG images show a generalized high blood pool and marrow distribution

in normal individuals. Liver uptake may be more marked than spleen uptake

and later images may show renal uptake, urinary excretion and gut activity.

99m

Tc-Infecton images

Technetium-99m Infecton accumulates at the sites of acute bacterial

infection in sufficient amounts to be imaged. No thyroid blockade is required.

CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

392

This procedure is contraindicated in patients who are sensitive to ciprofloxacin,

pregnant or suspected to be pregnant, lactating, have renal failure or are under

the age of 14. About 370 MBq (10 mCi) are injected intravenously. Anterior

and posterior regional or whole body images are acquired at 1 and 3–4 hours,

and in some patients at 24 hours after injection, using a large FOV gamma

camera set with a low energy, general purpose, parallel hole collimator, peaked

to 140 keV with a 15% window. Images with 500 000 counts are collected.

Infecton is a small highly diffusible molecule that enters sites of inflam-

mation non-specifically and leaves progressively as blood level falls through

renal clearance. Sites of acute inflammation, such as an active rheumatoid joint,

will show initial uptake that may be maintained for the first four hours or fade.

A 24 hour image will show fading in a rheumatoid joint or other active inflam

matory arthropathy but there will be persistence of uptake in septic arthritis,

since binding to the dividing bacteria persists. A 24 hour image is also helpful in

suspected endocarditis, vascular or orthopaedic prosthesis and fracture. One is

generally not required when the site of uptake is obvious on the early images or

when chest or abdominal infection is suspected.

Technetium-99m Infecton is excreted into the bladder, where activity

increases with time so that voiding should be undertaken between sets of

images. Initial blood pool activity in the liver and spleen decreases in the later

images. No activity is seen in normal bone marrow, bone, muscle or soft tissue.

Exceptions are bone epiphyses in children, where symmetrical uptake is seen.

Occasionally, scrotal activity may be observed in adults as a result of vascu

larity. Biological barriers prevent access of intravenously injected agents, while

bacterial flora does not usually divide actively. As a result, normal gastrointes

tinal flora does not take up Infecton. There is, however, occasional weak biliary

excretion with some caecal activity seen after 4 hours. Definite biliary or

intestinal uptake after 1 hour is pathological. Uptake in the gut after 4 hours is

considered as a normal variant in Asians, probably as a result of active ‘normal’

flora.

(d) Abnormal Infecton images

There is focal uptake on the first image around the site of an abscess (but

not in its centre where the pus contains dead bacteria) and at other infection

sites containing actively dividing bacteria. There may be 10

bacteria in 1 mL of

infected material, giving a great number of binding sites for ciprofloxacin.

The great advantage of

99m

Tc-Infecton imaging is the lack of normal bone

marrow uptake, so that sites of infection in the spine are positive even when a

white cell scan reveals a ‘cold’ defect. When infected, hip and knee prostheses

show uptake and beading around the prosthesis much more clearly than with

5.12. RADIOIMMUNOASSAY PROTOCOLS

393

white cells or

Ga. This is partly due to the easy penetration of the small

molecules of ciprofloxacin.

In the bowels, inflammatory but non-infective diseases such as Crohn’s

disease and ulcerative colitis show negative scan findings, but an associated

abscess may yield a positive image. Enteritis due to clostridia or salmonella will

usually be positive. It is interesting to note that gut bacterial infections tend to

be segmental rather than diffuse in parts of either the small or large intestine. It

has been found that active tuberculosis takes up Infecton.

Renal abscesses may be detected, provided enough time is allowed for the

renal excretion of the agent to be completed. Single photon emission tomography

may be helpful in hip prostheses. In diabetic feet, it can distinguish skin from bone

infections. In the heart, serial images will show persistent uptake in valve infections

as the blood pool clears. The technique is particularly useful in demonstrating

whether infection is present around a pacemaker, as well as in the sternal split after

open chest cardiac surgery. Given the normal lack of marrow uptake, persistent

sternal uptake indicates infection rather than a response to surgery.

BIBLIOGRAPHY TO SECTION 5.11

BECKER, W., “Immunoscintigraphy of infective lesions”, Immunoscintigraphy: Facts

and Fiction (MUNZ, D.L., EMRICH, D., Eds), Excerpta Medica International

Congress Series, Elsevier Science, Amsterdam (1990) 159–171.

COLEMAN, R.E., “Radiolabeled leukocytes”, Nuclear Medicine Annual 1982

(FREEMAN, L.M., WEISHMAN, H.S., Eds), Raven, New York (1982) 119–141.

JOSEPH, K., HOFFKEN, H., BOSSLET, K., SCHORLEMMER, H.U., Imaging of

inflammation with granulocytes labelled in vivo, Nucl. Med. Commun. 9 (1988) 763–769.

McAFEE, J.G., GAGNE, G., SUBRAMANIAN, G., SCHNEIDER, R.F., The locali-

zation of indium-111-leukocytes, gallium-67 polyclonal IgG and other radioactive agents

in focal inflammatory lesions, J. Nucl. Med. 32 (1991) 2126–2131.

5.12. RADIOIMMUNOASSAY PROTOCOLS

Protocol 1a: Preparation of hormone (T4/T3) free serum

The following procedure is used:

(a) Collect a pool of human serum, preferably from a range of donors. Each

donation must be individually tested for hepatitis B markers and anti-

CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

394

HIV antibodies (the antibodies to the AIDS virus). Sera found positive

should not be used. Note that serum pools may yield false results when

tested for viruses. Alternatively, donor horse serum may be used. This

avoids the potential infection risk.

(b) Filter the serum through a coarse filter (e.g. glass wool) to remove coarse

particles.

125

I-T4 to give approximately 2000 counts/(min·mL). Allow time for

equilibration and remove 1 mL for counting. This yields the initial counts

needed to monitor the removal of endogenous hormone.

(d) Assemble a 50 mL disposable syringe to serve as a column as illustrated in

Fig. 5.2.

(e) Weigh out Celite (Celite Hyflo-Super-Cel, Koch Light) and charcoal

(Norit PN5) in the ratio 1:4. Typically, using a 50 mL syringe, weights

would be 2 g Celite and 8 g charcoal.

(f) Mix the charcoal and Celite in a covered beaker or screw cap bottle and

add approximately 25 mL of distilled water. Continue to add water in

small amounts until a thick slurry is obtained that can be poured into the

column.

Serum

containing

125

I–T4

2000 Counts·min

–l

·mL

–l

Charcoal/Celite mix (4:1)

at least 4 cm

Celite 2–3 mm thick

Glass wool

Disposable syringe

Glass fibre filter

(Whatman GF/C)

FIG. 5.2. Column used for preparation of hormone (T4/T3)-free serum.

5.12. RADIOIMMUNOASSAY PROTOCOLS

395

(g) Using a Pasteur pipette, add the charcoal–Celite mixture to the column

and allow it to settle until the water is just visible at the top. Do not allow

to run dry.

(h) Gradually add approximately 50 mL of the serum to the column.

(i) Allow the column to run under gravity and discard the water void

volume. The first 1 mL of the eluted serum may also need to be

discarded.

(j) Collect the serum that has passed through the column. When all the

serum has entered the column, more water may be added to the top, to

facilitate the passage of the serum down the column.

(k) Count a 1 mL aliquot of the serum to monitor the removal of T4 and T3.

Normally, about 98% or more of endogenous hormone is removed by this

process.

Protocol 1b: Preparation of hormone-free serum

Preparation is by absorption by anion exchange resin stripping:

(a) Add

125

I-T3/T4 1000–2000 counts/(min·100 mL) of serum; incubate for

min for equilibration. Count an aliquot before addition of resin.

(b) Add AGI-X8 200–400 mesh Cl anion exchange resin (BioRad No. 140-

1451) in the labelled serum (300 mg/mL); stir at 37ºC for three hours.

(d) Count an aliquot of serum to monitor the stripping efficiency.

(e) Add further resin in the same proportions and repeat the operation for

another three hours.

(f) Filter the serum after 2 days of incubation with resin, followed by a coarse

filter (GF/D) and a finer filter (GF/B).

(g) Add 1:100 dilution of 10% sodium acid, store at –20ºC.

(h) Wash the used resin in 2 L of double distilled H

O; soak the washed resin

in double distilled H

(i) Check the stripping efficiency and count 100 mL serum for 1 min; repeat

the procedure if more than 5% of radioactivity is shown; the stripping

efficiency should be more than 95%.

Protocol 2: Preparation of T3/T4 standard

(a) Preparation

The following procedure is used:

CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

396

—Weigh: 3.255 mg (tri-iodothyronine, free acid, sigma) for T3

2.223 mg (L-thyroxine free acid, sigma) for T4.

—Dissolve with a small amount of ammonical (2N) ethanol (<0.5 mL) and

dilute in a 10 mL volumetric flask with a 50% propylene glycol aqueous

spectrophotometric check.

—Dilute stock standard with propylene glycol aqueous solution in a ratio of

1:10.

—Check that absorbency is: 320 nm for T3

325 nm for T4.

—Check that molar concentration (mm) is: OD/4658 for T3

OD/6210 for T4.

—Dilute the T3 solution to 1:20 with 0.05M phosphate buffer and 1:50 in

hormone-free serum. Finally, dilute the working solution to 1, 2, 4, 6 and

10nM.

—Dilute the T4 solution to 1:100 with 0.05M phosphate buffer and 1:10 in

hormone-free serum. Finally, dilute the working solution to 10, 50, 100,

150 and 300nM.

(b) Regeneration of resin

The following procedure is used:

—Rinse the resin with double distilled water.

—Stir with 1M NaOH (1 L NaOH = 300 g resin) for 2 hours at room

temperature.

—Filter and discard NaOH; rinse resin until the pH of the water returns to

7.0.

—Stir with 1M HCl (1 L HCl = 300 g resin) for two hours at room temper-

ature.

—Filter, discard HCl and rinse until the pH of the water returns to 7.0.

—Store the resin in a suitable dry container.

Protocol 3: Typical immunization schedule for production of polyclonal

antibodies to antigens in animals

(a) Preparation

The antigen should be as pure as possible since any impurities present

may also produce antibodies. This is especially important if the impurities are

structurally similar to the primary antigen (e.g. many steroids) or if the tracer

or standard used for the assay is likely to contain the same impurities.

5.12. RADIOIMMUNOASSAY PROTOCOLS

397

The high doses of antigen used for immunization may induce tolerance

and a poor antibody response. Do not exceed 100 mg/kg body weight of the

animal.

The first, and sometimes the second, immunization is given in Freund’s

complete adjuvant. Subsequent secondary immunizations are given in Freund’s

incomplete adjuvant. The complete adjuvant contains inactive TB and other

bacteria that are highly immunogenic. To make up the suspension, mix 1

mL of

adjuvant and antigen solution in a buffer in a syringe. Connect this syringe to

another syringe via a three way tap and pass the mixture several times from one

syringe to the other until it is completely emulsified. Good emulsion formation

is seen when a drop ‘floats’, rather than disperses, on the surface of the water.

Immunogens are always administered subcutaneously or intradermally,

never intravenously. The multisite injection technique is preferred. For subcu

taneous injection, 1 mL of emulsion at four to six sites along the back and neck

is used. Abscesses may form, and if these are causing great inconvenience, the

animal may need to be sacrificed. Where the intradermal route is used, the

back of the animal is shaved and 25–50 mL per injection of emulsion injected at

multiple (10–100) sites.

The more animals immunized, the greater the chances of obtaining

antisera. Generally, where rabbits are used, immunizing four to six animals may

yield at least two good antibody producers.

A small test bleed (about 2 mL from an ear vein in the case of rabbits or

guinea pigs) is taken two to four weeks after the first injection of immunogen.

If three test bleeds between four to twelve weeks do not provide any evidence

of antibody formation, it may not be worthwhile to continue and the animal

may be discarded.

Animals producing good antisera may be bled regularly, at twice weekly

intervals. About 20 mL can be collected from the ear vein of a rabbit, but with

guinea pigs, cardiac puncture will be necessary. The characteristics of

antibodies can change with time and each bleed needs to be tested separately.

If an animal producing good antibodies becomes ill, it should be carefully

observed and, if there is any likelihood of it dying, it should be sacrificed by

exsanguination under anaesthesia.

At each bleed, blood should be collected in glass tubes and allowed to

clot for one to two hours at room temperature and two to six hours at 4ºC. The

tubes should not be disturbed during the clotting process as haemolysis may

result. Centrifuge at 1500g for 15 min, preferably at 4ºC. If the clot is left for a

further period (e.g. overnight) at 4ºC, it may further retract, releasing more

antiserum.

CHAPTER 5. GUIDELINES FOR GENERAL IMAGING

398

(b) Immunization protocol

The following protocol is used:

(1) For primary immunization, use 50–100 mg/kg antigen in Freund’s

complete adjuvant. Inject subcutaneously or intradermally as described

above.

(2) Wait for two to four weeks.

(3) First make a test bleed, taking 2 mL of blood and testing for its binding

activity to the antigen.

(4) Then perform a secondary immunization using the same quantity of

antigen in Freund’s complete adjuvant.

(5) Bleed every two to four weeks, test the serum for binding activity; store

the serum as indicated in the next section.

(6) Repeat the secondary immunization every six to eight weeks as in step

(4), but using Freund’s incomplete adjuvant.

(7) Collect, test and store the serum as in step (5).

If a preservative such as 0.1% sodium acid (but not Merthiolate) is added,

antisera may be stored at 4ºC for several years.

Antisera are also stable for many years stored frozen at –20 or –40ºC.

Rapid (snap) freezing using a mixture of CO

and acetone is preferred. The

storage should be in aliquots that can be reconstituted just prior to use. Where

storage at –20ºC is convenient, the antiserum may be diluted in the ratio 1:10 in

buffer containing 0.1% sodium acid before aliquotting. At this temperature,

diluted antiserum is stable.

If freeze drying equipment is available, antisera may be lyophylized and

stored in aliquots for reconstitution immediately before use.

Protocol 4: Production of immunogens from haptens using the mixed

anhydride reaction

(a) Activation of hapten (steroid)

The following procedure is used:

—Add 40 mmol (5 mL) of N-methylmorpholine to 40 mmol of the steroid

derivate in 250 mL non-aqueous solvent (e.g. N,N-dimethylformamide

(DMF) or dioxane).

5.12. RADIOIMMUNOASSAY PROTOCOLS

399

—Cool to –15º C (+10ºC if using dioxane).

—Add 40 mmol (6 mL) isobutylchloroformate.

—React for 3 min at –15ºC.

(b) Conjugation to bovine serum albumin (BSA)

The following procedure is used:

—Add the activated steroid prepared as above slowly to 20 mg (0.3 mmol)

BSA in 800 mL DMF.

—React at –15ºC for one hour and then at 0ºC for three hours.

—Dialyse the product.

Protocol 5: Conjugation of T4 to BSA, using carbodiimide

The following procedure is used:

—Dissolve 20 mg of T4 in 5 mL of DMF.

—Dissolve 50 mg of BSA in 25 mL of distilled water.

—Add 30 mg of 1,1¢ carbonyl-di-imidazole (CDI) to the BSA solution and

adjust the pH to 5.5 with NaOH.

—Allow to react for 10 min.

—Add 10 mg more of CDI and adjust the pH to 5.5, if necessary.

—React overnight at room temperature in the dark.

—Dialyse and lyophylize the product.

Protocol 6: Antibody purification methods

(a) Preparation with ammonium sulphate

The following procedure is used:

—Dilute 3 mL of antiserum to 10 mL with 0.9% saline.

—Add 2.7 g of ammonium sulphate and stir mixture gradually (45%

saturation).

—Mix for one hour at room temperature.

—Centrifuge at 2000 rev./min for 30 min at room temperature.

—Re-dissolve the pellet in the minimum amount of phosphate buffer,

pH7.4 (or other buffer as may be used for the antibody coating).

—Dialyse against phosphate (or other coating) buffer.